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GEOGRAPHIC VARIATION IN THE SUSCEPTIBILITY OF FALSE CODLING MOTH, THAUMATOTIBIA

LEUCOTRETA, POPULATIONS TO A GRANULOVIRUS (CrleGV-SA)

Submitted in fulfilment of the requirements for the degree of Master of Technology Agriculture (Research) at the Nelson

Mandela Metropolitan University

By

JOHN KWADWO OPOKU-DEBRAH

Supervisor: Mr. Philip Retief Celliers

Co-Supervisor: Dr. Sean Douglas Moore

December 2008

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DECLARATION This is to certify that this dissertation represents entirely my own work and that all relevant

sources are duly acknowledged.

SIGNATURE DATE

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ABSTRACT The false codling moth (FCM), Thaumatotibia (=Cryptophlebia) leucotreta (Meyrick)

(Lepidoptera: Tortricidae) is a serious pest of citrus and other crops in Sub-Saharan

Africa. The introduction of the Cryptophlebia leucotreta granulovirus (CrleGV-SA)

Cryptogran and Cryptex (biopesticides) has proven to be very effective in the control of

FCM. However, markedly lower susceptibility of some codling moth (CM), Cydia

pomonella (L.) populations to Cydia pomonella granulovirus (CpGV-M), another

granulovirus product used in the control of CM’s in Europe have been reported. Genetic

differences between FCM populations in South Africa have also been established. It is

therefore possible that differences in the susceptibility of these geographically distinct

FCM populations to CrleGV-SA might also exist. To investigate this phenomenon, a

benchmark for pathogenecity was established. In continuation of previous work with

Cryptogran against the 1st and 5th instar FCM larvae, dose-response relationships were

established for all five larval instars of FCM. In surface dose-response bioassays, the

LC50 values for the 2nd, 3rd and 4th instars were calculated to be 4.516 x 104, 1.662 x 105

and 2.205 x 106 occlusion bodies (OBs)/ml, respectively. The LC90 values for the 2nd, 3rd

and 4th instars were calculated to be 4.287 x 106, 9.992 x 106 and 1.661 x 108 OBs/ml,

respectively. Susceptibility to CrleGV-SA was found to decline with larval stage and

increase with time of exposure. The protocol was used in guiding bioassays with field

collected FCM larvae. Laboratory assays conducted with Cryptogran (at 1.661 x 108

OBs/ml) against field collected FCM larvae from Addo, Kirkwood, Citrusdal and

Clanwilliam as well as a standard laboratory colony, showed a significant difference in

pathogenecity in only one case. This significant difference was observed between 5th

instars from the Addo colony and 5th instars from the other populations. Four

geographically distinct FCM colonies from Addo, Citrusdal, Marble Hall and Nelspruit

were also established. Since Cryptogran and Cryptex are always targeted against 1st

instar FCM larvae in the field, further comparative laboratory assays were conducted with

the Addo colony and an old laboratory colony. Cryptogran was significantly more

pathogenic than Cryptex against both the Addo and the old colony. However, a high level

of heterogeneity was observed in responses within each population.

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TABLE OF CONTENTS

Page number

Declaration………………………………………………………………………………………..II Abstract…………………………………………………………………………………………...III Table of contents……………………………………………………………………………….IV List of Figures…………………………………………………………………………………...XI List of Tables……………………………………………………………………………….......XV List of Abbreviations………………………………………………………………………...XVII Acknowledgements…………………………………………………………………………..XIX Dedication……………………………………………………………………………………….XX

CHAPTER ONE: BACKGROUND STUDY AND PROJECT PROPOSAL 1.1 INTRODUCTION……………………………………………………………………….……..1 1.2 THE SUBSTRATE (THE CITRUS FRUIT)……………………………………….…….….1

1.2.1 History of citrus in South Africa……………………………………….….……1

1.2.2 The citrus market………………………………………………………………….2

1.2.3 Cultivars grown……………………………………………………………..……..3 1.3 THE HOST (FALSE CODLING MOTH, Thaumatotibia leucotreta)…………………..3

1.3.1 History & Taxonomy……………………………………………………………...3

1.3.2 Economic Importance……………………………………………………………4

1.3.3 Seasonal history…………………………………………………………………..5

1.3.4 Distribution…………………………………………………………….…………..5

1.3.5 Nature and extent of injury………………………………………….…………..6

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1.3.6 Host range………………………………………………………….………………6 1.3.7 The Life cycle of FCM………………………………………..…………………...9

1.3.7.1 Egg………………………………………………………..……………….9 1.3.7.2 Larva………………………………………………………..……………10 1.3.7.3 Pupa……………………………………………………………………...12 1.3.7. 4 Adult……………………………………………………………………..13

1.3.8.1 Control Measures (Pre-harvest treatment)………………………………..14

1.3.8.1.1 Orchard sanitation…………………………………………………..14 1.3.8.1.2 Chemical control…………………………………………………….14

1.3.8.1.3 Biological control……………………………………………………15

1.3.8.1.3.1 Parasitoids…………………………………………………..15

1.3.8.1.3.2 Predators…………………………………………………….15

1.3.8.1.3.3 Pathogens…………………………………………………...15

1.3.8.1.4 Alternative control methods………………………………………16

1.3.8.1.4.1 Sterile Insect Technique (SIT)…………………………….16

1.3.8.1.4.2 Mating disruption / Pheromone technique……………….16

1.3.8.1.4.3 Attract and Kill………………………………………………16

1.3.8.2 Control measures (Post-harvest treatment)……………………………...17

1.3.8.2.1 Cold treatment……………………………………………………….17

1.4 THE PATHOGEN……………………………………………………………………………17

1.4.1 History and application of Baculoviruses…………………………….........17

1.4.2 Taxonomy…………………………………………………………………………18

1.4.3 Baculovirus Infection process………………………………………………...20

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1.4.4 Pathogenicity……………………………………………………………………..22 1.4.5 Virus variation……………………………………………………………………23

1.4.6 Host variation and disease resistance / low susceptibility………………25

1.4.7 Granuloviruses…………………………………………………………………..27

1.4.7.1 Host range………………………………………………………………27

1.4.7.2 Gross pathology and Symptomatology…………………………...28

1.4.7.2.1 Type 1 GV……………………………………………………..28

1.4.7.2.2 Type 2 GV..........................................................................28

1.4.7.2.3 Type 3 GV……………………………………………………..29

1.4.8 Cryptophlebia leucotreta granulovirus (CrleGV)…………………………..29

1.4.8.1 An overview of Cryptogran..………………………………………...30

1.5 BIOASSAY OF ENTOMOPATHOGENIC VIRUSES……………………………...........30

1.5.1 Bioassay Techniques…………………………………………………………...31

1.5.1.2 Mass dosing bioassay………………………………………………..33

1.5.1.2.1 Surface dosing bioassay……………………………………..33

1.5.1.2.2 Diet incorporation bioassay………………………………….34

1.5.1.2.3 Droplet feeding bioassay…………………………………….35

1.5.1.2.4 Egg-dipping Bioassay………………………………………..35 1.6 JUSTIFICATION…………………………………………………………………………….36 1.7 AIM……………………………………………………………………………………………37

1.7.1 Objectives…………………………………………………………………………37 1.8 EXPECTED OUTCOMES………………………………………………………………….38

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CHAPTER TWO: BENCHMARK DOSE-RESPONSE BIOASSAYS WITH FCM LARVAE 2.1 INTRODUCTION…………………………………………………………………………….39 2.2 MATERIALS AND METHODS………………………………………………………….....39

2.2.1 Virus purification protocol (using a glycerol gradient)…………………...39

2.2.2 Determination of virus concentration………………………………………..41

2.2.2.1 Protocol for virus enumeration…………………………………….…..41

2.2.3 Serial dilution technique for enumerated virus samples…………………43 2.2.4 Preparation of diet and polypots for viral inoculation……………………44

2.2.5 Conducting of bioassays using CrleGV-SA (Cryptogran) against FCM instar larvae……………………………………………………………….45

2.2.6 Statistical analysis………………………………………………………...........45

2.3 RESULTS……………………………………………………………………………………46

2.3.1 Virus purification………………………………………………………………..46

2.3.2 Incubation and determination of respective FCM larval instars………..46

2.3.3 Surface dose-response bioassays with 2nd instar FCM larvae………….47

2.3.4 Surface dose-response bioassays with 3rd instar FCM larvae…………..48

2.3.5 Surface dose-response bioassays with 4th instar FCM larvae…………..48

2.3.6 Combined dose-response bioassay data for all instars……………….…50 2.4 DISCUSSION………………………………………………………………………………..51 2.5 CONCLUSION………………………………………………………………………………54 CHAPTER THREE: DOSE-RESPONSE BIOASSAYS WITH CRYPTOGRAN AGAINST FIELD COLLECTED FCM LARVAE 3.1 INTRODUCTION……………………………………………………………………………55

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3.2 MATERIALS AND METHODS…………………………………………………………….55

3.2.1 Mass fruit collections from a range of geographic regions……………..55

3.2.2 Determination of parasitised larvae………………………………………….57 3.2.3 Diet preparation and conducting of assays………………………………...58

3.2.4 Surface dose - response bioassays with field collected FCM larvae…..59 3.2.5 Bioassays with laboratory reared 4th instar FCM larvae on a non-agar diet……………………………………………………….60

3.2.6 Statistical analysis………………………………………………………………60

3.3 RESULTS……………………………………………………………………………………60

3.3.1 Field collection of FCM larvae from citrus fruits…………………………..60

3.3.2 Parasitism…………………………………………………………………………62

3.3.3 Dose-response bioassays with laboratory reared 4th instar FCM larvae on a non-agar diet………………………………………………...62 3.3.4 Dose-response bioassays with field collected FCM larvae using an agar diet………………………………………………………………..63

3.3.5 Bioassays with field collected FCM larvae from Lone Tree Farm (Addo, Eastern Cape) using a non agar-based diet……………………….64

3.3.6 Bioassays with field collected FCM larvae from Tregaron Farm (Kirkwood, Eastern Cape) using a non agar-based diet…………..65

3.3.7 Bioassays with field collected FCM larvae from Rondegat Farm (Clanwilliam, Western Cape) using a non agar-based diet……….66

3.3.8 Bioassays with field collected FCM larvae from Jansekraal Farm (Citrusdal, Western Cape) using a non agar-based diet………….68

3.3.9 Susceptibility of field collected and laboratory reared 5th instar FCM larvae to CrleGV-SA using a non agar-based diet………………….69

3.4 DISCUSSION………………………………………………………………………………..71 3.5 CONCLUSION………………………………………………………………………………75

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CHAPTER FOUR: DOSE-RESPONSE BIOASSAYS WITH CRYPTOGRAN AND CRYPTEX AGAINST GEOGRAPHICALLY DISTINCT LABORATORY COLONIES OF FCM 4.1 INTRODUCTION…………………………………………………………………………….76 4.2 MATERIALS AND METHODS…………………………………………………………….76

4.2.1 Mass fruit collections for the establishment of geographically distinct FCM colonies…………………………………………………………..76

4.2.2 Determination of parasitised larva……………………………………………77

4.2.3 Laboratory rearing of field collected FCM larvae………………………….78

4.2.3.1 Small scale moth rearing using test-tubes…………………………...78

4.2.3.2 Large scale moth rearing using jam jars……………………………..79

4.2.4 Conducting of bioassays using Cryptogran and Cryptex against 1st instar FCM larvae………………………………………………….82

4.2.5 Statistical analysis………………………………………………………………83

4.3 RESULTS……………………………………………………………………………………83

4.3.1 Survival and development of field collected FCM larvae from Addo, Citrusdal, Marble Hall and Nelspruit…………………………83

4.3.2 Parasitism…………………………………………………………………………84

4.3.3 Relative humidity and temperature in the incubation chamber…………84

4.3.4 Bioassays with Cryptex against 1st instar FCM larvae from the old colony……………………………………………………………...85

4.3.5 Bioassays with Cryptogran against 1st instar FCM larvae from the old colony……………………………………………………………..86

4.3.6 Bioassays with Cryptex against 1st instar FCM larvae from the Addo colony………………………………………………………….88

4.3.7 Bioassays with Cryptogran against 1st instar FCM larvae from the Addo colony…………………………………………………………..89

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4.3.8 Comparison of assays between the Addo and the old FCM laboratory colonies with Cryptex and Cryptogran……………………….91

4.4 DISCUSSION……………………………………………………………………………….93 4.5 CONCLUSION………………………………………………………………………………96

CHAPTER FIVE: SUMMARY, RECOMMENDATION AND FUTURE RESEARCH

5.1 SUMMARY…………………………………………………………………………………..97 5.2 RECOMMENDATIONS…………………………………………………………………….98 5.3 FUTURE RESEARCH………………………………………………………………………99 REFERENCES…………………………………………………………………………………101 APPENDIX 1. PROBAN (Van Ark, 1995) output of probit analysis of surface-dose response bioassay data (LC) on an agar diet, with Cryptogran against laboratory reared 2nd, 3rd and 4th instar FCM larvae………………………………………………….....114 APPENDIX 2. SPPSS 11.0 Output data analysis of bioassays with laboratory reared and field collected FCM larvae from the Eastern Cape and Western Cape Provinces………………………………………………………………………………………..138 APPENDIX 3. PROBAN (Van Ark, 1995) output of probit analysis of surface-dose response bioassay data (LC) on a non- agar diet, with Cryptogran against laboratory reared 4th instar FCM larvae…………………………………………………………………..164 APPENDIX 4 PROBAN (Van Ark, 1995) output of probit analysis of surface-dose response bioassay data (LC) on an agar diet, with Cryptogran and Cryptex against 1st instar FCM larvae from the old colony……………………………………………………172 APPENDIX 5 PROBAN (Van Ark, 1995) output of probit analysis of surface-dose response bioassay data (LC) on an agar diet, with Cryptogran and Cryptex against 1st instar FCM larvae from the Addo colony…………………………………………………192 APPENDIX 6 Combined PROBAN (Van Ark, 1995) output of probit analysis of surface-dose response bioassay data (LC) on an agar diet, with Cryptogran and Cryptex against 1st instar FCM larvae from the old colony and the Addo colony….224 APPENDIX 7 Survival, development, incubation room temperature and humidity data for field collected FCM larvae from Addo, Citrusdal, Marble Hall and Nelspruit regions………………………………………………………………………………..235

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LIST OF FIGURES

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CHAPTER ONE Fig. 1.1 FCM (black arrow) feeding in a citrus fruit, marked by its characteristic frass (black granules discolouring the fruit – red arrow……………………………..6 Fig. 1.2 (a) a newly laid FCM egg; (b) two day old egg depicting the characteristic red coloration and (c); a fully developed neonate larvae about to hatch…………9 Fig. 1.3 First instar FCM larva boring into a citrus fruit……………………………………...10 Fig. 1.4 Pupae of FCM………………………………………………………………………….12 Fig. 1.5 An adult FCM on a citrus fruit………………………………………………………...13 Fig. 1.6 A diagrammatic representation of the taxonomy of baculoviruses……………….18 Fig. 1.7 (A) A typical granulovirus with single nucleocapsids per occlusion body (Bar = 0.5µm) (Tanada & Kaya, 1993) (B) a MNPV virus (Bar = 1.0µm) and (C) an SNPV with one nucleocapsid per virion embedded in the same occlusion body (Bar = 0.5µm)………………………………………………………...19 Fig. 1.8 The morphology of members of the Baculoviridae family of insect pathogenic viruses……………………………………………………………………..20 Fig. 1.9 A longitudinal section of the budded (A) and occluded virion (B) types. Note the characteristic thick protein coat enveloping the electron dense nucleocapsids of the occluded virus………………………………………...21 Fig. 1.10 Occlusion derived virus (ODV) and the budded virus, displaying their varying protein and lipid compostions. LPC - lysophosphatidylcholine; SPH – sphingomyelin; PC – phosphatidylcholine; PI – phosphatidyl inositol; PS – phosphatidylserine; PE – phosphatidylethanolamine……………………..21 Fig. 1.11 The life-cycle of a baculovirus through its host……………………………………22 Fig. 1.12 (A) Symptoms of a CrleGV infected fifth instar FCM (still alive), with inner body mass appearing whitish; (B) a 5th instar larva with brownish lesions due to infection (still alive); (C) a healthy 5th instar larvae; (D) a dead and distended virus infected 5th instar FCM larva……………………………………...29

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CHAPTER TWO Fig. 2.1 A 0.02 Thoma bacterial counting chamber. With the TL (top left), TR (top right), BL (bottom left), BR (bottom right) and one R (random) chamber, used in virus enumeration (virus particles: black arrow)……………….42 Fig. 2.2 A five-fold serial dilution of CrleGV-SA in distilled water [d (H2O)] used in surface dosage-response bioassays with 2nd instar FCM larvae……………..44 Fig. 2.3 Polypots with agar-based artificial diet being inoculated with CrleGV-SA for use in five-fold serial dilution surface dose-response bioassays with FCM larvae……………………………………………………………………………..44 Fig. 2.4 Comparison of dose-response probit lines for the 1st, 2nd, 3rd, 4th and 5th FCM instars. *L1 (first FCM instar), L2 (second FCM instar), *L3 (third FCM instar), *L4 (fourth FCM instar) and L5 (fifth FCM instar)…………………………………...50 Fig. 2.5 LC50 and LC90 for CrleGV-SA against a laboratory colony of FCM in a benchmark study…………………………………………………………………51 CHAPTER THREE Fig. 3.1 A map showing the citrus growing areas in South Africa, where FCM-infested fruit were collected……………………………………………………………………..57 Fig. 3.2 Preparation steps for the conducting of surface dose-response bioassays with CrleGV-SA at 1.661 x 108 OBs/ml against field collected FCM larvae. A size 000 paint brush, used in transferring larvae onto diet (blue arrow); individually cut diet plugs (black arrow); a glass pie dish (green arrow) and a polypot with its base removed used in cutting round diet plugs (red arrow). And an FCM infested fruit dissected for larval isolation (brown arrow)…………..58 Fig. 3.3 Total numbers of FCM larvae of each instar collected from a range of geographic regions in South Africa………………………………………………….61 Fig. 3.4 Percentage of parasitised FCM larvae of each instars collected from a range of geographic regions in South Africa………………………………………..62 Fig. 3.5 Mortality of field collected FCM larvae from Lone Tree Farm (Eastern Cape), in bioassays with CrleGV-SA at 1.661 x 108 OBs/ml……………………………...65 Fig. 3.6 Mortality of field collected FCM larvae from Tregaron Farm (Kirkwood, Eastern Cape) in bioassays with CrleGV-SA at 1.661 x 108 OBs/ml…………….66

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Fig. 3.7 Mortality of field collected FCM larvae from Rondegat (Western Cape) in bioassays with CrleGV-SA at 1.661 x 108 OBs/ml…………………………………67 Fig. 3.8 Mortality of field collected FCM larvae from Jansekraal (Western Cape) in bioassays with CrleGV-SA at 1.661 x 108 OBs/ml……………………………...68 Fig. 3.9 Control and CrleGV-SA treatment (at 1.661 x 108 OBs/ml) mortality for laboratory reared 5th instar FCM larvae…………………………………………….69 Fig. 3.10 Mortality for both field collected and laboratory reared FCM larvae in bioassays with CrleGV-SA at 1.661 x 108 OBs/ml………………………………..70 Fig. 3.11 False codling moth infestation on navel oranges (Addo, SRV, Eastern Cape): 2007 – 2008…………………………………………73 CHAPTER FOUR Fig. 4.1 (A) FCM infested navel oranges collected from the field for the establishment of FCM laboratory colonies. (B) A dissected navel orange revealing infestation of a 5th instar FCM larva. (C) Test-tubes (28 ml capacity) each containing an FCM larva on diet stored in an incubation chamber at 27o ± 1oC……………………………………...78 Fig. 4.2 Layout of an oviposition apparatus with; (a) a kitchen sieve (green arrow) inverted over a wax paper (black arrow) holding ovipositing moths; (b) a test -tube (28 ml capacity) in which a moth has recently eclosed (red arrow) (c) a test tube (blue arrow) transferring a freshly eclosed adult moth into the sieve…..79 Fig. 4.3 Jam jars (370 ml capacity) containing diet with larvae feeding in diet and pupating in cotton wool………………………………………………………….80 Fig. 4.4 A wax paper containing FCM eggs………………………………………………….81 Fig. 4.5 A custom built moth emergence and oviposition structure with wax paper sheets (black arrow) fitted to each compartment on which eggs are laid. Moist cotton wool is plugged at the top of each compartment – serving as a source of water for the moths (blue arrow)……………………………………81 Fig. 4.6 Percentage survival and development of field collected FCM larvae to adulthood………………………………………………………………………………..83 Fig. 4.7 Proportion of parasitised larvae recovered from field collected larvae from Addo……………………………………………………………………………….84 Fig. 4.8 Dose-response probit lines (four replicates) from bioassays conducted with Cryptex (CrleGV-SA) against 1st instar larvae from the old colony…………86

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Fig. 4.9 Dose-response probit lines (four replicates) from bioassays conducted with Cryptogran (CrleGV-SA) against 1st instar larvae from the old colony…….88 Fig. 4.10 Dose-response probit lines (three replicates) from bioassays conducted with Cryptex (CrleGV-SA) against 1st instar larvae from the Addo colony……..89 Fig. 4.11 Dose-response probit lines (four replicates) from bioassays conducted with Cryptogran (CrleGV-SA) against 1st instar larvae from the Addo colony…90 Fig. 4.12 Dose-response probit lines for the bioassays: Old – Cryptex, Old – Cryptogran, Addo – Cryptex and Addo – Cryptogran treatments………..92 Fig. 4.13 LC50 and LC90 of Cryptogran and Cryptex for two laboratory colonies of FCM (Addo and an old colony)……………………………………………………..92

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LIST OF TABLES

CHAPTER ONE Page number Table 1.1 Gross historical export value (Rands) of citrus varieties in South Africa………..2 Table 1.2 Cultivated plants reported as hosts of FCM………………………………………..7 Table 1.3 Wild plants reported as hosts of FCM………………………………………………7 CHAPTER TWO Table 2.1 Duration of FCM life cycle (egg - adult) at 27oC ± 1 room temperature……….46 Table 2.2 Mortality of 2nd instar larvae, in a dose response (five-fold) bioassay with CrleGV-SA……………………………………………………………………...47 Table 2.3 Mortality of 3rd instar larvae, in a dose response (five-fold) bioassay with CrleGV-SA……………………………………………………………………...48 Table 2.4 Mortality of 4th instar larvae, in a dose response (five-fold) bioassay with CrleGV-SA……………………………………………………………………...49 Table 2.5 Mean LC50 and LC90 for all FCM larval instars with CrleGV-SA………………..51

CHAPTER THREE Table 3.1 Passport data of FCM infested citrus fruit sampled from the Eastern Cape Province for the conducting of assays…………………………………………….56 Table 3.2 Passport data of FCM infested citrus fruit sampled from the Western Cape Province for the conducting of assays…………………………..56 Table 3.3 FCM larvae collected from navel oranges from a range of geographic regions from December 2007 to May 2008……………………………………….61 Table 3.4 Mortality of 4th instar FCM larvae in five-fold dose response bioassays with CrleGV-SA……………………………………………………………………...63 Table 3.5 Control mortality of FCM larvae in bioassays (on an agar-based diet), collected from Lane Late navel oranges at Lone Tree Farm (Addo, SRV)……63 Table 3.6 Treatment mortality of FCM larvae in bioassays (on an agar-based diet), collected from Lane Late navel oranges at Lone Tree Farm (Addo, SRV)…..64

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Table 3.7 Control and CrleGV-SA treatment (1.661 x 108 OBs/ml) mortality for field collected FCM larvae from Lone Tree Farm (Addo, Eastern Cape) in bioassays on an agar-based diet………………………………………………….64 Table 3.8 Control and CrleGV-SA treatment (1.661 x 108 OBs/ml) mortality for field FCM larvae from Tregaron Farm (Kirkwood, Eastern Cape) in bioassays on an agar-based diet…………………………………………………..66 Table 3.9 Control and CrleGV-SA treatment (1.661 x 108 OBs/ml) mortality for field FCM larvae from Rondegat Farm (Clanwilliam, Western Cape) in bioassays on an agar-based……………………………………………………67 Table 3.10 Control and CrleGV-SA treatment (1.661 x 108 OBs/ml) mortality for field FCM larvae from Jansekraal Farm (Citrusdal, Western Cape) in bioassays on an agar-based…………………………………………………..68 Table 3.11 Control and treatment mortality for laboratory reared 5th instar FCM larvae...69 Table 3.12 Mortality of field collected and laboratory reared 5th instar FCM larvae……...70

CHAPTER FOUR Table 4.1 Passport data of FCM infested citrus fruit sampled from the Eastern Cape, Western Cape and Mpumalanga Provinces for the establishment of laboratory colonies……………………………………………...77 Table 4.2 Mortality of 1st instar FCM larvae from the old colony in a dose-response bioassay with Cryptex………………………………………………………………85 Table 4.3 Mortality of 1st instar FCM larvae from the old colony in a dose-response bioassay with Cryptogran…………………………………………………………..87 Table 4.4 Mortality of 1st instar FCM larvae from the Addo colony in a dose-response bioassay with Cryptex…………………………………………….88 Table 4.5 Mortality of 1st instar FCM larvae from the Addo colony in a dose-response bioassay with Cryptogran………………………………………..90 Table 4.6 Comparison of probit line slopes from dose-response bioassays with Cryptex and Cryptogran against 1st instar FCM larvae from two different laboratory colonies…………………………………………………………………..91 Table 4.7 Mean LC50 and LC90 values for 1st instar FCM larvae from Addo and the Old colony treated with Cryptogran and Cryptex…………………………………93

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LIST OF ABBREVIATIONS

BV – budded virus

ºC – degrees Celsius

CM – Codling moth

CpGV – Cydia pomonella granulovirus

CpGV-M – Mexican isolate of Cydia pomonella granulovirus

CrleGV – Cryptophlebia leucotreta granulovirus

CrleGV-CV – Cape Verde isolate of Cryptophlebia leucotreta granulovirus

CrleGV-CV3 – strain or genotype number 3 of a Cape Verde isolate of Cryptophlebia

leucotreta granulovirus

CrleGV-SA – South African isolate of Cryptophlebia leucotreta granulovirus

DNA – Deoxyribose nucleic acid

e.g. – example

et al. – et alia (and others)

FCM – false codling moth

Fig. – figure

g – gram

GV – granulovirus

ha – hectare/s

HabrGV – Harrisina brillians granulovirus

HaGV – Helicoverpa armigera granulovirus

IPM – integrated pest management programme

LC – lethal concentration

LC50 – median lethal concentration

LC90 – 90 % lethal concentration

LC99.9 – 99.9 % lethal concentration

LD50 – median lethal dosage

LT – lethal time

LT50 – median lethal time

LT90 – 90 % lethal time

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Ltd – Limited

min – minutes

ml – millilitre

mm – millimetre

MNPV – multiply enveloped nucleopolyhedrovirus

NPV – nucleopolyhedrovirus

SNPV –single nucleocapsids NPVs

ODV – occlusion derived viruses

OB – occlusion body

PbGV – Pieris brassicae granulovirus

PrGV – Pieris rapae granulovirus

Rpm – revolutions per minute

SDS – sodium dodecyl sulphate

SE – standard error

SIT – sterile insect technique

SlNPV – Spodoptera littoralis nucleopolyhedrovirus

SNPV – singly enveloped nucleopolyhedrovirus

SpfrGV – Spodoptera frugiperdagranulovirus

TnGV - Trichoplusia ni granulovirus

sp. – species

µl – microlitre

µm – micron

XecnGV – Xestia c-nigrum granulovirus

º – degrees

% – percent

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ACKNOWLEDGEMENTS My sincere thanks to:

• My supervisor, Mr. P. R. Celliers for his excellent guidance, advice and support

through out this project. Dankie man!

• My co-supervisor, Dr. Sean Moore (CRI) for giving me a project topic and

sometimes taking extra work just to make this project a success. It has always

been an honour working with you.

• Prof. Pieter Van Niekerk, for your valuable assistance and encouragement.

• Dr. Jacques Pietersen, for your help with statistical analysis.

• Wayne Kirkman, for the technical assistance and guidance towards this project.

• Nyameka, for the technical assistance and support.

• Craig Chambers, technical assistance and support.

• Ursula Gutsche, for assistance.

• Citrus Research International (CRI) for funding this project. It has always been a

pleasure to work with such a reputable institution, the experience gained is

invaluable.

• River Bioscience (Pty) Ltd. for their technical assistance and providing virus

inocula for this study.

• All the citrus growers who provided citrus material for this studies.

• Nelson Mandela Metropolitan University, for providing a scholarship for this study.

• All the lecturers and staff at the Department of Agriculture and Game

Management.

• My parents, for their perpetual love and support throughout my entire career.

• My brother Kofi Kyei, for the huge financial support towards this study. Thanks bro,

I owe you one!

• The Almighty God, for giving me the spiritual wings to come this far.

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DEDICATION

To my late brother, KWAME AMOAH OPOKU-DEBRAH

1

CHAPTER ONE

BACKGROUND STUDY AND PROJECT PROPOSAL 1.1 INTRODUCTION The false codling moth (FCM), Thaumatotibia (=Cryptophlebia) leucotreta (Meyrick)

(Lepidoptera: Tortricidae) is a pest of citrus fruit, macadamias, avocadoes, stone fruit,

peppers and other crops in sub-Saharan Africa (Newton, 1998). In 2004, the estimated

annual loss incurred by the citrus industry of South Africa, as a result of FCM infestation

was about R100 million (US$14 million) (Moore et al., 2004a). Not only does the insect

damage citrus crops pre-harvest, but its phytosanitary status (quarantine status) is such

that the detection of a single larva in fruits marked for export could result in the entire

consignment being rejected (Moore, 2002; Hattingh, 2006). Conventional control methods

– using chemical insecticides are fraught with problems. The most common being the

high residues left on fruits (post harvest), warranting stricter residue restriction limits

imposed by overseas markets. Another big problem is the non-target effects of sprays –

which kills other beneficial organisms, leading to secondary pests outbreaks. Other

problems include the safety and environmental risks involved in their usage, coupled with

the recent development of resistance - of the pest, to some of these chemical insecticides

(Hofmeyr & Pringle, 1998). The introduction of biopesticides, Cryptophlebia leucotreta

granulovirus (CrleGV-SA) such as: Cryptogran (Moore, 2002; Moore & Kirkman, 2004;

Moore et al., 2004a; Moore et al., 2004b) and Cryptex (Kessler & Zingg, 2008) have

shown promise in controlling this pest. Studies on the susceptibility of geographically

distinct FCM populations in South Africa to these biopesticides will be the focus of this

study.

1.2 THE SUBSTRATE (THE CITRUS FRUIT) 1.2.1 History of citrus in South Africa According to Ida (2005), the exact origin of the genus citrus is unknown, although it is

believed to have originated from South-East China, the Malay Peninsula and Burma. Ida

2

(2005) adds on that, the first documented work on citrus dates as far back as 800 B.C,

314 B.C, 310 B.C. and 1650 A.D., in India, China, Europe and Southern Africa

respectively. Consequently, in South Africa the initial arrival of citrus was documented in

the journal of Jan van Riebeeck (the first governor of the Dutch colony in Cape Town)

(Ida, 2005). The first evidence of the growth of citrus trees in South Africa was in 1654

near Table Mountain. It was only after 300 years that the industry recorded some growth.

This was due to the high demand in export value (National Agricultural Directory of South

Africa NADSA, 2005).

1.2.2 The citrus market In 2001 the total contribution of the major citrus growing countries in southern Africa;

Zimbabwe, Mozambique, South Africa and Swaziland combined was about 1.5% of world

production (NADSA, 2005). In 2002 to 2003, total annual production of citrus in South

Africa was about 1.9 metric tonnes (Delien, 2005). By 2005 revenue from the industry

was about two billion Rands, which was approximately 4.5% of the total agricultural gross

production value for that year (NADSA, 2005).

Table 1.1 Gross historical export value (Rands) of citrus varieties in South Africa Year Soft citrus Grape fruit Lemons & limes Oranges Total 1995 158,093,472 122,162,177 61,285,061 835,069,349 1,176,610,059 1996 194,365,445 139,268,461 87,419,709 903,328,854 1,324,382,469 1997 216,633,295 82,515,798 98,922,393 861,757,134 1,259,828,620 1998 321,040,343 280,053,462 92,201,486 1,183,254,135 1,876,549,426 1999 456,876,238 282,408,807 124,384,871 1,468,413,948 2,332,083,864 2000 346,435,747 219,882,407 143,315,657 950,390,985 1,660,024,796 2001 363,201,728 325,772,077 155,713,593 1,845,045,379 2,689,732,777 2002 404,903,412 383,349,582 258,605,159 1,842,103,548 2,888,961,701 2003 503,324,474 341,556,082 276,489,545 2,417,244,571 3,538,614,672 2004 605,903,316 405,088,857 371,270,484 2,176,861,138 3,559,123,795 2005 406,599,000 256,475,000 482,708,678 1,479,255,000 2,625,037,678 2006 471,692,000 466,582,000 361,064,000 1,744,071,000 3,043,409,000 Source: (Hardman, 2007).

Most of the citrus produced in South Africa is primarily for export. Thus like most countries

the South African citrus industry continues to face large-scale competition and very

vigorous export quality control requirements by their respective ports of entry (Hattingh,

3

2006). The United Kingdom and the European Union are the main export market

destinations. But, of late Japan, South Korea, Russia, Australia, China and USA have

been added to the list (NADSA, 2005; Hattingh, 2008). The industry has also made

significant progress in increasing market access to Vietnam, Malaysia, Thailand, Israel,

Jordan, Syria, Lebanon and Malaysia (Hattingh, 2008). Export volume of citrus has also

shown a steady increase over the years (Table 1.1) (Hardman, 2007). Currently, South

Africa is the second largest exporter of citrus after Spain (Hardman, 2007).

1.2.3 Cultivars grown South Africa grows a wide range of citrus cultivars. The most popular growing areas for

citrus in South Africa can be found in Limpopo (Tzaneen, Hoedspruit, Senwes TVL,

Letsitele and Letaba), in Mpumalanga (Nelspruit, Onderberg, Groblersdal and Marble

Hall), the North West (Rustenburg, Vaalharts), Kwazulu Natal (Pongola and Nkwalini),

Western Cape (Clanwilliam and Citrusdal) and the Eastern Cape (Gamtoos River Valley,

Kat River, Petensie and Sundays River Valley) (Ida, 2005; NADSA, 2005; Hardman,

2007).

1.3 THE HOST (FALSE CODLING MOTH, Thaumatotibia leucotreta) 1.3.1 History & Taxonomy The first literature on FCM was documented by Fuller (1901). Fuller initially referred to it

as, the Natal codling moth, due to its development, appearance, habits and nature of

damage it caused on citrus in KwaZuluNatal. Eight years later it was recorded as the

orange codling moth, Enarmonia batrachopa, from the Transvaal by Howard (1909).

Thereafter it was generally referred to as the false codling moth and was described

taxonomically by Meyrick (1912) as Argyroploce leucotreta (Eucosmidae: Olethreutidae).

Later on, Clarke (1958) transferred it to Cryptophlebia leucotreta (Meyrick). Cryptophlebia

leucotreta is commonly referred to as the false codling moth (FCM), since its habits were

considered to be ‘akin to that of the codling moth’ Cydia pomonella (L.) (Reed, 1974). The

false codling moth is also known as the tea seed borer and the red bollworm of cotton

(Newton, 1998). At present the false codling moth formerly known as Cryptophlebia

4

leucotreta has been re-classified by Komai (1999). It is now referred to as Thaumatotibia

leucotreta (Venette et al., 2003; Stibick, 2006).

1.3.2 Economic Importance Smith (1936) and Myburgh (1965) reports that, FCM continues to be a pest of economic

importance to citrus. According to Smith (1936), in an experiment conducted on the extent

of damage caused by fruit-fly and FCM in two successive seasons in the Western

Transvaal in South Africa, the damage caused by FCM alone on the Washington navel

cultivar of citrus, was 83.2% as opposed to 0.8% by the fruit fly. He emphasises this when

he states that, ‘the damage done by fruit-fly alone was so slight that the insect could

hardly be considered as a pest of economic importance’ (Smith, 1936).

Although the dry season tends to limit FCM host availability, it continues to thrive due to

abundance of irrigation systems in citrus orchards in southern Africa (Reed, 1974). In

Samaru (Northern Nigeria) FCM became a major problem of cotton in 1967 due to the

introduction of irrigation regimes during the dry season (Reed, 1974). FCM continued to

have a preference for ripening citrus fruits, of which the Washington navel was most

susceptible (Newton, 1998). According to Newton (1998), FCM larval development in

limes and lemons was rarely completed, of which he attributed to their high acid content.

A single larva can destroy an entire orange and the subsequent moth produced - in a few

days, depending on temperature, could then lay more eggs leading to the build up of

large larval populations leading to the destruction of a large number of fruits. However,

the degree of fruit damage was highly variable from orchard to orchard and even between

seasons (Begemann & Schoeman, 1999).

Fruit losses as a result of FCM attacks, range from below 2% to as high as 90% (Newton,

1998). FCM causes an annual loss of about R100 million (US$14 million) to the South

African citrus industry (Moore et al., 2004a). Not only does the insect damage citrus crops

pre-harvest, but its phytosanitary status (quarantine status) is such that the detection of a

single larva in fruits marked for export could result in the entire consignment being

5

rejected. This is because the pest does not occur in countries where citrus is exported

(Moore et al., 2004a; Hattingh, 2006).

1.3.3 Seasonal history The false codling moth is known to breed throughout the year in orchards where out-of-

season fruit is present (Stofberg, 1954). Reed (1974) and Myburgh (1987) states that,

FCM has no resting stage (diapause) and thus breeds all year round. The pest can

maintain itself in citrus orchards (navel and Valencia oranges), since larvae escaping just

prior to the picking of navel oranges in May or June have a pupal stage lasting about 35

days (Stofberg, 1954). Moths emerging during July to August can oviposit on Valencia

oranges, which normally become more heavily infested from June onward (Stofberg,

1954). The first eggs are laid on in-season fruit between October and December, and

reach large population numbers towards late summer and then gradually decline with the

onset of low winter temperatures (Newton, 1998). Where no out-of-season fruit is present,

populations are extremely low or even absent until the setting of the new crop the

following season (Newton, 1998).

1.3.4 Distribution FCM is mostly confined to the hot tropics and sub tropics (Karvonen, 1983). Studies by

Bredo (1933), Catling (1969), CIBC (1984), Gunn (1921), Hargreaves (1922), Hepburn

(1947), Jack (1916), Meyrick (1930), Muck (1985), Pearson (1958), Stofberg (1954),

Sweeney (1962), Thompson (1946), and Wysoki (1986), found FCM from Ethiopia and

Congo, Swaziland, Madagascar, Reunioun and St. Helena, Kenya, Uganda, South

Africa, Zimbabwe, Mauritius, Cape Verde Islands, Ivory Coast, Mozambique, Malawi,

Nigeria and Somalia, and Israel respectively.

6

1.3.5 Nature and extent of injury The female false codling moth lays most of her eggs directly on fruits. The entrance of the

fruit is conspicuous due to the frass thrown out by the insect (Fig. 1.1) (Newton, 1990;

Myburgh, 1987).

Figure 1.1 FCM (black arrow) feeding in a citrus fruit, marked by its characteristic frass (black granules discolouring the fruit – red arrow) (Source: Moore, 2002). When fully-grown, the larva bores its way out of the fruit to seek a site for pupation

(Newton, 1998). Around the point of infestation, the rind takes on a yellowish-brown

colour as the tissue decays and collapses. In the early stages of decay the symptoms are

relatively easy to recognize. Infested green fruit ripen prematurely and the wounding

process leads to fruit abscission. Fruit already showing colour-break tends to be a deeper

tone than usual and the point of entry tends to be paler than the background colouration

(Newton, 1990).

Oviposition on physically damaged and early-ripening fruits is considered to be much

greater than that on healthy ones in their normal stage of development. Larval penetration

adversely affects the physiological state of these fruits, leading to premature abscission.

Once a fruit has dropped to the orchard floor it plays host to a wide range of fungal

invaders and vertebrate and invertebrate feeders (Newton, 1998).

1.3.6 Host range FCM has been reported to attack a wide range of cultivated and wild plants (Pinhey,

1975; Daiber, 1980; Newton, 1998; Vennette, et al., 2003) (Tables 1.2 & 1.3).

7

Table 1.2 Cultivated plants reported as hosts of FCM (Pinhey, 1975; Daiber, 1980; Newton, 1998; Vennette, et al., 2003).

Common Name Scientific Name Avocado Persea americana Apricot Prumus armeniciata Banana Musa paradisica Bean Phaseolus spp. Cacao Theobroma cacao Citrus Citrus sinensis, Citrus spp. Coffee Coffea Arabica, Coffea spp. Cola Cola nitida Corn Zea mays

Cotton Gossypium hirsutum Grape Vitis spp. Guava Psidium guyjava Litchi Litchi chinensis

Loquat Eribotrya japonica Macadamia nut Macadamia ternifolia

Mango Mangifera indica Olive Olea europaea subsp. Europaea

Pepper/pimeto Capsicum spp. Persimmon Diospyros spp.

Plum Prumus spp. Pineapple Ananas comosus

Pomegrade Punica granatum Sorghum Sorghum spp.

Tea Camellia sinensis Table 1.3 Wild plants reported as hosts of FCM (Schwartz, 1981; Vennette, et al., 2003).

Common Name Scientific Name Bur weed Triumfeta spp. Bluebush Diospyros lycoides Bloubos Royena pallens

Boerboon Schotia afra Buffalo thorn Zizyphus mucronata Carambola Averrhoa carambola Castorbean Ricinnus communis

Chayote Sechium edule Cowpea Vigna unguiculata, Vigna spp.

Custard apple Amona reticulate Elephant grass Pennisetum purpureum English walnut Juglans regia

Governors plum Flacourtia indica Indian mallow Abutilon hybridium

8

Jakkalsbessie Diospyros mespiliforms Jujube Zizyphus jujube Jute Abutilon spp.

(Wild) Kaffir plum Harpephyllum caffum Kapok/copal Ceiba pentrandra

Kei apple Dovyalis caffra Khat Catha edulis

Kudu-berry Pseudolachnostylis maprouneifolia Lima bean Phaseolus lunatus

Mallow Hibiscus spp. Mangosteen Garciania mangostana

Marula Sclerocarya caffra, sclerocarya birrea Monkey pod Cassia petersiana

Oak Quercus spp. Okra Ablemoschus esculentus

Peacock flower Caesalpinia pulcherima Pride of De Kaap Bauhimia galpini

Raasblaar Combretum zeyheri Red milkwood Mumisops zeyheri

Rooibos/Bushwillow Combretum apiculatum Sida Sida spp.

Snot apple Azanza agarkeana Stamvrugte Chrysophylulum palismontanum

Sodom apple Calotropis procera Soursop Ammona muricata Stemfruit Englerophytum magaliesmontanum

Surinum cherry Eugenia uniflora Suurpruim/large sour plum Ximenia caffra

Water-bessie Syzygium cordatum Wag’n bietjie Capparis tomentosa

Weeping boerboon Scotia brachypetala Wild fig Ficus capensis

Wild medlar Vangueria infausta Wing bean Xeroderris stuhlmannii

Yellow-wood berries Podocarpus falcatus Yellow-wood, real Podocarpus latifolius

FCM has also been reported on Schotia afra, Ricinnus communis, Crassula ovata,

Opuntia ficus-indica, Passiflora caerulea, Asparagus crassicladus and Albuca spp.

(Kirkman & Moore, 2007).

9

1.3.7 The Life cycle of FCM In Stofberg’s (1939) view the complete life cycle (egg to adult) of FCM in summer, lasts

between 48 to 65 days. However, in winter it takes a much longer period of between 70 to

90 days to complete its life cycle. Stofberg (1939) further explains that there is much

variation in the number of days for FCM to complete its life cycle, of which temperature

and probably humidity play a significant role. Stofberg (1939) found 66 days as the

average duration for the complete life cycle and between 51/2 to 6 generations per annum

in citrus. Under natural conditions FCM generations are not that distinct with all the stages

been present when citrus fruits are in season (Stofberg, 1939).

1.3.7.1 Egg

Figure 1.2 (a) a newly laid FCM egg; (b) two day old egg depicting the characteristic red coloration and (c); a fully developed neonate larvae about to hatch (Source: Moore, 2002). A single female FCM can lay as many as 300 eggs, with an average of probably 100 eggs

(Stofberg, 1939). Hatching occurs at all times during the day (Daiber, 1979a). On citrus,

the incubation period is between 9 - 12 days in winter and 6 - 8 days in summer

(Stofberg, 1939). However, in laboratory cultures the incubation period varies

considerably depending on the temperature the eggs are exposed to. If kept at 25oC the

incubation period lasts between 3 - 5 days (Daiber, 1979a). FCM eggs have also been

reported to be parasitized by trichogrammatid parasitoids. When parasitized, the eggs

appear quite black (Sishuba, 2003).

When the first eggs are laid they are; flat oval shaped discs, with a granulated surface

and with measurements varying from 0.77 mm in length by 0.60 mm in width and up to 1

mm in diameter. The eggs are white to cream colored when initially laid (Fig. 1.2 a). Some

(a) (b) (c)

10

days after being laid the fertile eggs turns reddish (Fig. 1.2 b) and shortly before hatching,

the eggs turn black (Fig. 1.2 c) as the head capsule forms and becomes visible through

the transparent egg shell under the chorion prior to hatching (Daiber, 1979a).

In laboratory cultures, eggs are laid on any clean flat surface whereas on oranges they

are laid inconspicuously in depressions of the rind (Newton, 1998). Oviposition on

physically damaged and early ripening oranges is much greater than on healthy navel or

Valencia oranges in their normal stage of development (Newton, 1998).

In the field however, eggs are mostly laid singly and are generally placed a little distance

from each other, although occasionally two eggs may be found touching one another. The

majority of eggs are deposited on the rind of the citrus fruit, but some are placed on

leaves and exceptionally a few can be found on the twigs (Newton, 1998). Up to 65 eggs

have been observed on a single fruit but such high numbers are rare (Stofberg, 1954). As

population size increases in citrus not only are more fruits infested, but also there is the

tendency for more eggs to be laid on each fruit (Catling & Aschenborn, 1978).

1.3.7.2 Larva FCM larvae are very similar to those of the common codling moth, with dark heads. Fully-

grown larvae have dark-brown heads and are 15 – 20 mm long, pinkish-red with less

intense colour on the underside. The legs and prolegs are the same colour as the

abdomen, and there are inconspicuous white hairs on the body (Fuller, 1901). FCM

larvae have a characteristic dark anal comb, which is not present in the codling moth

(CM), Cydia pomonella. Upon emergence from the egg, the young larva bores its way into

the rind and in most instances into the centre of the fruit (Fig. 1.3) (Newton, 1990). An

emerging larva usually eats its way out of the eggshell (Stofberg, 1954).

Stofberg (1939) asserts that, the larval development of FCM lasts between 25 to 35 days

and 40 to 60 days, for winter and summer respectively. Newton (1998) also found FCM

larval development, to range from 25 - 35 days and 35 - 67 days in winter and summer

respectively. FCM has five larval instars, of which the first instar (neonate larva) (Fig. 1.3)

is extremely delicate and suffers high mortality. Low humidity, results in high mortality for

11

the egg and the first instar in laboratory cultures. Low winter temperatures on the other

hand are also lethal to these life stages in the field (Catling & Aschenborn, 1978).

Figure 1.3 First instar FCM larva boring into a citrus fruit (Source: Moore, 2002).

The young larva is often cannibalistic towards eggs and larvae, and this behaviour has

been associated with the fact that rarely more than one larva completes its development

in a single fruit (Annecke & Moran, 1982). However, many hundreds of larvae can be

reared in single containers containing artificial diet in laboratory cultures (Moore, 2002;

Ludewig, 2003; Sishuba, 2003). Nevertheless, care should be taken since poor food

quality in laboratory cultures increases larval mortality (Daiber, 1979b; Moore, 2002).

Immediately after hatching, the first instar finds a host fruit and a suitable entry point. In

navel oranges, it has been reported to have preference for the navel end (Newton, 1998).

The first instar larva is approximately 1 - 1.2 mm in length, with a dark pinacula giving it a

spotted appearance, whilst the fifth instar larvae (Fig. 1.12 C) are orangey-pink, becoming

paler on the sides and yellow on the ventral region, 12 - 18 mm long, with a brown head

capsule and first thoracic segment (Catling & Aschenborn, 1978). The last instar leaves

the fruit through a conspicuous, frass-filled exit hole and commonly drops to the ground

on a silken thread or emerges after the fruit has fallen (Catling & Aschenborn, 1978). The

mean head capsule width for the first to the fifth instar larvae has been recorded as; 0.22,

0.37, 0.61, 0.94 and 1.37 mm, respectively (Daiber 1979b).

12

1.3.7.3 Pupa

Figure 1.4 Pupae of FCM (Source: Moore, 2002). The FCM pupal stage consists of two sub-stages, the pre-pupal and the pupal stage (Fig.

1.4) (Stofberg, 1954). On the ground the larva spins a silky cocoon of trash and soil

particles. Larvae in artificial media (laboratory) normally form cocoons in the cotton wool

stoppers fitted into the opening of the rearing containers (Daiber, 1979c; Moore, 2002;

Sishuba, 2003). Inside the fresh cocoon the pre-pupa is found, this being the fifth instar

larva that has stopped feeding. The pre-pupa then molts into a pupa (Fig. 1.4), which at

first appears light brown and soft skinned until the chitin hardens and becomes dark

brown. Pupae from which female moths emerge are larger than those from which male

moths emerge (Daiber, 1979c). The abdominal segments are flexible and each has a

number of small spines. Those on the terminal segment are considerably longer and

thicker than on the others (Gunn, 1921). Daiber (1979c) also observed that the average

duration of the pre-pupal stage was 2 days at 25oC. In addition, the duration of the pupal

stage for the male moths was longer than that for the female moths; females taking 11

days and males taking 12 days at 25oC (Daiber, 1979c). In the field, the pupal stage lasts

between 12 to 24 days in summer and from 29 to 40 days in winter (Stofberg, 1954).

13

1.3.7. 4 Adult

Figure 1.5 An adult FCM on a citrus fruit (Source: Moore, 2002).

The adult stage (Fig. 1.5) commences when the moth emerges from the cocoon. The

adult moths have a superficial likeness in size and tone of colour to the true codling moth,

except that, the false codling moth lacks the coppery patches on the wings that are

present in the true codling moth (Fuller, 1901). The adult is a rather small, inconspicuous,

dark brown to grey moth. It is active at night from dusk onwards and is therefore seldom

seen in citrus orchards (Annecke & Moran, 1982). The fore wings are mottled while the

hind wings have a paler, more even colour and are fringed with hairs. The male is smaller

than the female and is distinguished by densely packed elongated scales on the hind

tibia, an anal tuft of scales and a scent organ near the anal angle of each hind wing

(Catling & Aschenborn, 1978).

The adult body length is normally 6 - 8 mm. The wingspan of female and male moths is

between 17 – 20 mm and 15 - 18 mm, respectively. The antennae are setiform with

distinct segments. Forewing coloration of the moth is similar between the sexes with grey,

black, brown and orange-brown markings (Couilloud, 1988).

Daiber (1980) reported that the sex ratio of FCM field populations is close to unity.

Females are reported to mate shortly after emergence with pre-oviposition periods lasting

from 5 - 6 days in the field and 1 - 2 days in laboratory cultures respectively. Daiber

(1980) also found that, the pre-oviposition period could vary from, between 1 - 22 days,

with 50% of the eggs being laid within 6 and 23 days after oviposition at 25oC and 10oC

respectively. Oviposition occurs at the highest rate in the early evening near sunset. FCM

is predominantly a warm climate pest with its development being limited by cold

14

temperatures (Diaber, 1980; Van Der Geest, et al., 1991). Exposure to temperatures

below 10oC reduces survival or development of several life stages, and eggs have been

reported to be killed at temperatures below 1oC (Van Der Geest, et al., 1991).

In the field, adults live for a week or two (Annecke & Moran, 1982). In captivity the

average life span of a male can vary from 14 days at 25oC to 34 days at 15oC, and that

of females from 16 days at 25oC and 48 days at 15oC (Daiber, 1980). According to

Catling & Aschenborn (1978), the adult feeds little or not at all, with water being essential

for extending longevity. Total development period is approximately 2.5 to 4 months in

winter and 1.5 to 2 months in summer, with five to six poorly defined overlapping

generations per year (Newton, 1998). Daiber (1980) also observed that, the generation

time in artificial media was 32 and 114 days at average ambient temperatures of 26.3oC

and 13.7oC, respectively. However, Stofberg (1954) recorded 37 days for the shortest and

107 days for the longest generation time in the field.

1.3.8.1 Control Measures (Pre-harvest treatment) 1.3.8.1.1 Orchard sanitation Previous studies carried out by Ullyett, (1939) and Stofberg (1939) found that, regular and

vigorous orchard sanitation carried out on navel oranges proved to be quite effective in

controlling FCM during seasons of high infestation rates. According to Schwartz (1974),

noticeable success in orchard sanitation is achieved by pulping infested fruits in a

hammer mill. This measure ensures the destruction of FCM larvae which could serve as a

source of re-infestation.

1.3.8.1.2 Chemical control Through the years the main focus on the application of insecticides, has been to ‘achieve

the highest kill possible’ (Debach, 1974). This philosophy however, comes with its own

problems. According to Debach (1974), in the 1980’s, despite the adoption of DDT as the

main means of controlling the scale insect which was devastating the entire citrus industry

in California (USA), there was hardly any progress. Rather, the importation of a natural

15

enemy to the scale rather saved the citrus industry in California. McMurray et al. (1970)

also reported similar trends with mites. Currently five chemical products Cypermethrin, Alsystin (triflumuron), Nomolt

(triflubenzuron), Penncap-M (micro-encapsulated methyl parathion) and Meothrin

(fenpropathrin) are registered for the control of FCM on citrus in South Africa (Moore,

2002). However, none of these above mentioned products are entirely compatible with an

integrated pest management programme (IPM). They are all considered detrimental to

natural enemies and therefore prone to causing secondary pest repercussions. FCM has

been reported to have developed resistance to Alsystin (Hofmeyr & Pringle, 1998).

1.3.8.1.3 Biological control 1.3.8.1.3.1 Parasitoids: Moore & Richards (2001) report that, an estimated release of

about 100000 parasitoids per hectare could reduce FCM infestation by 61%. The

Trichogrammatoidea cryptophlebiae (egg parasitoid) proved to be of potential, for use as

a biological control agent on FCM (Schwartz, 1981; Moore & Fourie, 1999). Sishuba

(2003) found Agathis bishopi (Nixon) (larval parasitoids) and T. cryptophlebiae (an egg

parasitoid) predominant in the Eastern Cape (South Africa). Gendall (2006) speculated

the augmentative release of A. bishopi for the control of FCM.

1.3.8.1.3.2 Predators

: Bugs (Orius insduosus, & Rhynocorus albopunctatus) and mites

(Pediculoides sp.) have been reported to attack FCM (Nyiira, 1970; Moore, 2002).

1.3.8.1.3.3 Pathogens

: Entomopathogenic microbes have shown promise in the control of

FCM. Currently two commercially produced insect viruses, Cryptophlebia leucotreta

granulovirus (CrleGV-SA), Cryptogran (Moore, 2002; Moore et al., 2004a) and Cryptex

(Kessler & Zingg, 2008) are used in the control of FCM in South Africa. The fungus,

Aspergillus alliceus (Moore et al., 1999) and Beauveria bassiana (Begemann, 1989) have

been reported to attack FCM in South Africa. Laboratory experiments by Malan & Moore

(2006) have also shown nematodes as having potential for the control of FCM.

16

1.3.8.1.4 Alternative control methods

1.3.8.1.4.1 Sterile Insect Technique (SIT) In Canada SIT proved to be successful in the control of codling moths (Bloem & Bloem,

2000). With this method sterile males are released at an over flooding ratio to the wild

males, in order to ensure that the probability of a female mating with a sterile male is

greater than her mating with a wild male (Myburgh, 1963; Hofmeyr et al., 2004).

Laboratory experiments by Hofmeyr et al. (2004) showed promise in the use of SIT in the

control of FCM in the Western Cape (South Africa). In a 35 hectare field trial, Hofmeyr &

Hofmeyr (2006) reported a 94.4% reduction in FCM infestation.

1.3.8.1.4.2 Mating disruption / Pheromone technique This method involves the release of synthetic female moth pheromones into the orchard

thereby confusing the male moths to follow the false pheromone trails, thus making them

unavailable for mating. Male moths exposed to high concentrations of the synthetic

pheromones become so desensitized that they no longer smell the natural pheromones

released by the female moths (Minks & Cardé, 1988). Hofmeyr et al. (1991) found this

method to be quite useful in the disruption of FCM males through field trials conducted in

citrus orchards at Citrusdal, Western Cape.

1.3.8.1.4.3 Attract and Kill

The attract and kill method is similar to the mating disruption technique. The only

difference is the incorporation of a pyrethroid, Last Call FCM (brand name) to the

synthetic pheromone. The product, Last Call FCM is normally applied in a droplet form on

trees. However, the product has been reported to be not as effective as the contemporary

mating disruption since the treatment is considered to be density dependent, and thus will

require a high percentage of kill in males to achieve significance (Hofmeyr, 2003).

17

1.3.8.2 Control measures (Post-harvest treatment) 1.3.8.2.1 Cold treatment Myburgh (1965) in an experiment on the effectiveness of low temperature in the control of

FCM found that cold sterilization, although, was very effective against FCM - extreme

temperatures could on the other hand resort in injuries to fruits destined for export. The

maintenance of the pre-shipment and in transit temperatures played a vital role in

controlling FCM. Myburgh (1965) reports that, FCM eggs proved to be the most

susceptible with the larvae being fairly resistant and the pupae slightly more resistant than

the former, at varying temperatures of -0.6oC, 1.1oC and 4.4oC. The internationally

accepted phytosanitary standard for the disinfection of FCM on citrus bound for export is

a 22 - day cold treatment, at temperatures of -0.6oC. But unfortunately this measure also

affects the quality of the citrus fruit (Hattingh, 2006). Jelbert (2006) speculates that, a new

method that involves the inclusion of CO2 with the normal 22 - day at -0.6oC cold

treatment could reduce the detrimental effects on fruit quality, as well as enhancing the

killing-time of FCM in fruits.

1.4 THE PATHOGEN 1.4.1 History and application of Baculoviruses The first accounts of baculovirus infection were documented in ancient Chinese literature

on silkworm, Bombyx mori culture, where the initial symptoms in the silk worm where

described as being ‘jaundiced’ (Miller, 1997). But in western literature, the first symptoms

in insect larvae were documented in a poem by an Italian bishop, Marco Vida of

Cremona, in 1527. Marco referred to these larval symptoms as ‘melting’ or ‘wilting’ (Benz,

1986). Similar research conducted in the 20th century confirmed the usefulness of

baculoviruses (Miller, 1997). In recent years a lot of research has been carried out on

baculoviruses as compared to that of other insect viruses, due to their promising

performance (Hunter-Fujitta et al., 1998).

18

1.4.2 Taxonomy The name ‘baculovirus’ was first coined from the Latin word baculum, which means ‘a rod’

or ‘rod-shaped’. Thus the baculovirus families are classified as rod-shaped virions due to

the characteristic shape of their nucleocapsids (Francki et al., 1991). Consequently,

baculoviruses are described as a family of rod-shaped, enveloped viruses having a

circular double-stranded DNA genome (Winstanley & O’Reilly, 1999).

Figure 1.6 A diagrammatic representation of the taxonomy of baculoviruses (Source: Murphy et al., 1995).

The Baculoviridae family was initially divided into three subgroups: subgroup A, nuclear

polyhedrosis viruses (NPVs); subgroup B, granulosis viruses (GVs); and subgroup C,

non-occluded viruses. The NPVs were further divided into the (a) multiple nucleocapsid

NPVs (MNPVs), with several nucleocapsids within their viral envelope (Fig. 1.7 B) and

the; (b) single nucleocapsids NPVs (SNPVs) with a single virion (Fig. 1.7C) (Murphy et

al., 1995; Winstanley & O’Reilly, 1999; Van Regenmortel et al., 2000).

BACULOVIRIDAE

GRANULOVIRUS (GV)

SNPV (Single nucleocapsids)

MNPV (Multiple nucleocapsids)

GV Only one (rarely two) nucleocapsid per OB

NUCLEOPOLYHEDROVIRUS (NPV)

19

Figure 1.7 (A) A typical granulovirus with single nucleocapsids per occlusion body (Bar = 0.5µm) (Tanada & Kaya, 1993) (B) a MNPV virus (Bar = 1.0µm) and (C) an SNPV with one nucleocapsid per virion embedded in the same occlusion body (Bar = 0.5µm) (Source: Maramarosch, 1977).

Members of the genus Nucleopolyhedrovirus have a polyhedral shape (0.15 µm x 15 µm)

with numerous virons in their crystalline occlusion body, while the granuloviruses are

ovicylindrical or granule-like (0.3 µm x 0.5 µm) and may contain one or rarely two virions

per occlusion body (Fig. 1.7A). Currently, the Baculoviridae family have been re-classified

into only two genera namely; (i) Nucleopolyhedrovirus, NPVs (formerly nuclear

polyhedrosis virus), and (ii) Granulovirus, GVs (formerly granulosis virus) (Fig. 1.6)

(Murphy et al., 1995; Miller, 1997). According to the Organization for Economic Co-

operation and Development, OECD (2002), presently the nomenclature of baculoviruses

is based firstly on the host insect from which the baculovirus was isolated, and secondly

on the type of occlusion body associated with it.

20

Figure 1.8 The morphology of members of the Baculoviridae family of insect pathogenic viruses (Hunter-Fujita et al., 1998). 1.4.3 Baculovirus Infection process The replicative stage of baculoviruses results in the production of two virion phenotypes.

The first are the occluded virion phenotypes, occlusion derived viruses (ODVs), which are

noticeable by their characteristic thick crystalline protein coat enveloping their electron

dense nucleocapsids (Fig. 1.9B). The ODVs initiate the primary infection and are

responsible for the transmission of infection via one larva to another in a population

(horizontal infection). Their characteristic thick protein matrix, which gives them some

level of resistance against ultraviolet light and mechanical stress, enables them to persist

in the environment. Their primary mode of infection involves attacking the midgut

epithelial cells of their host (Van Regenmortel et al., 2000; OECD, 2002).

Nucleopolyhedrovirus (NPV)

Granulovirus (GV)

21

Figure 1.9 A longitudinal section of the budded (A) and occluded virion (B) types. Note the characteristic thick protein coat enveloping the electron dense nucleocapsids of the occluded virus (Winstanely & O’Reillly, 1999).

Figure 1.10 Occlusion derived virus (ODV) and the budded virus, displaying their varying protein and lipid compostions. LPC - lysophosphatidylcholine; SPH – sphingomyelin; PC – phosphatidylcholine; PI – phosphatidyl inositol; PS – phosphatidylserine; PE – phosphatidylethanolamine (Funk et al., 1997).

The second viral phenotype, the budded virus (BV) (Fig. 1.9A & 1.10) initiates a

secondary infection in the midgut created by the ODVs via the newly produced

nucleocapsids. These newly produced nucleocapsids will then traverse the nuclear

membrane, the cytosol and then bud through the basal lamina of the midgut cells into the

haemolymph (Fig. 1.11). By this time they will have acquired a new virion envelope,

consisting of a plasma membrane containing polymers of a viral encoded glycoprotein,

called gp64 (Fig 1.10). Hence the BVs are only responsible for cell-to-cell transmission of

viral infection within susceptible host tissues (Van Regenmortel et al., 2000; OECD,

2002).

22

Figure 1.11 The life-cycle of a baculovirus through its host (Hunter-Fujita et al., 1998).

1.4.4 Pathogenicity Baculovirus pathogenesis is considered to be varied, between and within families (NPVs

and GVs). These notable differences have been restricted to their tissue tropism.

However there is a general consensus on their initial infection pathway (Federici, 1997).

The normal route of infection is the oral ingestion of virus particles. Once virus particles

have been ingested, they are readily dissolved in the highly alkaline midgut of infected

larvae (Summers, 1971; Federici, 1997). The released virions, from the occlusion bodies,

23

pass through the peritrophic membrane (a protective lining secreted by the midgut)

(Federici, 1997).

Generally, there is little consensus on the mechanism of passage of baculoviruses

through the peritrophic membrane of many insect species (Federici, 1997). There is also

considerable variation in the nature of the peritrophic membranes of different insects

(Federici, 1997). For example, the peritrophic membranes of the Douglas fir tussock moth

(Orgyia pseudotsugata) larva are considered to be fibrous (allowing particles less than

800 nm to pass), whilst that of Trichoplusia ni are multilayered and channeled (Adang &

Spence, 1983; Federici, 1997).

Enzymes have also been reported to play a role in facilitating the passage of virions

(although not known for all baculoviruses) through the peritrophic membrane of most

insects (Derkseen & Granados, 1988; Wang et al., 1994; Federici, 1997). Studies by

Lepore et al. (1996) showed that enzymes (enhancins) released by GVs attacking T. ni

(TnGVs) facilitated localized digestion of the peritrophic membrane creating lesions for

virion passage.

However, the peritrophic membrane is not considered the critical barrier to infection, since

it is known to be shed during molting - making newly molted larvae more susceptible to

infection, since virions come into direct contact with midgut microvilli. It is speculated that

most virus are ingested by larvae during the intermolt period (Washburn et al., 1995;

Federici, 1997; Sun, 2005). According to Summers (1971) and Federici (1997), for GVs,

viral DNA is normally injected by the nucleocapsids into the nuclear pore of the host cell

to initiate replication. But unlike the GVs, the NPVs have been reported to enter directly

into the nuclear pore before uncoating to release viral DNA.

1.4.5 Virus variation According to Cory et al. (1997), although baculoviruses are considered to be the most

studied group of insect viruses, their interactions within their host and environment

(ecology) has received relatively little attention. Baculoviruses isolated from one insect, or

24

a group of insects from a single species at one time and location, are referred to as

‘isolates’ (Cory et al., 1997).

Cory et al. (1997) further state that the systematic naming of baculoviruses after insect

species from which they are isolated is quite problematic. For instance, baculoviruses

found in the same area have been reported to vary considerably within isolates (Goto et

al., 1992). Recent studies have also shown that baculoviruses isolated from a sinlge

insect host, could exhibit high levels of genotypic variation (Parnell et al., 2002; Cooper et

al., 2003). Cory et al. (2005) found that, an NPV isolated from a single pine beauty moth,

Panolis flammena larva had twenty four genetically different genotypes and that all these

genotypes differed in both virulence and pathogenicity.

Other studies report high levels of genetic variation and biological properties observed

between isolates collected from the same insect species in different geographic locations

(Cory et al., 1997; Cory et al., 2005). One notable example is the marked genetic

difference observed in two novel CrleGV-SA isolates (Cryptogran and Cryptex) as

reported by Goble (2007). Several authors have also reported the occurrence of field-

collected isolates frequently showing differences in virus genotype (Lee & Miller, 1978;

Knell & Summers, 1981; Smith & Crook, 1988; Maeda et al., 1990).

This marked genotypic variation observed in baculoviruses has been attributed to small

mutations, sequence duplication or acquisition of host DNA (Brown et al., 1985).

However, with regard to phenotypic variation, Cory et al. (1997) speculate that when

mixed wild-type isolates are collected from the field, each time for reapplication – it leads

to the selection of particular variants which could negatively impact on the efficacy of

commercially applied baculoviruses. Cory et al. (1997) suggests that to avoid loss in virus

efficacy and yield, the clonal diversity of isolates should be determined, and those with

defined activity profiles selected for specific biocontrol programmes.

Conversely, other studies have found that different baculovirus species can co-infect

more than one host (Fritsch et al., 1990; Jehle et al., 1992; Lacey et al., 2002). The Cydia

pomonella granulovirus, CpGV has been reported to infect both codling moths and FCM.

25

However CpGV is noted to be more virulent to CM than to FCM. On the other hand CM is

not susceptible to the FCM GV, CrleGV (Fritsch et al., 1990; Jehle et al., 1992).

1.4.6 Host variation and disease resistance / low susceptibility Of late, there have been some global concerns regarding insect resistance to some of

these very useful biopesticides. Insects of the same species collected from different

regions, infected with baculoviruses have been reported to show variation in susceptibility

(Fuxa, 1987 & 1993). Fuxa (1993) reports of some observed variation in response

between different Spodoptera frugiperda populations to three NPVs found in the same

area. The explanation given for this was that there had been some immigration of insects

with different levels of susceptibility to the different virus isolates.

Briese & Mende (1981), observed some variation in response between different

populations of the potato tuber moth, Phthorimaea operculella to Phthorimaea operculella

granulovirus. Laboratory strains of the tuber moths which were used as a standard

comparison to some field strains, exhibited significantly greater resistance to the virus

having an LC50 value 30 times greater than any of the field populations (Briese & Mende,

1981). There are a number of other reports where differences in susceptibility between

geographically distinct insect host populations to baculoviruses were observed (Briese,

1986).

However, recent reports indicate that some field populations of codling moths showed

markedly reduced susceptibility to a commercially produced baculovirus, CpGV-M

(Mexican isolate) (Fritsch et al., 2005; Sauphanor et al., 2006; Jehle et al., 2006, 2008a &

2008b). In 2002 and 2003, the first accounts of CpGV-M showing reduced efficacy was

observed in organic orchards (Jehle et al., 2008b). In 2005, two CM populations with up

to 1000-fold reduced susceptibility to CpGV were reported in southern Germany (Fritsch

et al., 2005). By 2006, another resistant population was recorded in France (Sauphanor et

al., 2006). Most recently, 30 orchards with CpGV-M resistance have been recorded

across Europe (Jehle, 2008b).

26

Further, studies by Jehle et al. (2006) confirmed that a laboratory strain of this field

population (reared for more than a year) showed no significant loss of resistance. They

found that resistance was not restricted to a specific instar (Jehle et al., 2006). They

speculated that, changes in the midgut cell structure (impairing entrance to midgut cells),

the midgut cell physiology (impairing primary infections of midgut cells) as well as the

immune status (impairing secondary infection in other tissues) of the host were all crucial

factors in conferring host resistance (Jehle et al., 2006).

Subsequent studies by Eberle & Jehle (2006) indicated that these CM populations

showed a 1000 to 10000 - times lower susceptibility to CpGV-M than other populations or

susceptible lab strains. Crossing experiments (reciprocal mass crossing and backcrosses

with F1 generations) between laboratory reared field resistant CM strains (reared for 2-

years) and virus free CpGV-M susceptible CM strains (reared for 9 years) revealed that,

the resistance was autosomal and incompletely dominant inherited (Eberle & Jehle,

2006).

Asser-Kaiser et al. (2007) alluded that the proposed resistant field populations (CpR) was

a mixture of both susceptible and resistant individuals. Asser-Kaiser et al. (2007)

observed that CpGV-M could still induce about 30-40% mortality (representing the

susceptible individuals within the CpR strain) in the CpR populations. However, they

concluded that a 1000 to 100000 fold resistance could still be expected in the remaining

60-70% CpR individuals (Asser-Kaiser et al., 2007).

According to Jehle et al. (2008a; 2008b) the CpGV–M which showed reduced efficacy in

controlling CM populations in Germany, has been replaced with CpGV-I12, an Iranian

isolate. Jehle et al. (2008a) reports that in a 7 day laboratory assay with CpGV-I12

against the CpR strain, the resistance ratio was 13.4 as opposed to a 104.7 increase with

CpGV-M application. However, complete mortality of the CpR strain for all instars was

achieved after a 9 day incubation period for the 5th instars when CpGV-I12 was applied

(Jehle et al., 2008a).

27

Martignoni & Schmid (1961) speculate that at a population level, susceptibility of

lepidopteran insects to baculoviruses changes at different stages of the population cycle.

Consequently, Myers (1988; 1990) put forward a “disease defense hypothesis”, which

suggests that a reduction in fecundity could be a trade-off for increased resistance to

disease in the host, leading to decreasing population numbers when exposed to virus

epizootics at high densities. It has been reported that field studies with the western tent

caterpillar, Malacosoma californicum pluviale, and its NPV, led to shifts in the frequencies

of large and small egg masses, with mean fecundity changing rapidly between different

sites (Myers & Kukan, 1995). However, resistance of caterpillars from small egg masses

was lower than that those from large egg masses. This was attributed to sub-lethal effects

of virus infection playing a role (Myers & Kukan, 1995; Rothman & Myers, 1996).

Sublethal effects such as changes in development time, reduction in fecundity, reduced

egg viability and changes in sex ratio have all been reported to impact on host-

baculovirus interactions (Cory et al., 1997).

1.4.7 Granuloviruses The initial account of a granulovirus infection was detected in the larva of the European

cabbageworm, Pieris brassicae (Paillot, 1926). The presence of very minute granules

found in infected host cells from this viral group, led viruses in this group to be referred to

as ‘granulosis’. However, studies in the molecular biology of the GVs have lagged behind

those of the NPVs due to the inherent difficulties in the setting up of tissue culture

systems for the former (Tanada & Kaya, 1993; Cory et al., 1997).

1.4.7.1 Host range Several accounts of granulovirus infections have been reported in more than 100 insect

species, mostly limited to insect members of the order Lepidoptera (Murphy et al., 1995).

Granuloviruses are generally considered to have a narrow host range, with infection being

confined to one or more species within the same family as the original host (Federici,

1997). Nevertheless, some four GVs: CpGV (Cydia pomonella GV), HaGV (Helicoverpa

armigera – cotton bollworm, GV), SpfrGV (Spodoptera frugiperda - fall armyworm, GV)

28

and XecnGV (Xestia c-nigrum – spotted cutworm, GV) have been documented as having

a relatively wide host range (Winstanley & O’Reilly, 1999). Despite this, a virus is almost

invariably more pathogenic to its homologous host. For instance the NPVs of Autographa

California (AcMNPV) and Anagrapha falcifera (AfMNPV) were reported to be 93 times

and 54 times less infectious to the neonate larvae of C. pomonella as compared to the

homologous GV, CpGV respectively (Lacey et al., 2002).

1.4.7.2 Gross pathology and Symptomatology With regard to disease caused by GVs three different types of GVs have been named,

based on their tissue tropism in their host namely: type 1, type 2 and type 3 GV (Federici,

1997).

1.4.7.2.1 Type 1 GV: With the type 1 GV, such as Tricloplusia ni (TnGV) and just like the

NPVs, once the host ingests the virus granules, only the fat body tissue is attacked via

the midgut epithelium (Federici, 1997). Because other tissues of the host are not

attacked, the infected larvae tend to live much longer (a week longer) than larvae of the

same stage infected with a similar amount of NPV. Infected larvae are known to last

between 10 to 14 days (post infection in the Trichoplusia ni fourth instar) and grow much

larger than uninfected larvae. They normally become sluggish within a day or two of death

with a creamy or yellowish appearance, due to the accumulation of viral granules in the

fat body (Federici, 1997). Since the epidermis is not affected there is no liquefaction,

hence infected larvae turn dark brown or black and may desiccate or disintegrate

(Federici, 1997).

1.4.7.2.2 Type 2 GV: The infection and gross pathology of the type 2 GV is parallel to that

of the typical lepidopteran NPV disease. Unlike the type 1 GV, the type 2 GV has a much

wider tissue tropism, with infection occurring in the midgut, epidermis, fat body, tracheal

matrix and possibly the malpighian tubules (Tanada & Kaya, 1993; Federici, 1997). The

type 2 GV is more acute than the type 1 GV, typically lasting between 5 - 10 days in

larvae infected during the fourth instar (Federici, 1997). The normal symptoms of the

disease involve the development of irregular white to yellow patches below the cuticle

29

(Fig. 1.12). Infected larvae swell and distend slightly (Fig. 1.12) and the body liquefies,

due to the injured epidermis (Federici, 1997). The Cryptophlebia leuotreta granulovirus

(CrleGV) and the Cydia pomonella granulovirus (CpGV) are examples of GVs that fall into

this category.

Figure 1.12 (A) Symptoms of a CrleGV infected fifth instar FCM (still alive), with inner body mass appearing whitish; (B) a 5th instar larva with brownish lesions due to infection (still alive); (C) a healthy 5th instar larvae; (D) a dead and distended virus infected 5th instar FCM larva (Source: Moore, 2002). 1.4.7.2.3 Type 3 GV: This is the last type of GV, and is unique to HabrGV (Harrisina

brillians, GV), which normally attacks the Western grapeleaf skeletoniser (Harrisina

brillians) (Federici, 1997). Again, unlike the type 1 & 2 GVs, this GVs tissue tropism is

restricted to the midgut epithelium. Thus, the HabrGV replicates only in the midgut with

virions and occlusion bodies attacking both the larvae and adult. The disease is very

acute and normally lasts between 4 to 7 days in the third or fourth instar. The larvae

shrivel after the fourth or fifth day post infection and do not liquefy (due to their intact

epidermis) (Federici, 1997).

1.4.8 Cryptophlebia leucotreta granulovirus (CrleGV) Previous studies have confirmed the existence of four distinct virus isolates of FCM

namely, the Cape Verde (Muck, 1985), Ivory Coast (Angelini et al., 1965) and two South

African isolates – CrleGV-SA (Moore, 2002; Singh, 2001; Singh et al., 2003; Goble,

A B

C D

30

2007). This recent study on the genetic differences in the South African isolate was

conducted by Goble (2007). Goble (2007) alluded that, although Cryptogran and Cryptex

were both CrleGV-SA isolates, genotypically they differed. In a DNA profile comparative

analysis, Goble (2007) showed that, whilst the Cryptogran isolate was identical to the

isolate characterized by Singh (2001) and Moore (2002), when using EcoRI and BamHI

digests, it differed significantly from Cryptex. Goble (2007) concluded that Cryptex

appeared to be identical to the CrleGV-SA isolate characterized by Fritsch (1989) using

the EcoRI, HindIII and BamHI.

1.4.8.1 An overview of Cryptogran Moore (2002) was the first to develop and evaluate Cryptophlebia leucotreta granulovirus

(CrleGV-SA), as a biological control agent for the management of FCM (Thaumatotibia

leucotreta) in South Africa. The granulovirus was identified from Goedehoop Citrus

Insectary at Citrusdal (Moore et al., 2004b).

According to Moore (2002), surface dose-response and time-response bioassays

conducted with the first and fifth larval instars of FCM revealed that CrleGV-SA had a high

infectivity and virulence against the FCM larval instars, hence making it a good candidate

for the biological control of FCM. The LT50 (time taken to kill 50% of the test insects) and

LT90 (time taken to kill 90% of the test insects) values with neonate larvae, using CrleGV-

SA were estimated to be 4 days 22 h and 7 days 8 h, respectively. The LT50 and LT90

values of the fifth instars were determined to be 7 days 17 h and 9 days 8 h, respectively

(Moore, 2002). Similar observations were also reported by Federici (1997) with fourth

instar Tricloplusia ni (another type 2 GV) with virulence lasting between 5 - 10 days.

The Cryptophlebia leucotreta granulovirus (CrleGV-SA) has subsequently been registered

and is now commercially produced and marketed by River Bioscience (Pty) Ltd. under the

brand name “Cryptogran” (the formulated virus product) (Moore, 2002; Moore et al.,

2004a).

31

1.5 BIOASSAY OF ENTOMOPATHOGENIC VIRUSES In toxicological studies, the effect of a “toxicant or stimulus” on a respondent is

investigated. The stimulus (CrleGV-SA) is applied to a subject (an FCM larvae) at a

specified dose (concentration, weight or time) (Finney, 1971). The subject which in our

case is an insect (FCM) will elicit a response (death or the growth of an organism or a

score), after receiving such a stimulus (Finney, 1971). Hawcroft et al. (1987) defines a

stimulus as, ‘a standard or test sample which contains the analyte or biologically active

substance to be applied to the biological system or subject.’ Consequently, a bioassay or

biological assay deals with the quantification of the responses that arise from the

application of such a stimulus to a biological system (Hawcroft et al., 1987).

1.5.1 Bioassay Techniques Bioassays generally, are used to measure dose-response or time-response relationships

(Finney, 1971). Assays have been proven to be essential in the assessment of the

virulence of a given virus preparation per se or in comparing different viral isolates or

similar batches and even different isolates (Hughes & Shapiro, 1997). In conducting dose-

response or time-response bioassays with viruses (stimulus) on insects (subjects) the

virus is either grown in the insect (in vivo) or in cell culture systems (in vitro) (Jones,

2000). Although there have been significant advancements with in vitro virus production

methods of late, the difficulty and costs associated with it have limited its usage.

Therefore, in vivo methods still remain the most preferred option (Black et al., 1997).

According to Jones (2000) the main advantage in the use of live insects over the cell

culture system is the ability to test for all stages involved in the infection process.

Nevertheless, there is always a problem with the inherent natural variability of the host,

coupled with the difficulties in controlling assay conditions which might influence host

susceptibility to the virus and consequently the outcome of the results (Jones, 2000). The

easiest way to overcome this problem is to ensure that, the supply of test insects is as

uniform as possible (in terms of age, weight and specific instar), and the insect rearing

diet kept under strict hygienic conditions (Jones, 2000).

32

In bioassays, the quantity of diet administered is normally used in determining the lethal

dose (LD) or lethal concentration (LC) of the virus sample (Hughes & Shapiro, 1997). For

instance with the LC method, a known concentration of a virus is administered onto the

diet surface or incorporated into it, but the larvae does not consume the entire diet hence

the precise dosage ingested by an individual larva is not known (Hughes & Shapiro, 1997;

Hunter-Fujita et al., 1998; Jones, 2000). Due to the nature of the LC method, it is

considered not to be effective in comparative studies, using different host species since

there will be variability in their intrinsic feeding rates leading to difficulties in the

determination of the precise dosage consumed by an individual larva (Hunter-Fujita et al.,

1998).

The LD method involves the administering of a precise amount of virus infected diet to the

larva, of which the larva consumes the entire diet-virus aliquot. Hence the exact dosage

of virus ingested by a single larva can be determined. The most common procedure used,

is the diet plug method and is considered to be most suitable for very small or gregarious

larvae (Hughes & Shapiro, 1997; Hunter-Fujita et al., 1998; Jones, 2000). The LD method

is considered to be more accurate than the LC method. However, it is labour intensive,

coupled with a high risk of injuring test larvae due to increased handling unlike the LC

method (Jones, 2000). Assays are mostly run to determine the LC50, the concentration

required to kill 50% of a given population of test insects or the LD50, the lethal dose

required to kill 50% of a given population of test insects (Finney, 1971; Hughes & Shapiro,

1997; Hunter-Fujita et al., 1998; Jones, 2000).

Essentially, with these two methods (LC and LD methods) the normal assumption is that,

the inoculum is evenly spread over the entire diet surface (Jones, 2000). A number of

techniques employed in the conducting of dosage-mortality bioassays on

entomopathogenic viruses are discussed by Hunter-Fujita et al. (1998), Jones (2000) and

Evans & Shapiro (1997) below.

33

1.5.1.2 Mass dosing bioassay The main objective of the mass dosing bioassay - is to estimate the potency of a virus

concentration on a test insect. Mass dosing bioassays are normally employed in LC

studies (Jones, 2000).

According to Hughes & Shapiro (1997), Hunter-Fujita et al. (1998) and Jones (2000), at

present the most commonly adopted mass dosing bioassay techniques are the:

(a) Surface dosing bioassay.

(b) Diet incorporation bioassay.

(c) Droplet feeding bioassay.

(d) Egg-dipping bioassay. Jones (2000) surmises that, the mass dosing bioassay is much preferred for insects that

feed gregariously and can be used in assaying a large number of insects of any age at

one time.

1.5.1.2.1 Surface dosing bioassay: With this method, the main idea is to mimic the

feeding habits of larvae that tend to feed on the surface of their substrate (diet). An

example is Spodoptera littoralis and Plodia interpunctella which feeds on the surface of

leaves (Hunter-Fujita et al., 1998; Jones, 2000). As a result, a known concentration of the

virus suspension is spread on the diet or leaf surface at a previously ascertained specific

volume required to adequately cover the entire diet surface. The virus medium could be

sterile distilled water or any suitable virus carrier medium (Jones, 2000). With leaf-eating

larvae, the leaves are maintained in a flat position for uniform spread of the virus

suspension (Jones, 2000). Afterwards, the inoculum is allowed to dry out thoroughly on

the diet surface. A single larva normally in LD or LT (lethal time) studies or more are

placed on the diet to feed until quantal response (death) or pupation (Hughes & Shapiro,

1997; Hunter-Fujita et al., 1998; Jones, 2000). However, with the diet plug method,

preliminary trials are carried out beforehand with the specific insect of interest to

determine the exact quantity of diet it consumes (Hunter-Fujita et al., 1998). Once this is

known, a known viral concentration is then administered onto the diet surface for the

34

larvae to consume the entire diet, hence the lethal dosage (LD) consumed by the larvae

is known (Jones, 2000). Hughes & Shapiro (1997) define a dosage as, ‘the quantity of

virus per millimeter (mm) of diet surface’.

Considering the nature of virus acquisition by field larva the surface dosing method was

preferred over the rest. For instance, it is unlikely that the FCM larva will come into

contact with the sprayed virus formulation when inside the fruit (as is the case with the

diet incorporation method) as it remains feeding in the citrus fruit until pupation (Newton,

1998). Its contact with the virus will be restricted to the fruit surface (Moore 2002; Moore

& Kirkman, 2004; Moore et al., 2004a; Moore et al., 2004b). In the field Cryptogran

(formulated virus product) is sprayed directly onto citrus trees, hence the FCM larva has

an increased likelihood of consuming the virus on the surface of fruits (Moore & Kirkman,

2006). In view of this the most justified means of mimicking what truly transpires in the

field was to inoculate the virus on the diet (substrate) surface (Moore, 2002). The LC

method was also preferred to the LD method on similar grounds, since ideally it will be

difficult to determine the exact dosage of virus ingested by an FCM larva in the field

(Moore, 2002).

1.5.1.2.2 Diet incorporation bioassay: Jones (2000) speculates that unlike the surface

dosing bioassay method, the diet incorporation assay rather mimics larvae that are

internal feeders and burrow into fruits, feeding on its internal contents (an example is the

FCM) (Jones, 2000). However, in terms of virus acquisition this may not be entirely so

since there is little chance of larvae ingesting virus inside a fruit, except on the fruit

surface. The diet incorporation assay is carried out in the same manner as that of the

surface dosing method. The only difference is the incorporation of the virus suspension

into the bulk diet (Hughes & Shapiro, 1997; Hunter-Fujita et al., 1998; Jones, 2000).

Hughes & Shapiro (1997) and Hunter-Fujita et al. (1998) explain that a known

concentration of the virus suspension is normally mixed thoroughly with the bulk diet after

it has cooled down to a temperature of about 45oC. Thereafter, one can dispense the diet

via an LC method or LD (diet plug method – larva consumes the entire diet-virus aliquot)

(Hughes & Shapiro, 1997; Hunter-Fujita et al., 1998; Jones, 2000). Depending on the

adopted methodology for both the LC and LD method, the test larvae can be retained on

35

the virus infested diet throughout the assay or transferred onto uncontaminated diet

afterwards until quantal response or pupation (Hunter – Fujita et al., 1998).

1.5.1.2.3 Droplet feeding bioassay: This method was first developed by Hughes and

Wood (1987), and is also known as the ‘synchronous peroral method’. Hunter-Fujita et al.

(1998) explains that, this method has the added advantage of reducing variability involved

in determining the dosage delivered and time taken for a test insect to consume that

dosage. As the name implies, instead of spreading or mixing the virus aliquot in or on diet

surfaces for the larvae to feed on, the inoculum is rather administered directly in droplets

of virus suspensions to the test larvae, hence increasing the probability of the inoculum

being ingested by the test insects (Hunter-Fujita et al., 1998; Jones, 2000). A dye (such

as Brilliant Blue R or food dye) is mixed with both the viral suspension and the control

(only distilled water and the die), hence it becomes easy to identify larvae that have

ingested the dye solution. The dye-virus solution is normally dispensed in droplets of

about 2 cm in diameter. Larvae that appear blue (presence of blue die in their guts) are

then transferred onto uncontaminated diet afterwards, until quantal response or pupation

(Hunter-Fujita et al., 1998; Jones, 2000). In addition Jones (2000) surmises that the

method has the added advantage of allowing a large number of test insects to be

assayed, plus the volume ingested by the test larvae remains virtually constant,

facilitating a good estimation of an LD value from a given LC value during an assay. It is

also preferred for LT studies (Jones, 2000).

1.5.1.2.4 Egg-dipping Bioassay: This bioassay method targets neonate larvae that tend to

eat the chorion of their eggs (Jones, 2000). A wetting agent (such as 0.1% Tween or

Teepol) is normally added to the virus suspension for ease of spread. For instance if eggs

are laid on a filter paper - after dipping them in a viral suspension, one should ensure that

the filter paper (containing eggs) dries adequately. Since most larvae naturally eat away

their chorion after hatching, the virus will be ingested along with the chorion (Jones,

2000). This method has two major disadvantages in that during dipping chances are there

will be increased variation in the amount of virus spread on the entire egg surface and

also not all the larvae will completely consume the chorion (Jones, 2000).

36

1.6 JUSTIFICATION Previous knowledge about field resistance of insects to baculoviruses was considered

unlikely and was only possible under laboratory conditions. This was normally induced by

continued selection with medium doses of the virus. In these cases, resistance was never

stable and was lost within a few generations, or even one generation (Briese & Mende,

1983; Jehle et al., 2006 & 2008b). However, the recent development of increased resistance by both field and laboratory

reared CM to CpGV-M is a cause for concern (Fritsch et al., 2005; Eberle & Jehle, 2006;

Jehle et al. 2006). Although CpGV-M (Mexican isolate) has been replaced with CpGV-I12

(Iranian isolate) the ecological implications of introducing yet another new isolate is still

not known (Asser-Kaiser et al., 2007; Jehle et al., 2008a &, 2008b).

It has also been reported that Cydia pomonella granulovirus (CpGV) just like

Cryptophlebia leucotreta granulovirus or Cryptogran (formulated CrleGV-SA) and Cryptex

(Another CrleGV-SA product, produced by Andermatt Biocontrol, Switzerland) are both

viruses that belong to the same family baculoviridae and genus granulovirus. These

viruses (CpGV and CrleGV) are noted to be very closely related (Wormleaton &

Winstanley, 2001).

According to Timm (2005a & 2005b), genetic studies on FCM populations in South Africa

revealed some marked differences. In her study on; (a) temporal patterns of genetic

variation among FCM populations as well as; (b) patterns of genetic variation among

geographic and host populations of FCM, the following findings were outlined by Timm:

1. FCM populations in both small and large geographic scales were distinct, with limited

dispersal between orchards.

2. Locally adapted populations may occur in regions or provinces in South Africa.

3. Patterns of genetic variation between these populations were stable during successive

seasons.

4. Populations found on different hosts may interbreed freely.

37

Considering the previous reports of increased resistance to commercially produced

baculoviruses in Europe, coupled with the marked genetic differences observed in some

FCM populations in South Africa, it is imperative that we investigate whether a similar

phenomenon occurs or could develop in South Africa, with FCM against CrleGV-SA

(unformulated virus product).

Furthermore, these studies will confirm or debunk any fears that may arise from the citrus

growers and the biopesticide industries in South Africa (such as, River Bioscience (Pty)

Ltd.), of any potential existence of FCM populations with lower susceptibility to the virus

products. It is even possible that certain populations may be more susceptible to

Cryptogran and others more susceptible to Cryptex.

Moreover, as scientists, it is our duty to investigate any potential scientific problem and

possibly find solutions to them quickly, before they get out of hand.

1.7 AIM In view of the above, the aim of this study is to investigate the susceptibility of various

field populations of FCM from a range of geographic areas to Cryptogran and Cryptex.

The following sub-objectives will be investigated in order to achieve our main objective.

1.7.1 Objectives 1. Establish a benchmark for pathogenicity, using laboratory reared FCM larvae against

CrleGV-SA. This will serve as a protocol for conducting future dose-response bioassays

with CrleGV-SA against field collected FCM larval instars. Consequently, any differences

or similarities in response between the field and laboratory reared FCM larval instars

against CrleGV-SA would be established to better understand their susceptibility.

2. Establish new and separate laboratory colonies of FCM using field collected FCM

larvae from a range of different regions throughout South Africa.

3. Conduct comparative surface dose-response bioassays with neonate FCM larvae, from

the established FCM laboratory colonies, using Cryptogran and Cryptex. This will enable

the detection of even small differences in host susceptibility and virus pathogenicity.

38

1.8 EXPECTED OUTCOMES 1. Any differences that might exist with regards to FCM susceptibility to Cryptogran or

Cryptex will be detected through this study.

2. This study will give us more insight into how these various locally adapted populations

of FCM “individually” respond to Cryptogran and Cryptex and thus aid us in the strategic

application of product formulations for specific citrus growing regions in South Africa.

3. Studies into the development of another CrleGV isolate which is more virulent, to a

particularly low susceptible FCM population in South Africa will be sought after if such a

phenomenon becomes apparent.

39

CHAPTER TWO

BENCHMARK DOSE-RESPONSE BIOASSAYS WITH FCM LARVAE 2.1 INTRODUCTION The main objective outlined in this study (see Chapter One) was to investigate the

susceptibility of geographically distinct FCM populations in South Africa to CrleGV-SA. In

order to achieve this objective, firstly a benchmark for pathogenecity had to be

established. However, a benchmark for pathogenecity with CrleGV-SA (Cryptogran)

against the 1st and 5th instar FCM larvae was previously established by Moore (2002).

Therefore it was necessary to establish another benchmark for pathogenecity, for the

other larval stages (2nd, 3rd and 4th). This benchmark will guide future bioassays with

CrleGV-SA against field collected FCM larval instars (see Chapter Three). Since most of

the FCM larval instars may be present at any one time in the field, due to overlapping

generations, stage-related susceptibility to CrleGV-SA will be of practical importance. This

protocol will enable us understand the effect (susceptibility) of host stage to baculovirus

infection. The protocol and results on the establishment of a benchmark for pathogenecity

for the 2nd, 3rd and 4th FCM instars using CrleGV-SA (Cryptogran) is discussed in this

chapter.

2.2 MATERIALS AND METHODS 2.2.1 Virus purification protocol (using a glycerol gradient) In order to remove impurities and formulation additives and resulting in a pure virus for

bioassays, the virus purification protocol as described by Hunter-Fujita et al. (1998) and

Moore (2002) using a glycerol gradient was adopted in this study. The two commercially

produced CrleGV-SA products, Cryptex and Cryptogran were purified accordingly. The

steps employed in the purification of the virus samples are outlined below.

1. Firstly, 2 ml each of Cryptex and Cryptogran were pipetted into two Sigma 3K20

centrifuge tubes. The tubes were then filled with 0.1% SDS. The resulting suspension

was spun for 15 minutes at 15000 rpm. The supernatant was discarded and the pellet

40

(containing the virus) resuspended in 0.1% SDS. The resulting solution was spun again

for 15 minutes at 15000 rpm. The resulting pellets were resuspended in 0.1% SDS and

held in microcentrifuge tubes (Eppindorf tubes) using an auto pipette.

2. A series of solutions of glycerol of differing densities was prepared using glycerol (30-

45% stock concentration) and 0.1% SDS. From this, six separate gradients solutions of

30 to 80% (v/v) glycerol in SDS rate zonal gradients were prepared. Therefore, for a 30%

(v/v) glycerol gradient, 35 ml of SDS was added to 15 ml of glycerol. For a 40% (v/v)

glycerol gradient, 30 ml of SDS was added to 20 ml glycerol. For a 50% (v/v) glycerol

gradient, 25 ml SDS was added to 25 ml glycerol. For a 60% (v/v) glycerol gradient, 20 ml

SDS was added to 30 ml glycerol. For a 70% (v/v) glycerol gradient, 15 ml SDS was

added to 35 ml glycerol. And for an 80% (v/v) glycerol gradient, 10 ml SDS was added to

40 ml glycerol.

3. The 30 to 80% (v/v) glycerol in SDS rate zonal gradients were then loaded on top of

each other starting with the most dense solution (80% v/v glycerol) placed at the bottom

and ending with the least dense solution (30% v/v glycerol) placed on top. The mixture

was held in four Ultra-Clear centrifuge tubes (25 x 89 mm). The solutions were then left

overnight at 8oC in order to form a clear gradient.

4. After 24 h, the pellets (in 0.1% SDS) were loaded on top of the 30-80% v/v glycerol

gradients. The gradient was then spun for 30 minutes at 15000 rpm in a Beckman L-70

Ultracentrifuge. The Ultracentrifuge tubes were then held in a dark room. By illuminating

the Ultracentrifuge tubes with a bright torch, clear band formations could be seen. The

bands (containing virus) were then collected in Eppindorf tubes using an auto pipette.

5. Thereafter, the virus bands were resuspended in 36 ml of d (H20) and spun for 20

minutes at 15000 rpm in Sigma 3K20 tubes. The supernatant was discarded and the

pellets resuspended in d (H20) using a vortexer. The process was repeated three times.

Finally, 2 ml of d (H20) was added to each resulting pellet and vortexed. The resuspended

pellets were then transferred into eppindorf tubes. The purity of the virus suspension was

checked under a light microscope.

41

6. The concentration of the virus suspension was determined using a Thoma bacterial

counting chamber at 400 X magnification under dark field light microscopy as explained

below.

2.2.2 Determination of virus concentration In order to determine the concentration of the virus samples light microscopy method for

enumeration of viruses in suspension was employed (Hunter-Fujita et al., 1998; Jones,

2000). A Thoma bacterial counting chamber (0.02 mm depth), at 400 times magnification

under dark field light microscopy was used in counting virus particles.

2.2.2.1 Protocol for virus enumeration 1. Firstly, the Thoma bacterial counting chamber slide and the lens tissue paper were

initially cleaned with 70% ethanol. Afterwards, the cover slip of the microscope was

partially placed on the Thoma chamber for ease of pipetting the virus suspension.

2. A stock virus sample (CrleGV-SA) was sonicated in an ultrasonic bath for about 60

seconds to reduce clamping of virus particles. Two samples of each virus suspension

were used for the procedures outlined below.

3. The sonicated sample was diluted with distilled water (dH20) at a 1:4 dilution rate (one

part virus and four parts distilled water). Thereafter, it was shaken to achieve a

homogenous suspension. The resulting suspension was further diluted at 1:4 (dilution

rate), with 1.0% SDS (sodium dodecyl sulphate). The resulting new suspension was

again shaken to achieve homogeneity.

4. A further 1:100 dilution of the virus-SDS solution was made. Afterwards, 5 µl of the

resulting sample was then pipetted onto the Thoma counting chamber. The cover slip was

firmly secured, by breathing on it and applying pressure on both sides until the

appearance of Newton’s rings (to confirm that the cover slip was secure). The slide was

allowed to stand for 5 minutes before counting - allowing the Brownian motion of particles

to settle.

42

. .. .

. . . . ..

.. …

. . .

.. .

. . ..

…

.

.. . .. .

. . .. . …

… . .. .

. . .

Figure 2.1 A 0.02 Thoma bacterial counting chamber. With the TL (top left), TR (top right), BL (bottom left), BR (bottom right) and one R (random) chamber, used in virus enumeration (virus particles: black arrow). 5. Each block in the Thoma bacterial counting chamber had sixteen squares (Fig. 2.1).

Four blocks from the top left (TL), top right (TR), bottom left (BL) and bottom right (BR)

corners of the chamber were counted at 400 times magnification (40 lens). Another single

block (R) from the middle, of the chamber was selected at random and counted (Fig. 2.1).

Therefore in all, five blocks were counted with four from the edge of the chamber (TL, TR,

BL and BR) and one from the middle (R).

6. Only moving virus particles were counted. The fine focus of the light microscope was

used to move in and out of focus, in order to count all virus particles moving at different

depths in the chamber. Again only occlusion bodies found on the top and right-hand line

(or left and bottom-line) of the large square were counted.

7. The whole process was repeated (at least twice) and the mean value of the counts was

fixed into the equation below, to determine the number of OBs. The equation used in

enumerating the virus concentration was as follows (Hunter-Fujita et al., 1998; Jones,

2000).

TL TR

BR BL

R R

R R

43

Virus sample in OBs/ml = D x X N x V Where: D = Dilution of suspension; X = Number of OBs counted; N = Number of small squares counted and; V = Volume in ml

2.2.3 Serial dilution technique for enumerated virus samples Once the concentration of the virus had been determined, serial dilutions, using a five-fold

serial dilution technique was employed. The procedure used in deriving diluted

concentrations of the virus stock was adopted from Moore (2002). Determination of the

most concentrated treatment (1.34 x 106 OBs/ml) that gave a good dose-response curve

for the 2nd FCM instars is described below.

To formulate the most concentrated treatment the stock virus suspension (5.0 x 1010

OBs/ml) was diluted into five parts with d (H2O) (making up 1.0 x 1010 OBs/ml).

Thereafter, 5 µl of the resulting virus suspension was diluted in 5000 µl of d (H2O) –

making a concentration of 1.0 x 107 OBs/ml (B). Afterwards, 2010 µl of (B) was diluted in

12990 µl of d (H2O). A final concentration of 1.34 x 106 OBs/ml was achieved. One-fifth

(1/5) proportion of the virus: d (H2O) suspension was then transferred from test tube (I) to

(II) and so on, ending with test tube (V) in order to achieve a five fold serial dilution (Fig.

2.2). The same procedure was used in deriving the required concentrations for carrying

out bioassays with the 3rd and 4th instar, with their required volumes adjusted accordingly.

Therefore the method used in determining the ideal series of concentrations for the

assays involved a lot of trial and error.

44

(B)

5.0 x 1010 OBs/ml of CrleGV-SA (Cryptogran stock suspension)

(I) (II) (III) (IV) (V)

2,010ul of (B) 3,000ul of (I) 3,000ul of (II) 3,000ul of (III) 3,000ul of (IV) + + + + +

12,990ul d(H2O) 12,000ul d(H2O) 12,000ul d(H2O) 12,000ul d(H2O) 12,000ul d(H2O)

(1.34 x 106) (2.68 x 105) (5.36 x 104) (1.07 x 104) (2.14 x 103)

Figure 2.2 A five-fold serial dilution of CrleGV-SA in distilled water [d (H2O)] used in surface dosage-response bioassays with 2nd instar FCM larvae. 2.2.4 Preparation of diet and polypots for viral inoculation Once a five-fold serial dilution of the virus sample was achieved, diets and polypots were

prepared accordingly for inoculation. In establishing a dose-response relationship for the

laboratory reared 2nd, 3rd and 4th larval instars, an agar-based diet was used.

Figure 2.3 Polypots with agar-based artificial diet being inoculated with CrleGV-SA for use in five-fold serial dilution surface dose-response bioassays with FCM larvae.

45

It was established by Moore (2002) that, it was necessary to use polypots (30 ml capacity

- Evron, South Africa) with holes (± 15mm diameter) cut in the centre of each lid for these

bioassays. Cotton wool was plugged into each hole, to ensure good ventilation in the

polypots (Fig. 2.3) and prevent larvae from escaping (Moore, 2002).

2.2.5 Conducting of bioassays using CrleGV-SA (Cryptogran) against FCM instar larvae Upon reaching the required instar, larvae were gently removed from their diets by

dissecting and breaking the diet with the aid of sterilised knives (sterilised in 2% sodium

hypochlorite). A size 000 paint brush (sterilised in 2% sodium hypochlorite) was used in

transferring larvae onto diets in each polypot (Fig. 2.3). To minimise contamination; two

000 size paint brushes were used, and each brush was rotated between treatments

(Moore, 2002). Brushes were rinsed off in distilled water, and dried between sheets of

paper towelling. Individual larvae were transferred onto the surface of each diet, starting

with the control and ending with the most concentrated virus treatment. A total of 150

larvae were used per trial, (with 25 larvae per treatment, including the control). Inoculated

diet with larvae was then transferred to the incubation chamber until evaluation. The 2nd,

3rd and 4th FCM larvae were evaluated after 8, 8 and 10 days respectively. The larvae or

pupae (if they had pupated) were recorded as dead or alive.

2.2.6 Statistical analysis Data from the dose-response bioassays were analysed using PROBAN (Van Ark, 1995),

a software programme used for conducting probit analyses with bioassay data. PROBAN

corrected the control mortality according to Abbott’s formula (Abbott, 1925). From this, the

LC50 and LC90 values were calculated. PROBAN transformed the doses to log10 and the

percentage response to empirical probits. Using this information, the fit of the probit

(regression) lines were calculated, as were the fiducial limits. Bartlett’s test (P < 0.01) was

employed in the comparison of probit lines (Van Ark, 1995).

46

According to Abbott:

% Corrected Mortality = [% Treatment Mortality - % Control Mortality] x 100

(100 % - Control mortality) Results from each bioassay were replicated three times, for the 2nd, 3rd & 4th instars. Only

good bioassays with control mortalities not exceeding 20% were used for data analysis.

2.3 RESULTS 2.3.1 Virus purification The initial concentration of the unpurified Cryptex and Cryptogran products was

determined to be 4.88 x 1011OBs/ml and 2.47 x 1011 OBs/ml respectively. However, after

purification the Cryptex and Cryptogran concentrations had reduced to 7.15 x 1010 OBs/ml

and 1.88 x 1010 OBs/ml respectively. It is quite common to obtain low concentrations of

virus samples after purification, due to loss of virus material (Hunter-Fujita et al., 1998).

2.3.2 Incubation and determination of respective FCM larval instars In the incubation chamber it normally took between 3 to 4 days for the FCM eggs to hatch

(emergence of the 1st instar). Again, it took another 2 days for the FCM larvae to grow

and molt into the next larval stage. Hence on the 4th, 6th, 8th, 10th and 12th days, after

hatching, one was likely to obtain a high population of 1st, 2nd, 3rd, 4th and 5th instar FCM

larvae in each jam jar respectively.

Table 2.1 Duration of FCM life cycle (egg - adult) at 27oC ± 1 room temperature

FCM Stage Average duration of life stage

Egg to 1st Instar 3 – 4 days 2nd Instar 2 days 3rd Instar 2 days 4th Instar 2 - 3 days 5th instar 2 days

pupa to adult 12 – 14 days Total duration (Egg - Adult)

28 – 30 days

47

2.3.3 Surface dose-response bioassays with 2nd instar FCM larvae In a five-fold serial dilution with 2nd instar FCM larvae against CrleGV-SA concentrations

ranging between 2.14 x103 and 1.34 x 106 OBs/ml, a dose-response relationship was

established. The lowest and highest larval mortality ranged from 0.0% to 4.0% and 16.0%

to 84.0% for the control and treatments respectively (Table 2.2). The ideal mortality range

(for treatments) for dose-response bioassays is recommended to range within 10% to

90% mortality (Finney, 1971; Jones, 2000).

Table 2.2 Mortality of 2nd instar larvae, in a dose response (five-fold) bioassay with CrleGV-SA.

Treatment (CrleGV in OBs/ml)

Replicate 1 Replicate 2 Replicate 3 Larval

mortality (%)

Mortality

corrected for control

mortality (%

)

Empirical

probit

Larval mortality (%)

Mortality

corrected for control

mortality (%

)

Empirical

probit

Larval mortalit

y (%)

Mortality

corrected for control

mortality (%

)

Empirical

probit

d (H2O) Control

4.00 - - 0.00 - - 4.00

2.14 x103 28.00 25.00 4.326 16.00 16.00 4.005 16.00 12.50 3.850 1.07 x 104 44.00 41.67 4.790 48.00 48.00 4.905 24.00 20.83 4.188 5.36 x 104 56.00 54.17 5.105 64.00 64.00 5.359 48.00 45.83 4.895 2.68 x 105 68.00 66.67 5.431 68.00 68.00 5.468 76.00 75.00 5.674 1.34 x 106 84.00 83.33 5.967 84.00 84.00 5.995 80.00 79.17 5.812 The regression lines for the data from the three replicates had the equations y = 2.493 +

0.556x (SE of slope = 0.1309), y = 2.207 + 0.628x (SE of slope = 0.1284) and y = 1.176 +

0.784x (SE of slope = 0.1482). Using Bartlett’s test for homogeneity of residual variances,

the residual variances of the three lines were homogenous making the chi-squared test

more applicable than would have been the case if deviations were heterogeneous. The

slopes of the three lines were parallel and therefore comparable. The elevations of the

lines did not differ significantly from one another. G for fiducial limits was calculated to be

0.2131, 0.1608 and 0.1372 for the three lines, respectively. According to Van Ark (1995),

values greater than 0.025 indicated a large variation in mortality, but the experimental

procedure or the value of the probit line was questionable if G exceeded 0.25. The mean

LC50 value for the 2nd instars was calculated to be 4.516 x 104 OBs/ml. The LC90 value

was also calculated to be, 4.287 x 106 OBs/ml.

48

2.3.4 Surface dose-response bioassays with 3rd instar FCM larvae In a five-fold serial dilution with 3rd instar larvae against CrleGV-SA concentrations

ranging between 1.60 x103 and 1.00 x 106 OBs/ml, a dose-response relationship was

established. Control mortality was zero percent (0.0%) for all replicates. However,

mortality of the larvae receiving treatment for the three replicates ranged from, 4.0% to

80% for the lowest and highest concentrations respectively (Table 2.3).

Table 2.3 Mortality of 3rd instar larvae, in a dose response (five-fold) bioassay with CrleGV-SA.

Treatment (CrleGV in OBs/ml)

Replicate 1 Replicate 2 Replicate 3 Larval

mortality (%)

Mortality

corrected for control

mortality (%

)

Empirical

probit

Larval mortality (%)

Mortality

corrected for control

mortality (%

)

Empirical

probit

Larval mortalit

y (%)

Mortality

corrected for control

mortality (%

)

Empirical

probit

d (H2O) Control

0.00 - - 0.00 0.00

1.60 x103 8.00 8.00 3.595 4.00 4.00 3.249 8.00 8.00 3.595 8.00 x 103 20.00 20.00 4.158 12.00 12.00 3.825 16.00 16.00 4.005 4.00 x 104 28.00 28.00 4.417 36.00 36.00 4.641 32.00 32.00 4.532 2.00 x 105 48.00 48.00 4.950 56.00 56.00 5.151 60.00 60.00 5.253 1.00 x 106 68.00 68.00 5.468 72.00 72.00 5.583 80.00 80.00 5.842 The regression lines had the equations y = 1.523 + 0.650x (SE of slope = 0.1351), y =

0.650 + 0.839x (SE of slope = 0.1476) and y = 0.769 + 0.839x (SE of slope = 0.1444).

The residual variances of the three lines were determined to be homogenous and the

slopes of the lines parallel and comparable. The elevations of the lines did not differ

significantly from one another. G for fiducial limits was calculated to be 0.1659, 0.1189

and 0.1137 for the three lines, respectively. The mean LC50 value for the 3rd instars was

determined to be 1.662 x 105 OBs/ml. The LC90 value was also calculated to be, 9.992 x

106 OBs/ml.

2.3.5 Surface dose-response bioassays with 4th instar FCM larvae In a five-fold serial dilution with 4th instar larvae against CrleGV-SA concentrations

ranging between 5.34 x104 and 3.34 x 107 OBs/ml, a dose-response relationship was

established. Control mortality was zero percent (0.0%) for all replicates. However,

49

mortality of the larvae receiving treatment for the three replicates ranged from, 8.0% to

84% for the lowest and highest concentrations respectively (Table 2.4).

Table 2.4 Mortality of 4th instar larvae, in a dose response (five-fold) bioassay with CrleGV-SA.

Treatment (CrleGV in OBs/ml)

Replicate 1 Replicate 2 Replicate 3 Larval

mortality (%)

Mortality

corrected for control

mortality (%

)

Empirical

probit

Larval mortality (%)

Mortality

corrected for control

mortality (%

)

Empirical

probit

Larval mortalit

y (%)

Mortality

corrected for control

mortality (%

)

Empirical

probit

d (H2O) Control

0.00 - - 0.00 - - 0.00 - -

5.34 x104 12.00 12.00 3.825 8.00 8.00 3.595 12.00 12.00 3.825 2.67 x 105 28.00 28.00 4.417 32.00 32.00 4.532 28.00 28.00 4.417 1.34 x 106 40.00 40.00 4.747 44.00 44.00 4.849 52.00 52.00 5.050 6.68 x 106 56.00 56.00 5.151 64.00 64.00 5.359 68.00 68.00 5.468 3.34 x 107 76.00 76.00 5.706 84.00 84.00 5.995 80.00 80.00 5.842 The regression lines had the equations y = 0.873 + 0.636x (SE of slope = 0.1295), y =

0.124 + 0.777x (SE of slope = 0.1364) and y = 0.493 + 0.725x (SE of slope = 0.1331).

The residual variances of the three lines were determined to be homogenous and the

slopes of the lines parallel and comparable. The elevations of the lines did not differ

significantly from one another. G for fiducial limits was calculated to be 0.1591, 0.1185

and 0.1295 for the three lines, respectively. The mean LC50 value for the 4th instars was

determined to be 2.205 x 106 OBs/ml. The LC90 value was also calculated to be, 1.661 x

108 OBs/ml.

The regression equations calculated from the mean of the three replicates for the 2nd, 3rd

and 4th instar FCM larvae had the equations, y = 0.6627x + 1.9158 (R2 – 0.9941), y =

0.7611x + 1.0672 (R2 – 0.9982) and y = 0.7252x + 0.4096 (R2 – 0.9913) respectively (Fig.

2.6).

50

2.3.6 Combined dose-response bioassay data for all instars In surface dose-response bioassays with laboratory reared 2nd, 3rd and 4th instars with

CrleGV-SA, the mean LC50 values were calculated to be; 4.516 x 104, 1.662 x 105 and

2.205 x 106 OBs/ml respectively. The LC90 values for the 2nd, 3rd and 4th instars were also

calculated to be, 4.287 x 106, 9.922 x 106 and 1.661 x 108 OBs/ml respectively (Table.

2.5, below). The LC50 and LC90 values for the 1st and 5th FCM larval instars established by

Moore (2002) were calculated to be 4.095 x 103, 2.678 x 107 and 1.185 x 105, 9.118 x 109

respectively. In consequence, the full regime of the dose-response relationship for all

instars was successfully established (Table 2.5).

Figure 2.4 Comparison of dose-response probit lines for the 1st, 2nd, 3rd, 4th and 5th FCM instars. *L1 (first FCM instar), L2 (second FCM instar), *L3 (third FCM instar), *L4 (fourth FCM instar) and L5 (fifth FCM instar). * Probit lines for the 1st and 5th FCM instar were established by Moore (2002).

L2 y = 0.6627x + 1.9158R2 = 0.9941

L3 y = 0.7611x + 1.0672R2 = 0.9982

L4 y = 0.7252x + 0.4096R2 = 0.9913

L1 y = 0.879x + 1.8755R2 = 0.9339

L5 y = 0.5481x + 1.1013R2 = 0.9072

1.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.5

0.5 1.5 2.5 3.5 4.5 5.5 6.5 7.5 8.5 9.5 10.5

Log Dose

Em

pir

ical

Pro

bit

s

L2L3L4L1L5Linear (L2)Linear (L3)Linear (L4)Linear (L1)Linear (L5)

51

Table 2.5 Mean LC50 and LC90 for all FCM larval instars with CrleGV-SA.

FCM Instar

LC50

LC90

Exposure time (days)

1st 4.095 x 103 1.185 x 105 7 2nd 4.516 x 104 4.287 x 106 8 3rd 1.662 x 105 9.922 x 106 8 4th 2.205 x 106 1.661 x 108 10 5th 2.678 x 107 9.118 x 109 14

*LC50 and LC90 values for 1st & 5th FCM instar were established by Moore (2002).

Figure 2.5 LC50 and LC90 for CrleGV-SA against a laboratory colony of FCM in a benchmark study. *LC50 values for the 1st and 5th FCM instars were established by Moore (2002).

2.4 DISCUSSION It was noted that changes in the incubation room temperature, coupled with competition

for food between larvae within a jar, played an important role in determining the number

of individuals to be expected per jar. Temperature is noted to play a vital role in speeding

the growth rate of FCM larvae (Daiber, 1980). At a temperature of 28oC larval

development was faster and as such resulted in greater synchronisation of larval instars.

However extreme (very high and low) temperatures could also prove detrimental to the

larvae (Daiber, 1980).

1.000E+00

1.000E+02

1.000E+04

1.000E+06

1.000E+08

1.000E+10

1st 2nd 3rd 4th 5th Instar stage

Con

cent

ratio

n (O

Bs/

ml)

LC90 LC50

52

In spite of the influence of temperature on the number of FCM larvae produced, the two-

day interval between instars proved to be quite reliable in determining the correct FCM

larval stage. Ideally the most accurate means of determining larval age (instar) is by

measuring the head capsule. However, it was not practicable to do so considering the

large number of larvae involved (Catling & Aschenborn, 1978; Daiber, 1979b & 1979c).

As a result, determination of the correct instar was judged by familiarity (experience) in

handling larva.

It has been reported that the duration of the interval between infection and death

correlates with insect stage (Escribano et al., 1999). In previous assays by Moore (2002)

the incubation time (exposure time) for the 1st and 5th instars were established as 7 and

14 days respectively (Table 2.5). Therefore an incubation time for the middle instars (2nd,

3rd and 4th instar larvae) had to be established. Therefore, in these studies, using a series

of assays, an evaluation time of 8, 8 and 10 days was determined to be ideal for the 2nd,

3rd and 4th instar FCM larvae respectively (Table 2.5).

LC50 and LC90 values increased with larval stage (Table 2.5 & Fig. 2.5), reflecting a

marked difference in susceptibility of larva with age. This phenomenon is described as

‘maturation resistance’ (Jones, 2000). This increasing ‘resistance’ (lower susceptibility)

with larval stage has been reported by several authors (Hughes & Shapiro, 1997; Hunter-

Fujita et al., 1998; Escribano et al., 1999; Jones, 2000; Moore, 2002).

Similar observations were made by Escribano et al. (1999), on Spodoptera frugiperda. In

droplet feeding bioassays with S. frugiperda against its NPV (Nicaragua isolate),

Escribano et al. (1999), observed a significant increase in the LC50 values for all instars.

The LC50 values increased with larval stage from 2.03 x 105 OBs/ml for the 2nd instars to

1.84 x 108 OBs/ml for the 5th instars.

In this study, the LC50 values established for the FCM instars, increased by 11, 3.6, 14

and 12 fold from one larval stage to the next (1st to 5th instars). There was a very small

increase in the LC50 value from the 2nd to 3rd instars, showing only 3.6 fold increase.

Escribano et al. (1999) also observed a marginal, 3.9 fold increase in the LC50 value from

53

the 2nd to 3rd S. frugiperda larval instar. The LC50 values increased with larval stage from

2.04 x 105 OBs/ml for the 2nd instars to 8.05 x 105 OBs/ml for the 3rd instars (Escribano et

al., 1999). This observation may indicate that susceptibility of lepidopteran larvae to

baculovirus reduces to a lesser extent from the 2nd to the 3rd larval stage than between

any of the other stages.

Another, difference was observed with the 3rd instar. Preliminary surface dose-response

bioassays conducted (data not shown) with the 3rd instars at a CrleGV-SA concentration

of 1.34 x 106 OBs/ml showed an almost total response (100% mortality) in all batches,

after an exposure time of 9 and 10 days. As a result, susceptibility of CrleGV-SA

appeared to increase dramatically in the 3rd instar after 9 and 10 days due to the instars

marked increased susceptibility to the virus at longer exposure times. According to

Marcus & Eaves (2000) as time of exposure increases, smaller and smaller

concentrations will be required to elicit larval mortality. The LC50 value for the 4th instar

was established after 10 days exposure. This phenomenon has been reported by Federici

(1997) with type 2 GVs (such as CrleGV-SA), typically lasting between 5 - 10 days in

larvae infected during the 4th instar stage. The LC50 value for the 5th instars (2.678 x 107

OBs/ml), on the other hand was established after 14 days (longest exposure time)

(Moore, 2002).

According to some authors, when larvae move from one instar to the next (molting), their

midgut epithelial cells are normally sloughed off (Federici, 1997; Sun, 2005; Jehle et al.,

2006). As a result, the new epithelial cells generated, are rather thin and easily accessible

by the virus, resulting in infection (Federici, 1997). However, unlike the early instars, the

late instars (such as the 5th instar FCM larvae) tend to have a much thicker peritrophic

membrane which makes it difficult for the virus to penetrate and get access to the midgut

epithelium in order to initiate infection (Federici, 1997). This phenomenon, explains why

the 5th instar is more resistant to CrleGV-SA than the earlier instars. For instance the LC50

for the 5th instars was 2.678 x 107 OBs/ml, much higher than the LC90 values established

for the 1st, 2nd and 3rd instars. The LC90 values for the 1st, 2nd, 3rd and 4th instars,

increased by 36, 2.3 and16.7 fold respectively from one instar to the next. However, the

LC90 values from the 4th to the 5th instar increased by an appreciable 54.89 fold.

54

2.5 CONCLUSION A benchmark for pathogenicity has been established with CrleGV-SA (Cryptogran)

against FCM larvae. Lower susceptibility to CrleGV-SA has been found to decline with

larval stage and increases with time of exposure. This protocol was used in guiding

bioassays with field collected FCM larvae (see Chapter three).

55

CHAPTER THREE DOSE-RESPONSE BIOASSAYS WITH CRYPTOGRAN AGAINST FIELD COLLECTED FCM LARVAE 3.1 INTRODUCTION Insects of the same species collected from different regions, have been reported to show

variation in susceptibility to baculovirus infection (Briese & Mende, 1981; Briese, 1986;

Fuxa, 1987 & 1993). This chapter investigates the susceptibility of field collected FCM

larvae from a range of geographic regions to Cryptogran. There is little information on

bioassays with field collected insect larvae. This is probably due to the difficulty in

obtaining enough larvae, as well as, forecasting the required larval populace - from a

given batch of infested fruits for assays. As a result laboratory populations established

from field lines are normally preferred (Briese & Mende, 1981; Eberle & Jehle, 2006;

Jehle et al., 2006).

3.2 MATERIALS AND METHODS 3.2.1 Mass fruit collections from a range of geographic regions Mass collections of citrus fruits for conducting assays with field collected FCM larvae,

commenced in December 2007 and ended in May 2008. In the Eastern Cape Province

two orchards, namely Lone Tree Farm in Addo and Tregaron Farm in Kirkwood, were

both earmarked for mass fruit collections (Table 3.1). In the Western Cape Province, two

other orchards Rondegat in Clanwilliam and Jansekraal in Citrusdal were also selected

for mass fruit collections (Table 3.2). Although other mass collections of fruits were

conducted in the Nelspruit and Marble Hall regions (both in the Mpumalanga Province),

the number of larvae obtained from these fruits was inadequately small to warrant reliable

assays (Fig. 3.1).

56

Table 3.1 Passport data of FCM infested citrus fruit sampled from the Eastern Cape Province for the conducting of assays.

Farm name Lone Tree Tregaron

Area Addo – Sundays

River Valley (SRV) Kirkwood – Sundays River Valley (SRV)

Geographic coordinates 33o34’S,25o41’E 33o25’S,25o27’E Farmer Danie Bouwer Shane Atwell Orchard 36 9

Variety and cultivar

Lane Late navel oranges

Palmer navels oranges

Tree spacing (rows x trees)

6 m x 3 m 3 m x 6 m

Rootstock - Swingle Year planted 1999 1996 Age of Trees 8 yrs 12 yrs

Number of trees 2590 860 Size(hectares) 4.660 1.547

(-) Orchard detail not available. FCM infested citrus fruits were handpicked either off the ground or from trees, normally if

the characteristic FCM penetration marks (normally filled with frass) were observed on the

fruit. All fruit collections in the Eastern Cape were carried out early in the morning by

vehicular transport. Therefore it was possible to conduct assays on larvae, the same day.

Table 3.2 Passport data of FCM infested citrus fruit sampled from the Western Cape Province for the conducting of assays.

Farm name Rondegat Jansekraal

Area Clanwilliam Olifantsrivierberg,

Citrusdal Geographic coordinates 32o11’S,18o54’E 32o36’S,19o01’E

Farmer Oubaas Van Zyl George Grib Orchard 1, 2, 5 & 7 13

Variety and cultivar

Washington navel Oranges

Australian Summer navel Oranges

Tree spacing (rows x trees)

- -

Rootstock - - Year planted - - Age of Trees 30 years 10 years

Number of trees - - Size(hectares) - -

(-) Orchard detail not available.

57

In contrast, fruit collections conducted in the Western Cape and Mpumalanga Provinces

rather had to be couriered by air hence larvae could only be subjected to assays after 3 to

4 days. All assays were conducted in the laboratories of Citrus Research International in

Port Elizabeth (Eastern Cape).

Figure 3.1 A map showing the citrus growing areas in South Africa, where FCM-infested fruit were collected (Source: http://www.maps.yellowpages.co.za)

3.2.2 Determination of parasitised larvae A number of parasitised larvae were recovered from the field collected larvae. FCM larval

instars that turned ‘prematurely’ pinkish and smaller than normal were recorded as being

parasitised. Agathis bishopi was the predominant parasitoid. The sex of the parasitoids

was not determined.

Citrusdal

Mable Hall

Kirkwoood

58

3.2.3 Diet preparation and conducting of assays The same diet formulation as previously described in Chapter Two (incorporating agar)

was initially used for conducting assays with the field collected FCM larvae. However, the

diet was modified to exclude agar due to the extremely high level of mortality observed for

larvae on the untreated control (Table 3.5). The protocol adopted was a diet consisting of

200 g dry dietary ingredients plus 200 ml distilled water, uniformly mixed to form a paste

(Moore, 2002). The ingredients were mixed in a glass pie-dish and baked in an oven at

180oC. After 25 minutes, the diet was allowed to cool in a laminar flow cabinet. Once cool,

individual diet plugs were cut using a polypot which had the bottom removed (Fig. 3.2).

Individual polypots were sterilized in 2% sodium hypochlorite and rinsed under a running

tap water, and latter on dried under a laminar flow hood. The individually cut diet plugs,

were then transferred into each polypot using sterilized (dipped in 2% sodium

hypochlorite) forceps (Fig 3.2) (Moore, 2002).

Figure 3.2 Preparation steps for the conducting of surface dose-response bioassays with CrleGV-SA at 1.661 x 108 OBs/ml against field collected FCM larvae. A size 000 paint brush, used in transferring larvae onto diet (blue arrow); individually cut diet plugs (black arrow); a glass pie dish (green arrow) and a polypot with its base removed used in cutting round diet plugs (red arrow). And an FCM infested fruit dissected for larval isolation (brown arrow). The diet plugs were dipped in the CrleGV-SA suspension (CrleGV-SA diluted in

autoclaved distilled water) at a concentration of 1.661 x 108 OBs/ml (Fig 3.2). Afterwards,

59

infested fruits were individually cut open with the aid of sharp knives to locate larvae. A

size 000 paint brush (sterilised in 2% sodium hypochlorite), was used in transferring

larvae onto the diet plugs (Moore, 2002).

Individual larvae were transferred onto the surface of each diet starting with the control

and ending with the virus inoculated diets (Hunter-Fujita et al., 1998; Jones, 2000; Moore,

2002). All assays were carried out within a laminar flow cabinet. Afterwards, both the

inoculated and untreated control diets (containing larvae) were transferred to an

incubation chamber with temperature 27o ± 1oC. All larvae or pupae were recorded as

dead or alive, 10 days post-infection.

3.2.4 Surface dose-response bioassays with field collected FCM larvae In surface dose-response bioassays with field collected larvae, only the 2nd, 3rd, 4th and 5th

instars were used for conducting assays. Only healthy FCM larvae were used in

conducting assays. This measure was considered crucial in reducing stress induced

mortality (Finney, 1971; Jones, 2000). A single concentration of the virus was employed

in all assays with field collected FCM larvae. As it was not possible to predict the instars

obtained from a particular sample (fruits), and their numbers, a concentration which was

useful for all instars had to be selected. A concentration that would not elicit a total

response in all instars had to be selected. Therefore a single CrleGV-SA concentration of

1.661 x 108 OBs/ml (LC90 for the 4th instar, on an agar diet) was adopted.

Bioassays were replicated twice or thrice, where possible, for the filed collected 2nd, 3rd,

4th and 5th FCM instars. In the previous assays conducted with laboratory reared FCM

larvae (see Chapter Two) the accepted control mortality was 20%. However, due to the

high control mortality observed with the field collected larvae it was decided that control

mortalities not exceeding 35% will be used for data analysis.

60

3.2.5 Bioassays with laboratory reared 4th instar FCM larvae on a non-agar diet In the benchmark study with 4th instars, the LC90 value (established on an agar diet) was

used in guiding these bioassays with field larvae, therefore another dose-response

relationship had to be established for the 4th instars using a non-agar diet (see Appendix

3).

3.2.6 Statistical analysis Data from the dose-response bioassays were analysed using PROBAN (Van Ark, 1995),

a software programme used for conducting probit analyses with bioassay data. PROBAN

corrected the control mortality according to Abbott’s formula (Abbott, 1925). From this the

LC50 and LC90 values were calculated. PROBAN transformed the doses to log10 and the

percentage response to empirical probits. Using this information, the fit of the probit

(regression) lines were calculated, as were the fiducial limits. Bartlett’s test (P < 0.01) was

employed in the comparison of probit lines (Van Ark, 1995) (see Appendix 3). Differences

between treatments were determined with SPSS 11.0 statistical package, using a chi-

square goodness of fit test (see Appendix 2).

3.3 RESULTS 3.3.1 Field collection of FCM larvae from citrus fruits A total of 8637 citrus fruits were collected from the Mpumalanga, Western and Eastern

Cape provinces (from December 2007 to May 2008) for larval collection. The percentage

of 1st, 2nd, 3rd, 4th and 5th instar FCM larvae collected from the infested fruits was recorded

to be; 2.24%, 15.22%, 30.21%, 17.69% and 34.63% respectively (Table 3.3 & Fig. 3.3).

61

Table 3.3 FCM larvae collected from navel oranges from a range of geographic regions from December 2007 to May 2008

Date of fruit

collection

Area

Province

Total

number of fruits

collected

Number of FCM

larvae parasitized

Total number of FCM

larvae collected

1st 2nd 3rd 4th 5th 1st 2nd 3rd 4th 5th 19/12/2007 Addo Eastern Cape 852 0 0 8 1 0 6 63 188 113 80 8/01/2008 Addo Eastern Cape 366 0 0 1 0 0 3 46 64 62 59 15/01/2008 Addo Eastern Cape 286 0 0 1 0 0 0 11 58 49 32 19/01/2008 Nelspruit Mpumalanga 318 0 0 0 0 0 0 2 7 3 5 23/1/2008 Addo Eastern Cape 458 0 0 1 0 0 8 32 65 34 73 30/01/2008 Addo Eastern Cape 543 0 0 3 0 0 12 49 73 54 125 15/02/2008 Citrusdal Western Cape 902 0 0 0 0 0 0 2 4 5 26 15/02/2008 Clanwilliam Western Cape 350 0 0 0 0 0 0 0 0 3 8 16/02/2008 Addo Eastern Cape 661 0 1 19 0 0 0 19 62 25 108 20/02/2008 Kirkwood Eastern Cape 892 0 1 14 5 0 6 44 125 94 246 28/02/2008 Clanwilliam Western Cape 287 0 0 0 0 0 0 6 24 22 83 29/02/2008 Citrusdal Western Cape 396 0 0 0 0 0 0 20 27 21 96 5/03/2008 Marble Hall Mpumalanga 506 0 0 0 0 0 0 4 13 9 25 11/03/2008 Kirkwood Eastern Cape 525 0 0 5 1 0 3 19 45 27 46 18/04/2008 Addo Eastern Cape 399 0 0 5 1 0 5 60 101 22 46 24/04/2008 Citrusdal Western Cape 178 0 0 0 0 0 23 55 10 1 2 25/04/2008 Clanwilliam Western Cape 189 0 0 0 0 0 9 38 39 11 21 9/05/2008 Kirkwood Eastern Cape 258 0 0 12 3 0 2 31 73 20 46 21/05/2008 Clanwilliam Western Cape 155 0 0 0 0 0 0 7 22 23 52 21/05/2008 Citrusdal Western Cape 116 0 0 0 0 0 0 16 40 11 13

Figure 3.3 Total numbers of FCM larvae of each instar collected from a range of geographic regions in South Africa.

0

100

200

300

400

500

600

700

1st 2nd 3rd 4th 5th FCM larval instar

Addo (Eastern Cape)

Kirkwood (Eastern Cape) Clanwilliam (Western Cape)

Citrusdal (Western Cape)

Nelspruit (Mpumalanga)

Marble Hall (Mpumalanga) Num

ber o

f lar

vae

62

3.3.2 Parasitism Some level of parasitism was observed with larvae collected from Addo and Kirkwood

(Eastern Cape). The highest number of parasitized larvae was recorded in the 3rd instars.

The 3rd instars from the Addo and Kirkwood populations recorded 92.68% and 75.61%

parasitism respectively (Fig. 3.4). The 2nd instars from the Addo and Kirkwood

populations (with 2.44% each) recorded the lowest percentage of parasitised larvae (Fig.

3.4). There was no parasitism in the 5th instars. Agathis bishopi was the predominant

parasitoid. No FCM larvae collected from the Western Cape and Mpumalanga provinces

were parasitised.

0.0010.0020.0030.0040.0050.0060.0070.0080.0090.00

100.00

1st 2nd 3rd 4th 5th

FCM larval instar

Perc

enta

ge o

f par

asiti

sed

larv

ae

Addo (Eastern Cape)Kirkwood (Eastern Cape)Clanwilliam (Western Cape)Citrusdal (Western Cape)Nelspruit (Mpumalanga)Marble Hall (Mpumalanga)

Figure 3.4 Percentage of parasitised FCM larvae of each instars collected from a range of geographic regions in South Africa.

3.3.3 Dose-response bioassays with laboratory reared 4th instar FCM larvae on a non-agar diet In a five-fold serial dilution surface inoculated dose-response bioassay on a non-agar diet,

using CrleGV-SA concentrations ranging between 8.0 x103 and 5.00 x 106 OBs/ml

against 4th instars, a dose-response relationship was established. Control mortality was

zero percent (0.0%) for all three replicates. However, mortality of larvae receiving

treatment for the three replicates ranged from, 28.0% to 92.0% for the lowest and highest

concentrations respectively (Table 3.4).

63

Table 3.4 Mortality of 4th instar FCM larvae in five-fold dose response bioassays with CrleGV-SA.

Treatmen

t (OBs/ml)

Replicate 1 Replicate 2 Replicate 3 Larval

mortality (%)

Corrected

mortality (%

)

Empirical

probit

Larval mortality

(%)

Corrected

mortality (%

)

Empirical

probit

Larval mortalit

y (%)

Corrected

mortality (%

)

Empirical

probit

Distilled water

(control)

0.00

-

-

0.00

-

-

0.00

-

-

8.0 x 103 36.00 36.00 4.641 36.00 36.00 4.641 28.00 28.00 4.417 4.0 x 104 52.00 52.00 5.050 48.00 48.00 4.950 40.00 40.00 4.747 2.0 x 105 64.00 64.00 5.359 72.00 72.00 5.583 68.00 68.00 5.468 1.0 x 106 80.00 80.00 5.842 84.00 84.00 5.995 84.00 84.00 5.995 5.0 x 106 84.00 84.00 5.995 88.00 88.00 6.175 92.00 92.00 6.405

* 25 individual larvae were tested per treatment and replicated three times. The regression (probit) lines had the equations y = 2.7062 + 0.5040x (SE of slope =

0.1258), y = 2.2836 + 0.6017x (SE of slope = 0.1323) and y = 1.4111 + 0.7536x (SE of

slope = 0.1392). The residual variances of the three lines were determined to be

homogenous and the slopes of the lines parallel and comparable. The elevations of the

lines did not differ significantly from one another. G for fiducial limits was calculated to be

0.2392, 0.1857 and 0.1312 for the three lines. The mean LC50, LC90, LC95, LC99 and

LC99.9 values for the 4th instars, using a non-agar based diet were determined to be 4.21 x

104, 6.58 x 106, 3.06 x 107, 5.93 x 108 and 1.78 x 1010 OBs/ml respectively (Appendix 3).

3.3.4 Dose-response bioassays with field collected FCM larvae using an agar diet Fruit collections were carried out at Lone Tree Farm (Addo). Assays were conducted with

CrleGV-SA (at 1.661 x 108 OBs/ml) against the 2nd, 3rd and 4th instar FCM larvae using an

agar based diet.

Table 3.5 Control mortality of FCM larvae in bioassays (on an agar-based diet), collected from Lane Late navel oranges at Lone Tree Farm (Addo, SRV)

FCM instar stage

Total number of larvae used

Percentage mortality after 10 days

2nd 35 81.67% 3rd 39 82.23% 4th 38 84.17%

* Data from two replicates.

64

Table 3.6 Treatment mortality of FCM larvae in bioassays (on an agar-based diet), collected from Lane Late navel oranges at Lone Tree Farm (Addo, SRV)

FCM instar stage

Total number of larvae used

Percentage mortality after 10 days

2nd 67 100% 3rd 147 100% 4th 119 100%

* Data from two replicates. The percentage response recorded in the control was extremely high (Table 3.5). The

percentage mortality for all instars (2nd, 3rd and 4th instars) was over 80%. Therefore, data

for the treatment mortality (Table 3.6) was considered inaccurate and unusable. Apart

from the extremely high mortality, most of the diet was contaminated with fungus –

accompanied by rapid decay. As a result, future assays with field collected larvae were

conducted on diet excluding agar.

3.3.5 Bioassays with field collected FCM larvae from Lone Tree Farm (Addo, Eastern Cape) using a non agar-based diet Mortality of the entire field collected 2nd to 5th instars, from Lone Tree Farm ranged from

13.00 to 29.00% and 43.50 to 100.00% for the control and treatments respectively (Table

3.7).

Table 3.7 Control and CrleGV-SA treatment (1.661 x 108 OBs/ml) mortality for field collected FCM larvae from Lone Tree Farm (Addo, Eastern Cape) in bioassays on an agar-based diet

FCM instar stage

Total number

of larvae

used as control

Control Mortality

(%)

Total number of larvae used in

treatment

Treatment mortality

(%)

Treatment mortality corrected for control Mortality

Significant difference

test

2nd 17 29.00 66 96.97 95.71 a 3rd 42 28.60 103 100.00 100.00 a 4th 56 28.60 68 100.00 100.00 a 5th 69 13.00 85 43.50 35.06 b

* Data from three replicates, pooled together. Values followed by the same letter are not significantly different (P<0.05) (see Appendix 2).

65

0

20

40

60

80

100

120

140

2nd 3rd 4th 5th

Larval instar

% M

orta

lity a a a

b

Figure 3.5 Mortality of field collected FCM larvae from Lone Tree Farm (Eastern Cape), in bioassays with CrleGV-SA at 1.661 x 108 OBs/ml. (*Bars with the same letter are not significantly different, P<0.05). There was no significant difference (Χ2 = 5.23, dƒ = 2, p = 0.73 Cramer’s V = 0.15) in the

mortality of the 2nd, 3rd and 4th instar FCM larvae from Lone Tree Farm to CrleGV-SA

(1.661 x 108 OBs/ml). However, there was a significant difference (Χ2 = 147.92, dƒ = 3, p

< 0.001, Cramer’s V = 0.68) in the mortality of the 5th instars to the single virus dose

(Table 3.7 & Fig. 3.5). Corrected treatment mortality for the 5th instars was 35.06%, whilst

that of the lower instars (2nd to 4th instars) ranged from 95.71 to 100%.

3.3.6 Bioassays with field collected FCM larvae from Tregaron Farm (Kirkwood, Eastern Cape) using a non agar-based diet Mortality of the entire field collected 2nd to 5th instar FCM larvae from Tregaron Farm,

ranged from 6.67 to 14.55% and 64.62 to 100.00% for the control and treatments

respectively (Table 3.8). There was no significant difference (Χ2 = 1.701, dƒ = 2, p = 0.43

Cramer’s V = 0.086) observed in the mortalities of the 2nd, 3rd and 4th instar FCM larvae

from Tregaron Farm to CrleGV-SA (at 1.661 x 108 OBs/ml). However, there was a

significant difference (Χ2 = 74.149, dƒ = 3, p < 0.001, Cramer’s V = 0.50) in the mortality

of the 5th instars to the single virus dose (Table 3.8 & Fig. 3.6).

66

Table 3.8 Control and CrleGV-SA treatment (1.661 x 108 OBs/ml) mortality for field FCM larvae from Tregaron Farm (Kirkwood, Eastern Cape) in bioassays on an agar-based diet

FCM instar stage

Total number

of larvae

used as control

Control Mortality

(%)

Total number of

larvae used in

treatment

Treatment mortality

(%)

Treatment mortality corrected for control Mortality

Significant difference

test

2nd 15 6.67 42 97.62 97.45 a 3rd 25 12.00 109 98.12 97.92 a 4th 38 7.89 81 100.00 100.00 a 5th 55 14.55 65 64.62 58.60 b

* Data from three replicates, pooled together. Values followed by the same letter are not significantly different (P<0.05) (see Appendix 2).

Figure 3.6 Mortality of field collected FCM larvae from Tregaron Farm (Kirkwood, Eastern Cape) in bioassays with CrleGV-SA at 1.661 x 108 OBs/ml. (*Bars with the same letter are not significantly different, P<0.05). Corrected treatment mortality for the 5th instars was 58.60%, whilst that of the lower

instars (2nd to 4th instars) ranged from 97.45 to 100%.

3.3.7 Bioassays with field collected FCM larvae from Rondegat Farm (Clanwilliam, Western Cape) using a non agar-based diet Mortality of the entire field collected 2nd to 5th instar FCM larvae from Rondegat Farm,

ranged from 10.00 to 26.47% and 69.80 to 100.00% for the control and treatments

respectively (Table 3.9).

0

20

40

60

80

100

120

2nd 3rd 4th 5th Larval instar

% M

orta

lity

a a a

b

67

Table 3.9 Control and CrleGV-SA treatment (1.661 x 108 OBs/ml) mortality for field FCM larvae from Rondegat Farm (Clanwilliam, Western Cape) in bioassays on an agar-based

FCM instar stage

Total number

of larvae

used as control

Control Mortality

(%)

Total number of larvae used in

treatment

Treatment mortality

(%)

Treatment mortality corrected for control Mortality

Significant difference

test

2nd 23 26.09 21 100.00 100.00 a 3rd 34 26.47 38 100.00 100.00 a 4th 19 10.53 31 100.00 100.00 a 5th 50 10.00 93 69.80 66.54 b

* Data from three replicates, pooled together. Values followed by the same letter are not significantly different (P<0.05) (see Appendix 2).

Figure 3.7 Mortality of field collected FCM larvae from Rondegat (Western Cape) in bioassays with CrleGV-SA at 1.661 x 108 OBs/ml (*Bars with the same letter are not significantly different, P<0.05). There was no significant difference (Χ2 = 7.947, dƒ = 5, p = 0.159 Cramer’s V = 0.206)

observed in the mortalities of the 2nd, 3rd and 4th instar FCM larvae from Rondegat Farm

to CrleGV-SA (at 1.661 x 108 OBs/ml). However, there was a significant difference (Χ2 =

31.992, dƒ = 3, p < 0.001, Cramer’s V = 0.418) in the mortality of the 5th instars to the

single virus dose (Table 3.9 & Fig. 3.7). Corrected mortality for the 5th instars was

66.54%, whilst that of the lower instars (2nd to 4th instars) was 100%.

0

20

40

60

80

100

120

2nd 3rd 4th 5th Larval instar

% M

orta

lity

a a a

b

68

3.3.8 Bioassays with field collected FCM larvae from Jansekraal Farm (Citrusdal, Western Cape) using a non agar-based diet Mortality of the entire field collected 2nd to 5th instar FCM larvae from Jansekraal Farm,

ranged from 11.54 to 20.00% and 70.15 to 100.00% for the control and treatments

respectively (Table 3.10).

Table 3.10 Control and CrleGV-SA treatment (1.661 x 108 OBs/ml) mortality for field FCM larvae from Jansekraal Farm (Citrusdal, Western Cape) in bioassays on an agar-based

FCM instar stage

Total number of larvae used as control

Control Mortality

(%)

Total number of

larvae used in

treatment

Treatment mortality

(%)

Treatment mortality corrected for control Mortality

Significant difference

test

2nd 42 16.67 41 100.00 100.00 a 3rd 22 18.18 35 100.00 100.00 a 4th 35 20.00 21 95.24 94.05 a 5th 26 11.54 67 70.15 66.26 b

* Data from two replicates, pooled together. Values followed by the same letter are not significantly different (P<0.05) (see Appendix 2).

Figure 3.8 Mortality of field collected FCM larvae from Jansekraal (Western Cape) in bioassays with CrleGV-SA at 1.661 x 108 OBs/ml (*Bars with the same letter are not significantly different, P>0.05). There was no significant difference (Χ2 = 7.947, dƒ = 5, p = 0.159 Cramer’s V = 0.206)

observed in the mortalities of the 2nd, 3rd and 4th instar FCM larvae from Rondegat Farm

to CrleGV-SA (at 1.661 x 108 OBs/ml). However, there was a significant difference (Χ2 =

0

20

40

60

80

100

120

2nd 3rd 4th 5th Larval instar

% M

orta

lity

a a a

b

69

29.813 dƒ = 3, p < 0.001, Cramer’s V = 0.426) in the mortality of the 5th instars to the

single virus dose (Table 3.10 & Fig. 3.8). Corrected mortality for the 5th instars was

66.26%, whilst that of the lower instars (2nd to 4th instars) ranged from 94.05 to 100%.

3.3.9 Susceptibility of field collected and laboratory reared 5th instar FCM larvae to CrleGV-SA using a non agar-based diet Further assays were conducted with 5th instars from a laboratory colony. This was

necessary since control mortality for the laboratory colony was generally low therefore

improving accuracy. Furthermore, treatment mortality was well below 100% and well

above 0%, making reliable comparison between field populations and the lab colony a

distinct probability.

Table 3.11 Control and treatment mortality for laboratory reared 5th instar FCM larvae

FCM instar stage

Total number of

larvae used as control

Control Mortality

(%)

Total number of

larvae used in treatment

Treatment mortality

(%)

5th 90 0.00 150 69.33 * Data from three replicates were pooled together; with 30 larvae used per treatment for the untreated control and 50 larvae used per treated diet. There was no correction for natural mortality, since control mortality was zero (0). There was no mortality of the control larvae. The average percentage mortality for the 5th

instars was 69.33% (Table 3.11 & Fig. 3.9).

0.00

69.33

01020304050607080

Control Treatment

5th Instar

% M

orta

lity

Figure 3.9 Control and CrleGV-SA treatment (at 1.661 x 108 OBs/ml) mortality for laboratory reared 5th instar FCM larvae.

70

Table 3.12 Mortality of field collected and laboratory reared 5th instar FCM larvae.

Source of FCM larvae Total number of 5th instars treated with CrleGV-SA (at 1.661

x 108 OBs/ml)

Corrected mortality

(%)

Significant difference test

Addo (Eastern Cape) 85 35.06 b Kirkwood (Eastern Cape) 65 58.60 a

Clanwilliam (Western Cape) 93 66.54 a Citrusdal (Western Cape) 67 66.26 a

Laboratory colony 150 69.33 a *Values followed by the same letter are not significantly different (P<0.05) (see Appendix 2).

There was a significant difference (Χ2 = 15.02, dƒ = 1, p < 0.001, Cramer’s V = 0.253) in

mortality between the field collected 5th instar FCM larvae, from Lone Tree Farm and the

laboratory colony (Table 3.12 & Fig. 3.9).

Figure 3.10 Mortality for both field collected and laboratory reared FCM larvae in bioassays with CrleGV-SA at 1.661 x 108 OBs/ml. (*values followed by the same letter are not significantly differently, P>0.05). However, there was no significant difference between the field collected 5th instar FCM

larvae, from Tregaron (Χ2 = 0.463, dƒ = 1, p = 0.496, Cramer’s V = 0.046), Rondegat (Χ2

= 0.008, dƒ = 1, p = 0.927, Cramer’s V = 0.006), Jansekraal (Χ2 = 0.015, dƒ = 1, p =

0.904, Cramer’s V = 0.008) and the laboratory colony to CrleGV-SA (at 1.661 x 108

OBs/ml) (Table 3.12 & Fig. 3.10).

0

10

20

30

40

50

60

70

80

Laboratory Addo Kirkwood Clanwilliam Citrusdal

Source of FCM larvae

% M

orta

lity

b

a a a a

71

3.4 DISCUSSION Sishuba (2003) found Agathis bishopi (Nixon) (larval parasitoids) and T. cryptophlebiae

(an egg parasitoid) predominant in the Eastern Cape (South Africa). Similarly, in this

study all the parasitised larvae were collected from the Eastern Cape (Addo and

Kirkwood), with Agathis bishopi being the predominant parasitoid. The highest number of

parasitised larvae was recorded with the 3rd instars (Fig. 3.4). Gendall et al. (2006) also

recorded the highest number of parasitised larvae with the 3rd instars. Gendall (2006)

speculated that, the augmentative release of A. bishopi for the control of FCM is probable.

The number of 1st instar larvae isolated from field collected fruits, was inadequately small

to enable the execution of bioassays (Table 3.4). Sishuba (2003), Moore & Kirkman

(2004) and Gendall et al. (2006) also reported similar trends with regard to low

populations of 1st instar larvae isolated from field collected fruits. Also, the 1st instar larvae

proved to be delicate and exhibited a very high level of mortality (Stofberg, 1939; Catling

& Aschenborn, 1978). In addition the methodology used in isolating larvae (by dissecting

infested fruits with the aid of sharp knives to isolate larvae) also proved to be more fatal

for the 1st instar than the other larvae, hence their exclusion.

It was observed that all the field collected larvae exhibited some degree of mortality on

the untreated control diet. It was concluded that the sudden change in diet from a citrus

fruit to an artificial diet contributed to larval mortality. This phenomenon was also

observed by Moore (2002) when establishing laboratory colonies from field lines. In

contrast, it was possible to obtain zero percent (0%) mortality of untreated control larvae

when using laboratory reared FCM larvae fed on an agar-based diet (see Chapter Two)

and a non agar-based diet (Table 3.3). The difficulty in obtaining very low or zero mortality

results for the untreated field collected FCM larvae has been reported by other

researchers (Sishuba, 2003; Moore & Kirkman, 2004; Gendall et al., 2006).

The mean LC50 and LC90 values for the 4th instars using a non-agar diet were determined

to be 4.21 x 104 and 6.58 x 106 OBs/ml respectively. However, the LC50 and LC90 values

for the 4th instars, previously established using an agar based diet were 2.205 x 106 and

1.661 x 108 OBs/ml (see Chapter Two). Moving from a non agar diet to an agar diet the

72

LC50 and LC90 values for the 4th instars increased by 52.43 and 25.25 fold respectively. In

bioassays, changes in diet have been reported to affect host susceptibility to baculovirus

infection (Hunter-Fujita et al., 1998; Jones, 2000). This increased mortality with CrleGV-SA treatments when using a non-agar diet could

generally be attributed to the different methodologies employed in these assays (Hunter-

Fujita et al., 1998; Jones, 2000). With the non-agar diet, the diet plugs were wholly dipped

into the virus suspension – which enabled the virus suspension to cover the entire diet

surface, hence increasing the probability of larvae ingesting more virus particles.

However, with the agar diet the virus suspension was dispensed only onto the diet. Hence

the probability of a larva ingesting virus was limited to the diet surface (Moore, 2002).

In addition, the coarseness of the diet surface (due to the exclusion of agar), for the non

agar diet ensured the adequate absorption of the inoculum. The methodology used in

dispensing the virus inoculum had a significant effect on virus pathogenecity. According to

Jones (2000) using different substrates in administering virus dosage as well as different

assay techniques could contribute to assay variation.

In laboratory assays with field collected 2nd, 3rd and 4th instar FCM larvae from Addo,

Kirkwood, Citrusdal and Clanwilliam, there was no significant difference in their response

(94.05 to 100% mortality) to CrleGV-SA at 1.661 x 108 OBs/ml. However, it was believed

that the virus concentration used was too high to pick up any small or relatively small

differences in susceptibility between instars. Conversely, mortality of 5th instars collected from Addo was significantly lower than for

those collected from Kirkwood, Clanwilliam, Citrusdal and for the laboratory colony. The

5th instars from Addo exhibited a low percentage mortality of 35.0% in contrast to the

58.60 to 69.33% mortality observed in the other populations. Intrinsic differences in

individual larvae could contribute to variation in assay results (Jones, 2000). Yet again,

the low number of individual larvae tested per treatment, per replicate probably

contributed to this significantly low mortality recorded with the 5th instars from the Addo

population. According to Jones (2000) an absolute minimum of 20 larvae should be tested

73

at each dose, with each trial being replicated at least three times. However, this was

difficult to achieve with the field collected larvae. This was probably due to the difficulty in

obtaining enough larvae, as well as, forecasting the required larval populace - from a

given batch of infested fruits for assays. A field trial conducted by Moore & Kirkman (unpublished) in the same orchard (Lone Tree

Farm - Addo) from which the larvae were collected for the bioassays - showed an 86.7%

reduction in fruit infestation with a Cryptogran application. Cryptogran was applied on

some selected citrus trees, with an untreated control. Thereafter, field evaluation trials

(individual fruits were scored for the presence of FCM) were conducted weekly for two

months (January and February) to determine infestation rate (Fig. 3.11) (Moore &

Kirkman, unpublished).

Figure 3.11 False codling moth infestation on navel oranges (Addo, SRV, Eastern Cape): 2007 - 2008. *Source: Moore & Kirkman (unpublished). In a study on genetic variation among geographic populations of FCM by Timm (2005b),

high levels of polymorphism were observed among FCM populations sampled from the

Western Cape, Eastern Cape and Mpumalanga provinces. However, genetic diversity

was reported to be much higher within the Western Cape populations than both the

Eastern Cape and Mpumalanga populations (Timm, 2005a & 2005b). Timm (2005a &

2005b) found that populations sampled from the same province appeared to be more

closely related to one another than those collected from other provinces. The inference

86.7%

January February

74

drawn was that, FCM populations were more or less locally adapted populations (Timm,

2005a). Timm (2005a) speculated that these locally adapted FCM populations could vary

in their response to insecticide resistance or virus susceptibility (Timm, 2005a).

Interestingly, Timm (2005a) found that population genetic differentiation was similar in the

Western Cape and Mpumalanga populations, but significantly lower in the Eastern Cape

populations. She concluded that the Eastern Cape populations were more heterogeneous

than the Western Cape and Mpumalanga populations (Timm, 2005a). However in this

study, there was a notable difference in virus susceptibility even between 5th instars

collected within the same province (Addo and Kirkwood). On the other hand, all field

applications of CrleGV-SA commercial products (such as Cryptogran) are targeted

against 1st instar larvae (other larval stages cannot be reached with the virus) (Moore,

2002; Moore & Kirkman, 2004).

Even if this difference in susceptibility of 5th instars in different populations is real and

repeatable, it may be irrelevant, as field usage of the virus is exclusively against 1st

instars. Therefore these comparisons must be conducted between 1st instars, if any

meaningful conclusion is to be drawn (see Chapter Four).

Despite the significantly lower susceptibility observed with 5th instars from Addo, 5th

instars (especially late 5th instars) are normally not accepted as good candidates for

bioassays analyses. Hence it is speculated that small differences in larval age could also

contribute to variation in assays. Especially when working with field larvae which vary in

age due to difficulty in synchronizing rearing conditions unlike in laboratory cultures. For

instance it is noted that there is a higher probability of early 5th instars ingesting diet than

is the case for late 5th instars, due to the increased tendency to enter into the pre-pupal

stage (see Chapter One section 1.3.73) observed with the latter (Catling & Aschenborn,

1978; Briese, 1981; Moore, 2002). In a similar experiment, Briese (1981) saw it justifiable

to exclude the 4th instars of the potato tuber moth, Phthorimaea operculella (Zeller), which

has only four instar stages, in laboratory assays.

75

There are a number of reports where differences in susceptibility between geographically

distinct insect host populations to baculoviruses were observed. Briese (1981)

investigated variation in the susceptibility of 16 field populations of P. operculella to its

granulovirus in Australia. Briese (1981) recorded a 11.6-fold increase in resistance

between the least and most susceptible field strains, whilst a laboratory strain (used as a

standard) showed more than a 30-fold increased resistance to the virus than any of the

field strains. The inference drawn was that this was as a result of natural selection for

resistant individuals during virus epizootics (Briese, 1981; Cory et al., 1997). In

consequence, this situation reflects some dangers associated with extrapolating

laboratory results as possible field scenarios.

3.5 CONCLUSION Conducting of bioassays with field collected FCM larvae on a non-agar based diet is more

suitable than an agar-based diet. Changes in diet and methodology could contribute to

variation in assays. Although mortality of 5th instar FCM larvae from Addo was

significantly lower in laboratory assays, a field trial conducted in the same orchard as the

larvae were collected showed excellent control of FCM. However, in order to obtain

reliable results and ascertain this scenario, further assays were conducted with laboratory

colonies of 1st instar FCM larvae from Addo (Lone Tree Farm) established from field lines,

as well as a standard laboratory colony (see Chapter Four).

76

CHAPTER FOUR DOSE-RESPONSE BIOASSAYS WITH CRYPTOGRAN AND CRYPTEX AGAINST GEOGRAPHICALLY DISTINCT LABORATORY COLONIES OF FCM 4.1 INTRODUCTION Previous studies have shown that virus isolates collected from the same insect species

can have different genotypes and that these genotypes can differ in their pathogenicity

(Smith & Crook, 1988; Maeda et al., 1990; Cory et al., 1997; Cory et al., 2005). A recent

study by Goble (2007) showed that although Cryptogran (Moore, 2002; Moore & Kirkman,

2004) and Cryptex (Kessler & Zingg, 2008) are both CrleGV-SA isolates they differ

significantly in their pathogenicity and genetic profile. Goble (2007) showed that

Cryptogran was significantly more pathogenic than Cryptex. However, this comparative

study by Goble (2007) was carried out on a single FCM laboratory colony reared for

numerous generations. Furthermore, virus isolates are known to be more pathogenic to

their homologous host (Lacey et al., 2002). For instance CpGV has been reported to

infect both codling moths (CM) and FCM. But, CpGV is noted to be more virulent in CM

than FCM. However, CM is not susceptible to the FCM GV, CrleGV (Jehle et al., 1992).

Significant genetic differences within geographically distinct FCM populations have also

been reported (Timm, 2005a & 2005b). In this study, four new and separate laboratory

colonies have been successfully established from field collected FCM larvae from Addo

(Eastern Cape), Citrusdal (Western Cape), Marble Hall and Nelspruit (both from the

Mpumalanga Province). Comparative bioassays were conducted with Cryptogran and

Cryptex against the old FCM colony and against the newly established Addo colony.

4.2 MATERIALS AND METHODS 4.2.1 Mass fruit collections for the establishment of geographically distinct FCM colonies Mass collections of FCM infested citrus fruit for the establishment of field lines

commenced in April 2007 and ended in June 2008. Fruit were collected either from the

floor of the orchard or on the trees. Only fruit showing the characteristic frass-filled (see

77

Fig. 1.1, Chapter One) penetration holes or discolouration around the penetration holes

were selected. Fruit were collected from individual orchards (each representing a

geographically distinct population) from Addo, Citrusdal, Marble Hall and Nelspruit for the

establishment of new and separate laboratory colonies (Table 4.1). Unlike the fruits

collected from Addo, those collected from Citrusdal, Nelspruit and Marble Hall had to be

couriered by air. The old colony was established in 1996 (Sean Moore pers. comm.). It is

estimated that there are over 144 FCM generations to date, that is, if one generation lasts

for a month (see Chapter Two). The parent material was obtained from Citrusdal,

Zebediela (Limpopo Province) and the Eastern Cape (Sean Moore, pers. comm.).

Therefore, the old population was of heterogeneous origin.

Table 4.1 Passport data of FCM infested citrus fruit sampled from the Eastern Cape, Western Cape and Mpumalanga Provinces for the establishment of laboratory colonies

Details Addo colony

Citrusdal colony

Nelspruit colony Marble Hall colony

Farm name Lone Tree Farm Noordhoek Crocodile Valley Schoeman Boerdery

Area

Addo – Sundays River Valley (SRV)

Citrusdal (bo-rivier)

Nelspruit Marble Hall

Geographic coordinates

33o34’S, 25o41’E

32o36’S, 19o01’E

25o28’S, 30o58’E

24o58’S, 24o18’E

Farmer Danie Bouwer Hardy van der Merwe

Johan Kellerman Oubaas van Zyl

Orchard 36 - Bridge 3 Block J11 Variety Lane Late navel

oranges Washington

navel oranges Lane late navel

oranges Washington

navel oranges Tree spacing (rows x trees)

6 m x 3 m 6 m x 4 m 8.3 m x 2.8 m 5.5 m x 5.5 m

Rootstock - Rough lemon Empress mandarin

Rough lemon

Year planted 1999 1978 1978 1968 Age of trees 8 yrs 30 yrs 30 yrs 40 yrs

Number of trees 2590 458 586 - Size(hectares) 4.66 1.1 1.36 3.84

4.2.2 Determination of parasitised larva

A number of larvae were found to be parasitised, the same criteria as described in

Chapter Two for the identification of parasitoids was adopted.

78

4.2.3 Laboratory rearing of field collected FCM larvae 4.2.3.1 Small scale moth rearing using test-tubes This procedure was employed in the establishment of an FCM laboratory colony from field

collected individuals. The moth rearing process was carried out concurrently in separate

rooms, with each room hosting one colony. The initial diet used for rearing the field

collected larvae consisted of, 200 g dry ingredients of the standard diet (Moore, 2002)

plus 200 ml distilled water, uniformly mixed to form a paste. The contents were mixed in

glass pie-dishes and heated in an oven at 180oC. After 25 minutes, the cooked diet was

allowed to cool in a laminar flow cabinet. Once cool, individual diet plugs (approximately 5

to 7 mm thickness) were cut using the lip of a glass test-tube (28 ml capacity) (Fig. 4.2).

Thereafter, the diet plugs were inserted into the tubes (using sterilized glass rods) and

pushed to the bottom of the tube. Fruit were individually cut open with the aid of sharp

knives to locate larvae (Fig. 4.1 B). A size 000 paint brush (sterilised in 2% sodium

hypochlorite) was used in transferring individual larvae onto the diet plugs (Moore, 2002).

Afterwards the test-tubes were corked with cotton wool. Thereafter, the larvae (held on

the diets in the test-tubes) were sent to an incubation chamber with a temperature of

about 27o ± 1oC for development and growth to take place (Fig. 4.1 C).

Figure 4.1 (A) FCM infested navel oranges collected from the field for the establishment of FCM laboratory colonies. (B) A dissected navel orange revealing infestation of a 5th instar FCM larva. (C) Test-tubes (28 ml capacity) each containing an FCM larva on diet stored in an incubation chamber at 27o ± 1oC. Upon emergence of adult moths the cotton wool stoppers were removed. To collect

emerging moths a second test-tube was inverted over the first one to allow the moths to

climb to the top of the inverted test-tube. Thereafter, all the moths emerging from the

A B C

79

individual test-tubes were transferred into a sieve (approximately 15.5 cm in diameter)

through a hole (approximately 25 mm diameter) cut in the middle of the sieve (Fig. 4.2 a).

The sieve was inverted onto a wax paper (Fig. 4.2 a). The bottom of the test-tube was

gently tapped with a finger to encourage the moths to fly or drop into the sieve (Fig. 4.2

c). The hole in the sieve was then fitted with cotton wool soaked in water which served as

a source of nourishment for the moths. The sieve was finally secured at both edges with

sellotape in order to prevent moths from escaping through the space between the edge or

mouth of the sieve and the wax paper.

Figure 4.2 Layout of an oviposition apparatus with; (a) a kitchen sieve (green arrow) inverted over a wax paper (black arrow) holding ovipositing moths; (b) a test-tube (28 ml capacity) in which a moth has recently eclosed (red arrow) (c) a test tube (blue arrow) transferring a freshly eclosed adult moth into the sieve.

4.2.3.2 Large scale moth rearing using jam jars This procedure was employed as a continuation of the small scale moth rearing process,

as well as, for the maintenance of stable laboratory colonies of FCM (Moore, 2002). For

large scale moth rearing, 370 ml jam jars (Fig. 4.3) were used instead of the individual

test-tubes (Fig. 4.2b). This was because each jam jar could hold enough larvae

(approximately 300 to 400 FCM eggs), and was also found to be suitable for large scale

80

moth rearing (Moore, 2002). FCM diet was prepared beforehand. Therefore, a mixture of

50 g standard dry diet and 50 ml distilled water (dH20) were weighed and dispensed into

each jam bottle. Afterwards individual bottles were then stoppered with cotton wool and

autoclaved at 121oC for 20 minutes and placed under a laminar flow hood to cool (Moore,

2002). After 3 to 4 days most of the moths held in the sieve (Fig. 4.2) had started to lay

eggs. Initially only a few eggs were laid (mostly singly) and were scattered on the wax

sheet. In order to maximize the use of each available egg, the egg sheets had to be cut

into several pieces before sterilizing in 10% formaldehyde solution (from 35 to 40%

formaldehyde stock solution). The egg sheets were then transferred into the 370 ml jam

jars (containing diet) using sterilized forceps (Moore, 2002). The jam bottles were finally

sent to an incubation chamber with temperature of about 27o ± 1oC for development and

growth to take place.

After the first filial generation (F1) the number of larvae pupating in the cotton wool

stoppers fitted in the jam jars (Fig. 4.3) was inadequately small to maintain a stable

laboratory colony. Therefore, individual pupae were gently removed (leaving a few cotton

wool strands on the pupa) from the cotton wool by hand. The pupae were then held singly

in the test-tubes (Fig. 4.2 b) until moth emergence.

Figure 4.3 Jam jars (370 ml capacity) containing diet with larvae feeding in diet and pupating in cotton wool. At this stage, moths emerging from the pupa (attached to the cotton wool strands inside

the test-tubes) were always held in the oviposition apparatus for egg laying (Fig. 4.2).

81

Eggs laid during the preceding 24 h on the wax paper sheets, in the oviposition apparatus

(Fig. 4.2) were reared using the jam jars as previously outlined.

Figure 4.4 A wax paper containing FCM eggs.

This process was repeated until the F4 generation. After the F4 generation the egg density

and moth numbers had improved significantly, thus the use of the oviposition apparatus

(Fig. 4.2) was discontinued. Instead an emergence box (Fig. 4.5) consisting of a ten

compartment facility specially designed for the moths was used.

Figure 4.5 A custom built moth emergence and oviposition structure with wax paper sheets (black arrow) fitted to each compartment on which eggs are laid. Moist cotton wool is plugged at the top of each compartment - serving as a source of water for the moths (blue arrow). Several pieces of egg sheets (containing approximately 300-400 eggs) from the F4

generation were cut into approximately 10 mm by 10 mm squares with a pair of scissors.

The eggs were sterilized in 10% formaldehyde solution using sterilised forceps (Moore,

2002). One egg sheet was then placed into each jam bottle (containing diet) using sterile

forceps. The jam bottles were then sent to the incubation chamber for development and

growth to take place (Fig. 4.3). When all the larvae in the jam jars had reached pupation,

the jam bottles were finally sent to the moth emergence box (Fig. 4.5) (Moore, 2002).

82

Thereafter, subsequent eggs laid during the preceding 24 h on the wax paper sheets (Fig.

4.4) fitted inside the wire mesh of the moth emergence box (Fig. 4.5) were always

collected for the maintenance of the laboratory colonies or for use in conducting

bioassays.

4.2.4 Conducting of bioassays using Cryptogran and Cryptex against 1st instar FCM larvae Upon reaching the F5 generation, eggs laid (Fig. 4.4) during the preceding 24 h on the

wax paper sheets, from the emergence box, were cut out with a pair of scissors and then

kept in empty sterile jam jars. The jam jars were covered with a lid and placed in the

incubation chamber. After 3 to 4 days most of the eggs had hatched out with the

emerging 1st instars congregating on the underside of the lids. Individual larvae were then

transferred from the lids using a size 000 paint brush into polypots filled with virus

inoculated diet (Cryptogran or Cryptex, treatments) or dH20 (control) (see Chapter Two).

To minimise contamination new lids were used for each treatment. The polypots were

then transferred to the incubation chamber until evaluation.

A five-fold serial dilution technique was employed (see Chapter Two). Fifteen polypots

were used per treatment, including the control. Two 1st instar FCM larvae were held in

each polypot, making a total of 30 larvae per treatment (including the control). Therefore,

a total of 90 polypots with 180 larvae were used per trial. Each batch (180 larvae) was

tested separately using Cryptogran or Cryptex. Considerable manipulation was required

to determine the appropriate series of concentrations to produce a good dose-response

curve (see Chapter Two). This was therefore quite a lengthy process. After 7 days, larvae

were recorded as dead or alive. Results from each bioassay were replicated at least three

times. Bioassays were conducted, only, with the old colony and the Addo colony since

there were not enough larvae from the other colonies (Citrusdal, Marble Hall and

Nelspruit colonies) to warrant any reliable assays during that period. Several bioassays

had to be conducted before an adequate number of acceptable results could be obtained.

In consequence some bioassays were replicated three times whilst others were replicated

four times.

83

4.2.5 Statistical analysis Data from the dose-response bioassays were analysed using PROBAN (Van Ark, 1995)

PROBAN corrected the control mortality according to Abbott’s formula (see Chapter Two)

(Abbott, 1925). From this, the LC50 and LC90 values were calculated. PROBAN

transformed the doses to log10 and the percentage response to empirical probits. Using

this information, the fit of the probit (regression) lines were calculated, as were the fiducial

limits. Bartlett’s test (P < 0.01) was employed in the comparison of probit lines (Van Ark,

1995).

4.3 RESULTS 4.3.1 Survival and development of field collected FCM larvae from Addo, Citrusdal, Marble Hall and Nelspruit A total of 1266, 663, 406 and 704 citrus fruits were sampled from individual orchards from

Addo, Citrusdal, Marble Hall and Nelspruit respectively. The highest percentage of field

collected larvae emerging into moths (first generation) was recorded with the 5th instars

from all the field lines (Fig. 4.6, Appendix 7). This indicates that 5th instars adapt easily

than do the other instars to laboratory conditions (such as changes in diet), hence making

them a good candidate for the establishment of a laboratory colony.

0.0010.0020.0030.0040.0050.0060.0070.0080.0090.00

1st

2nd

3rd

4th

5th

1st

2nd

3rd

4th

5th

1st

2nd

3rd

4th

5th

1st

2nd

3rd

4th

5th

Addo Citrusdal Marble Hall Nelspruit

Instar stage

% S

urvi

val

Figure 4.6 Percentage survival and development of field collected FCM larvae to adulthood.

84

4.3.2 Parasitism

0.00

10.00

20.00

30.00

40.00

50.00

60.00

1st 2nd 3rd 4th 5th

Larval Instar

Perc

enta

ge p

aras

itise

d (%

)

Addo

Figure 4.7 Proportion of parasitised larvae recovered from field collected larvae from Addo With the exception of the larvae collected from Addo, there was no parasitism recorded

with the other colonies. More 3rd instar larvae were parasitised than any other instar

(53.85%) with the 2nd instars recording the lowest (7.69%) (Fig. 4.7) (see Appendix 7).

There was no parasitism in the 1st and 5th instars. Agathis bishopi was the predominant

parasitoid. The sex of the parasitoids was not determined.

4.3.3 Relative humidity and temperature in the incubation chamber There were fluctuations in the temperature and humidity readings in the incubation

chambers in which the Addo, Citrusdal, Nelspruit and Marble Hall colonies were being

reared (see Appendix 7). Conditions such as size of each incubation chamber, human

movement, external day and night temperatures (influenced by the season of the year)

could have caused these fluctuations. The lowest and highest room relative humidity

readings in the chambers were recorded during the months of September and November

respectively, for all colonies (see Appendix 7). An average incubation room temperature

of 27o ± 1oC and 44.5% relative humidity was considered to be ideal for the growth and

development of the newly established FCM colonies. However, ideal humidity for moths is

a lot higher (in the region of 70%) (Moore, 2002).

85

4.3.4 Bioassays with Cryptex against 1st instar FCM larvae from the old colony In a five-fold serial dilution surface inoculated dose-response bioassay on an agar-based

diet using Cryptex, against 1st instars from the old colony a dose-response relationship

was established. Cryptex concentrations ranging between 1.60 x102 and 1.00 x 105

OBs/ml were used. Control mortality ranged from 5.00% to 17.00% for all four replicates.

However, mortality of larvae feeding on treated diet (Cryptex) ranged from 13.33% to

83.33.0% for the lowest and highest concentrations respectively (Table 4.2).

Table 4.2 Mortality of 1st instar FCM larvae from the old colony in a dose-response bioassay with Cryptex.

Treatment

(OBs/ml)

Replicate 1 Replicate 2 Replicate 3 Replicate 4

Larval m

ortality (%)

Corrected

mortality (%

)

Empirical

probit

Larval m

ortality (%)

Corrected

mortality (%

)

Empirical

probit

Larval m

ortality (%)

Corrected

mortality (%

)

Empirical

probit

Larval m

ortality (%)

Corrected

mortality (%

)

Empirical

probit

d (H2O) Control

5.00 - - 13.00 - - 13.00 - - 17.00 - -

1.60 x102 13.33 8.77 3.645 26.67 15.71 3.994 20.00 8.05 3.598 30.00 15.66 3.991 8.0 x 102 23.33 19.30 4.133 33.33 23.37 4.273 33.33 23.37 4.273 36.67 23.69 4.284 4.0 x 103 43.33 40.35 4.756 43.33 34.87 4.611 40.00 31.03 4.505 50.00 39.76 4.740 2.0 x 104 50.00 47.37 4.934 53.33 46.36 4.909 50.00 42.53 4.812 63.33 55.82 5.146 1.0 x 105 76.67 75.44 5.688 80.00 77.01 5.739 76.67 73.18 5.618 83.33 79.82 5.839

The regression (probit) lines for the four replicates (Fig. 4.8) had the equations y = 2.1422

+ 0.6911x (SE of slope = 0.1383), y = 2.4603 + 0.6156x (SE of slope = 0.1500), y =

2.1861 + 0.6567x (SE of slope = 0.1624) and y = 2.3529 + 0.6734x (SE of slope =

0.1591), respectively.

86

2

2.5

3

3.5

4

4.5

5

5.5

6

6.5

7

1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5

Log dose

Em

piri

cal p

robi

ts

Rep 1

Rep 2

Rep 3

Rep 4

Figure 4.8 Dose-response probit lines (four replicates) from bioassays conducted with Cryptex (CrleGV-SA) against 1st instar larvae from the old colony. Using Bartlett’s test for homogeneity of residual variances, the residual variances of the

four lines were shown to be homogenous, making the chi-squared test more applicable

than would have been the case if deviations were heterogeneous. The slopes of the four

lines were parallel and therefore comparable. The elevations of the lines did not differ

significantly from one another. According to Van Ark (1995), values of G greater than

0.025 indicate a large variation in mortality, but the experimental procedure or the value of

the probit line was questionable if G exceeded 0.25. G for fiducial limits was calculated to

be 0.1538, 0.2281, 0.2348 and 0.2145 for lines 1, 2, 3 and 4 respectively. The mean LC50

and LC90 values for the four lines were determined to be 1.37 x 104 and 1.25 x 106 OBs/ml

respectively (see Appendix 4 & 5).

4.3.5 Bioassays with Cryptogran against 1st instar FCM larvae from the old colony Control mortality ranged from 5.00% to 17.00% for all four replicates. However, mortality

of larvae feeding on treated diet (Cryptogran) ranged from 16.67% to 96.67% for the

lowest and highest concentrations respectively (Table 4.3).

87

Table 4.3 Mortality of 1st instar FCM larvae from the old colony in a dose-response bioassay with Cryptogran.

Treatment

(OBs/ml)

Replicate 1 Replicate 2 Replicate 3 Replicate 4

Larval m

ortality (%)

Corrected

mortality (%

)

Empirical

probit

Larval m

ortality (%)

Corrected

mortality (%

)

Empirical

probit

Larval m

ortality (%)

Corrected

mortality (%

)

Empirical

probit

Larval m

ortality (%)

Corrected

mortality (%

)

Empirical

probit

d (H2O) Control

5.00 - - 17.00 - - 13.00 - - 7.00 - -

1.60 x102 26.67 22.81 4.255 30.00 15.66 3.991 16.67 4.21 3.273 30.00 24.73 4.317 8.0 x 102 36.67 33.33 4.569 40.00 27.71 4.408 33.33 23.37 4.273 46.67 42.65 4.815 4.0 x 103 53.33 50.88 5.022 46.67 35.74 4.635 60.00 54.02 5.101 63.33 60.57 5.268 2.0 x 104 76.67 75.44 5.688 56.67 47.79 4.945 73.33 69.35 5.506 70.00 67.74 5.460 1.0 x 105 96.67 96.49 6.811 86.67 83.94 5.992 93.33 92.34 6.428 93.33 92.83 6.463

The regression (probit) lines for the four replicates (Fig. 4.9) had the equations y = 2.1949

+ 0.8358x (SE of slope = 0.1386), y = 2.3220 + 0.6744x (SE of slope = 0.1606), y =

1.3590 + 0.9987x (SE of slope = 0.1803) and y = 2.8155 + 0.6733x (SE of slope =

0.1304), respectively.

The residual variances of the four lines were determined to be homogenous. The slopes

of the four lines were parallel and comparable. The elevations of the lines did not differ

significantly from one another. G for fiducial limits was calculated to be 0.1057, 0.2180,

0.1253 and 0.1441 for lines 1, 2, 3 and 4 respectively. The mean LC50 and LC90 values for

the four lines were determined to be 4.45 x 103 and 2.62 x 105 OBs/ml respectively (see

Appendix 4 & 5).

88

22.5

33.5

44.5

55.5

66.5

77.5

8

0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5

Log dose

Empi

rical

pro

bits

Rep 1

Rep 2

Rep 3

Rep 4

Figure 4.9 Dose-response probit lines (four replicates) from bioassays conducted with Cryptogran (CrleGV-SA) against 1st instar larvae from the old colony.

4.3.6 Bioassays with Cryptex against 1st instar FCM larvae from the Addo colony Control mortality ranged from 3.00% to 13.00% for all three replicates. However, mortality

of larvae feeding on treated diet (Cryptex) ranged from 10.00% to 93.33% for the lowest

and highest concentrations respectively (Table 4.4). Table 4.4 Mortality of 1st instar FCM larvae from the Addo colony in a dose-response bioassay with Cryptex.

Treatment (OBs/ml)

Replicate 1 Replicate 2 Replicate 3 Larval

mortality (%)

Corrected

mortality (%

)

Empirical

probit

Larval mortality (%)

Corrected

mortality (%

)

Empirical

probit

Larval mortalit

y (%)

Corrected

mortality (%

)

Empirical

probit

d (H2O) Control

10.00 - - 13.00 - - 5.00 - -

1.60 x102 16.67 7.41 3.554 30.00 19.54 4.142 10.00 5.26 3.380 8.0 x 102 36.67 29.63 4.465 33.33 23.37 4.273 20.00 15.79 3.997 4.0 x 103 73.33 70.37 5.535 43.33 34.87 4.611 33.33 29.82 4.470 2.0 x 104 76.67 74.07 5.645 56.67 50.19 5.005 50.00 47.37 4.934 1.0 x 105 90.00 88.89 6.221 93.33 92.34 6.428 66.67 64.91 5.383

The regression (probit) lines for the three lines (replicates) (Fig. 4.10) had the equations y

= 1.9592 + 0.8836x (SE of slope = 0.1543), y = 1.9754 + 0.7825x (SE of slope = 0.1600)

and y = 1.9782 + 0.6853x (SE of slope = 0.1473), respectively.

89

The residual variances of the three lines were determined to be homogenous and the

slopes of the lines parallel and comparable. G for fiducial limits was calculated to be

0.1171, 0.1605 and 0.1776 for lines 1, 2, and 3 respectively. The mean LC50 and LC90

values for lines 1, 2 and 3 was determined to be 1.19 x 104 and 7.67 x 105 OBs/ml

respectively (see Appendix 4 & 5).

Figure 4.10 Dose-response probit lines (three replicates) from bioassays conducted with Cryptex (CrleGV-SA) against 1st instar larvae from the Addo colony.

4.3.7 Bioassays with Cryptogran against 1st instar FCM larvae from the Addo colony Control mortality ranged from 3.00% to 20.00% for all four replicates. However, mortality

of larvae feeding on treated diet (Cryptogran) ranged from 20.00% to 96.67% for the

lowest and highest concentrations respectively (Table 4.5).

The regression (probit) lines for the four replicates (Fig. 4.11) had the equations y =

2.6256 + 0.6167x (SE of slope = 0.1306), y = 2.4896 + 0.7194x (SE of slope = 0.1520), y

= 3.1655 + 0.7934x (SE of slope = 0.1820) and y = 2.8935 + 0.5029x (SE of slope =

0.1241), respectively. The residual variances of the four lines were determined to be

homogenous and the slopes of the lines parallel and comparable. G for fiducial limits was

calculated to be 0.1724, 0.1714, 0.2022 and 0.2338 for lines 1, 2, 3, and 4 respectively.

0 1 2 3 4 5 6 7 8 9

0 1 2 3 4 5 6 7 8 Log dose

Empi

rical

pro

bits

Rep 1

Rep 2

Rep 3

90

Table 4.5 Mortality of 1st instar FCM larvae from the Addo colony in a dose-response bioassay with Cryptogran.

Treatme

nt (OBs/ml)

Replicate 1 Replicate 2 Replicate 3 Replicate 4

Larval m

ortality (%)

Corrected

mortality (%

)

Empirical

probit

Larval m

ortality (%)

Corrected

mortality (%

)

Empirical

probit

Larval m

ortality (%)

Corrected

mortality (%

)

Empirical

probit

Larval m

ortality (%)

Corrected

mortality (%

)

Empirical

probit

d (H2O) Control

7.00 - - 17.00 - - 20.00 - - 5.00 - -

1.60 x102 16.67 10.39 3.740 33.33 19.68 4.147 53.33 41.67 4.790 20.00 15.79 3.997 8.0 x 102 43.33 39.07 4.723 46.67 35.74 4.635 76.67 70.83 5.548 30.00 26.32 4.366 4.0 x 103 46.67 42.65 4.815 56.67 47.89 4.945 90.00 87.50 6.150 40.00 36.84 4.664 2.0 x 104 53.33 49.82 4.996 76.67 71.89 5.580 96.67 95.83 6.731 56.67 54.39 5.110 1.0 x 105 83.33 82.08 5.918 90.00 87.95 6.173 96.67 95.83 6.731 66.67 64.91 5.383

Figure 4.11 Dose-response probit lines (four replicates) from bioassays conducted with Cryptogran (CrleGV-SA) against 1st instar larvae from the Addo colony. The mean LC50 and LC90 values for lines 1, 2, 3 and 4 was determined to be 6.45 x 103

and 1.63 x 106 OBs/ml respectively. Several bioassays had to be conducted before an

adequate number of acceptable results could be obtained (see Appendix 4 & 5).

2

3

4

5

6

7

8

1 2 3 4 5 6 7

Log dose

Empi

rical

pro

bits

Rep 1 Rep 2 Rep 3 Rep 4

91

4.3.8 Comparison of assays between the Addo and the old FCM laboratory colonies with Cryptex and Cryptogran Data from each of the replicates from the bioassays conducted with each of the old and

Addo colonies were pooled together, in order to compare their susceptibility to both

Cryptogran and Cryptex (Table 4.6, Appendix 6). There were four treatments-subject

combinations in total. Old - Cryptex: Old colony treated with Cryptex Old - Cryptogran: Old colony treated with Cryptogran Addo - Cryptex: Addo colony treated with Cryptex Addo - Cryptogran: Addo colony treated with Cryptogran Table 4.6 Comparison of probit line slopes from dose-response bioassays with Cryptex and Cryptogran against 1st instar FCM larvae from two different laboratory colonies

Line (Slope) Addo - Cryptex

Addo - Cryptogran

Old - Cryptex

Old - Cryptogran

Addo - Cryptex 2.40 0.55 2.23 Addo - Cryptogran 0.42 0.23 0.93

Old - Cryptex 1.82 4.35* 4.05* Old - Cryptogran 0.45 1.08 0.25

* Asterisk indicates a significant difference (P<0.05): Multiple comparisons of elevations with Bonferroni method. Using Bartlett’s test for homogeneity of residual variances, the slopes of the probit lines

cof the ombined replicates for each bioassay were all determined to be homogenous and

therefore comparable. However there was a significant difference (F = 10.043, dƒ = 3, p <

0.001) between the slopes of the probit lines from the four bioassay combinations (Table

4.6, Appendix 6).

The slopes of the probit lines from the Old - Cryptex and the Addo - Cryptogran bioassays

were significantly different (P<0.05) from each other. There was also a significant

diference (P<0.05) between the slopes of the probit lines from the Old - Cryptex and the

Old – Cryptogran bioassays. There was no significant difference (P<0.05) between the

other treatments.

92

2

3

4

5

6

7

8

1 2 3 4 5 6 7Log dose

Empi

rical

pro

bits Old -

Cryptex

Old -Cryptogran

Addo -Cryptex

Addo -Cryptogran

Figure 4.12 Dose-response probit lines for the bioassays: Old – Cryptex, Old – Cryptogran, Addo – Cryptex and Addo – Cryptogran treatments.

1.00E+02

1.00E+03

1.00E+04

1.00E+05

1.00E+06

1.00E+07

Cryptogran Cryptex Cryptogran Cryptex

Old colony Addo colony

FCM population

Con

cent

ratio

n (O

Bs/

ml)

LC90

LC50

Figure 4.13 LC50 and LC90 of Cryptogran and Cryptex for two laboratory colonies of FCM (Addo and an old colony) The regression equations calculated from the slopes of the Old – Cryptex, Old –

Cryptogran, Addo – Cryptex and Addo – Cryptogran treatments (Fig. 4.14) had the

equations y = 2.2047 + 0.6741x (SE of slope = 0.0793), y = 2.2288 + 0.7790x (SE of

slope = 0.0735), y = 2.1381 + 0.7375x (SE of slope = 0.0845) and y = 2.9957 + 0.5769x

(SE of slope = 0.0671), respectively (see Appendix 6).

93

Table 4.7 Mean LC50 and LC90 values for 1st instar FCM larvae from Addo and the Old colony treated with Cryptogran and Cryptex.

FCM Colony CrleGV-SA Product

LC50 LC90

Old Colony

Cryptex 1.37 x 104 1.25 x 106

Cryptogran 4.45 x 103 2.62 x 105

Addo Colony

Cryptex 1.19 x 104 7.67 x 105

Cryptogran 6.45 x 103 1.63 x 106

The LC50 value for the old colony treated with Cryptex increased by 1.15-fold in

comparison to the Addo colony also treated with Cryptex. There was no significant

difference (P<0.05) between these treatments. For the Addo colony treated with

Cryptogran the LC50 values was 1.45 times higher in comparison to the old colony also

treated with Cryptogran. There was no significant difference (P<0.05) between these

treatments. For the old colony, the LC50 value of Cryptex was 3.8 times higher than the

LC50 value of Cryptogran. There was a significant difference (P<0.05) between these

treatments. The LC50 value of Cryptex against the Addo colony was 1.84 times higher

than that of Cryptogran against the same colony. However there was no significant

difference (P<0.05) between these treatments (see Appendix 6).

4.4 DISCUSSION Although it was possible to establish all instars of field collected FCM larvae on artificial

diet, obtaining high numbers of fifth instar larvae would be highly advantageous, due to

their higher survival rate than the other instars. An incubation room temperature of about

27o ± 1oC was critical for the establishment and maintenance of the new colonies.

Temperature is noted to play a vital role in speeding the growth rate of FCM larvae

(Diaber, 1980; Van Der Geest, et al., 1991; Moore, 2002). On average the establishment

of stable laboratory colonies from field lines was achieved by the fourth generation of

FCM. Once the new laboratory colonies had become established, it was determined that

their life-cycles (1st to 6th generation) were approximately 14 days longer than that of the

old colony (see Chapter Two, Table 2.1). But after the 7th generation, the duration of the

life cycle had reverted back to that of the old colony. It was also found that only by the F5

94

generation was there sufficient oviposition to obtain numbers of 1st instars for conducted

bioassays.

In a laboratory study using surface-dose response bioassays with 1st instar FCM larvae of

the old colony, Goble (2007) showed that Cryptogran was significantly more pathogenic

than Cryptex. Goble (2007) determined the LC50 and LC90 for Cryptogran to be 4.054 x

103 OBs/ml and 7.372 x 104 OBs/ml whilst that for Cryptex was 8.460 x 103 OBs/ml and

1.950 x 105 OBs/ml respectively. However, this significant difference in pathogenicity was

established with the LC90 values (Goble, 2007).

In this study, the LC50 value for Cryptex against 1st instar larvae of the old colony was

5.38 times higher than that determined by Goble (2007). Also the LC50 value of the Addo

colony treated with Cryptogran showed a marginal decrease (1.59-fold) in pathogenecity

in comparison to that reported by Goble (2007) with the old colony. It is quiet common to

observe variation between assays conducted by different researchers in different

laboratories (Jones, 2000). However, Hunter-Fujita et al. (1998) implores that when

assays are conducted frequently, variation between and within assays becomes less.

According to Dulmage (1973) up to 3-fold differences can be expected for virus assays

conducted under identical conditions.

Similarly, in these studies, the LC50 values of Cryptogran were determined to be

significantly lower than those of Cryptex, indicating that Cryptogran was more pathogenic

than Cryptex against both FCM colonies (the Addo and Old FCM colony). This

observation further confirms that genotypically distinct virus isolates (Cryptogran and

Cryptex) show high levels of variation in pathogenecity (Cory, 1997; Cory et al., 2005).

The slopes of the probit lines from some of the bioassays against the Addo colony

differed significantly for Cryptex (F6, 27 = 3.761) (Fig. 4.10) and Cryptogran (F5, 23 =

19.517) (Fig. 4.11, Appendix 5). This suggests that there was a high level of variation in

the response of the Addo colony to the two virus isolates. For instance, Cryptogran was

significantly more pathogenic than Cryptex, based on their LC50 values. However, based

on their LC90 values, Cryptex appeared to be slightly (but not significantly) more

95

pathogenic than Cryptogran (Table. 4.7). The LC90 value, for Cryptogran was 2.13 times

higher than that for Cryptex, against the Addo colony. Results with the old colony showed

the inverse (significant difference in pathogenicity was established based on both the

LC50 and LC90 values). However, LC50 values (not LC90 values) have been accepted as

the most accurate means of determining the potency of a virus (Hunter-Fujita et al., 1998;

Jones, 2000).

Timm (2005a) observed significantly high levels of genetic heterogeneity within FCM

populations found in the Eastern Cape. Therefore these differences in pathogenecity in

the Addo colony could explain why some members of the population were more

susceptible to Cryptogran at lower concentrations (LC50) than Cryptex; whilst other

members appeared to be more susceptible to Cryptex than Cryptogran at higher

concentrations (LC90) of the virus (Fig. 4.15 & Table 4.7).

In another study Asser-Kaiser (2007) found that codling moth (CM) populations that

developed resistance to a Cydia pomonella granulovirus, CpGV-M (Mexican isolate)

products, were actually a heterogeneous population (a mixture of both susceptible and

resistant individuals). It was discovered that CpGV-M could still induce about 30-40%

mortality, representing the susceptible individuals within the resistant populations (Asser-

Kaiser et al., 2007).

However, some authors contend that, the determination of the potency of a baculovirus

using quantal response (mortality) alone does not fully reflect the efficacy of a virus (Sait

et al., 1994; Goulson & Cory, 1995). Other factors such as latent infection and sub-lethal

effects have been noted to be of significant importance in this regard (Cory et al., 1997).

For instance, sub-lethal effects such as altered host development and growth are all

essential parameters for determining the potency of a baculovirus. However, there is little

knowledge on the mechanisms of sub-lethal infections (Cory et al., 1997).

It might even be possible that the application of Cryptex and Cryptogran against 1st instar

FCM larvae from both the Addo and old colonies could trigger other viruses (possibly new

96

CrleGV-SA isolates yet to be discovered) out of their latent forms, thus influencing overall

mortality results.

To date, five FCM colonies have been established in the laboratory. It is not known

whether these colonies carry any CrleGV-SA isolates in their latent forms, which could be

present in their field populations. Although, a number of diseased FCM larvae were

recovered from the field material used in this study, the causative agent (pathogens) of

this infection is not yet known, and this should be investigated. Previous studies by Moore

(2002) speculated on the possible existence of other South African isolates of CrleGV-SA

that needed to be confirmed by future work.

It is becoming quite commonly believed that insects could be co-infected with different

viruses with the ‘parent’ viruses only manifesting themselves after being triggered by

other newly introduced viruses or through stress related factors encountered by the host

(Longworth & Cunningham, 1968). For instance, in one study Cory et al. (2005) found

that, an NPV isolated from a single pine beauty moth, Panolis flammena larva had twenty

four (24) genetically different genotypes and that all these genotypes differed in both

virulence and pathogenicity.

Longworth and Cunningham (1968) state that in order to ensure that larval mortality is as

a result of an artificially induced infection and not a resident latent infection; one should

ensure that the stock insect cultures are as disease free as possible.

4.5 CONCLUSION Four geographically distinct FCM colonies from Addo, Citrusdal, Marble Hall and Nelspruit

have been successfully established. In surface dose-response bioassays Cryptogran has

proven to be significantly more pathogenic than Cryptex on two FCM populations (Addo

colony and an old colony). These findings may indicate that under laboratory conditions

there is a high level of variation in susceptibility within, as well as between, certain

geographically distinct FCM populations in South Africa. Even though it was not possible

(due to time constraints) to carry out comparative assays using Cryptex and Cryptogran

against the other colonies, this should be investigated in the near future.

97

CHAPTER FIVE

SUMMARY, RECOMMENDATIONS AND FUTURE RESEARCH

5.1 SUMMARY The main aim of this project (see Chapter One) was to investigate the comparative

susceptibility of geographically distinct false codling moth (FCM) populations to

Cryptophlebia leucotreta granulovirus (CrleGV-SA) products, Cryptogran (Moore, 2002;

Moore & Kirkman, 2004; Moore et al., 2004a; Moore et al., 2004b) and Cryptex (Kessler &

Zingg, 2008). In order to achieve this, three objectives were proposed. The objects were

to:

1. Establish a benchmark for pathogenicity, using laboratory reared FCM larvae against

CrleGV-SA. This will serve as a protocol for conducting dose-response bioassays with

CrleGV-SA against field collected FCM larval instars. Consequently, any differences or

similarities in response between the field and laboratory reared FCM larval instars against

CrleGV-SA would be established to better understand their susceptibility.

2. Establish new and separate laboratory colonies of FCM using field collected FCM

larvae from a range of different regions throughout South Africa

3. Conduct comparative surface dose-response bioassays with neonate (1st instar) FCM

larvae, from the established FCM laboratory colonies, using Cryptogran and Cryptex. This

will enable the detection of even small differences in host susceptibility and virus

pathogenicity.

The first objective was successfully achieved. In continuance of previous work carried out

by Moore (2002), a complete dose-response relationship was established for all five FCM

larval instars. This will serve as a standard reference point for pathogenecity.

Comparative assays conducted with Cryptogran (at 1.661 x 108 OBs/ml) against an old

laboratory colony and field collected larvae (2nd, 3rd, 4th and 5th instars) from four

geographic areas, namely Addo, Kirkwood, Clanwilliam and Citrusdal, showed some

variation in larval susceptibility to the virus. This difference was restricted to the 5th

98

instars. The 5th instars from the Addo population showed a significantly lower

susceptibility to the virus than the other populations. However, a follow up field trial

confirmed the efficacy of Cryptogran. All applications of Cryptogran and Cryptex are

always targeted against the 1st instars as they are the only live stage that can be reached

by the virus. Therefore, subsequent assays using Cryptogran and Cryptex against newly

established field lines were carried out only against 1st instar larvae (see Chapter Four).

The second objective was also achieved. Four new and separate laboratory colonies of

FCM, from a range of geographic regions were established. These colonies (over six

generations) were established with field material from Addo, Citrusdal, Marble Hall and

Nelspruit.

The third objective was not completed due to time constraints. However, assays

conducted with Cryptogran and Cryptex against 1st instar larvae from the old colony and

the Addo colony showed marked differences in virus pathogenecity. Cryptogran was

determined to be more pathogenic than Cryptex against both populations (the Addo and

old FCM colony). There were also differences in virus pathogenecity within the Addo

population. This is probably indicative of a high level of heterogeneity within these

populations.

Therefore, there was no indication of any possible emergence of some ‘resistant’ FCM

populations or populations simply with a lower susceptibility, as initially proposed (see

Chapter One). Most importantly, the findings in this study do not represent a detailed

comprehensive or conclusive study on variation in susceptibility to baculoviruses between

FCM populations, but rather an indication of its likelihood, which needs to be confirmed by

a more thorough and detailed study.

5.2 RECOMMENDATIONS Although in this study Cryptogran showed a high level of pathogenecity, requiring lower

concentrations of the virus to achieve equal infectivity in comparison to Cryptex, the

potency of Cryptex cannot be ruled out. According to Ridout et al. (1993) the potency of

baculoviruses might not be dependent on the application of high concentrations of virus

99

material, for that matter, but rather on the ability of each virus particle to initiate infection

independently of each other.

Again, the continuous application of baculoviruses (especially single isolates) could lead

to the promotion of particular variants (via natural selection) (Cory et al., 1997). Therefore,

it might be a good idea to apply both Cryptogran and Cryptex simultaneously in the field

to avoid the development of resistance by some FCM populations in South Africa to the

virus, especially considering the genetically heterogeneous nature of these populations

(Timm, 2005a).

5.3 FUTURE RESEARCH In view of the above, the following are proposed. 1. More detailed studies on geographic variation in the susceptibility of FCM populations

to CrleGV-SA, using droplet feeding as well as detached fruit assay techniques in dose-

response and time response studies should be investigated. Field trials should also be

conducted in this regard.

2 The effect of the combined usage (mixture) of Cryptogran and Cryptex in spray

formulations, both in laboratory and field studies.

3. Moore (2002) obtained indications in his studies of the probable existence of other

South African isolates of CrleGV-SA and stated that this needed to be confirmed by future

work. It is therefore imperative that the existence of these suggested new CrleGV-SA

isolates be investigated. The already established new laboratory colonies from the

different field populations would serve as a strategic stock for the initial investigation of

such new isolates. Consequently, it might even be possible to formulate multiple isolates

of the CrleGV-SA thereby minimising the possibility of some FCM populations developing

resistance to a particular isolate.

4. Sub lethal effects of these baculoviruses on their hosts should be investigated. These

include reduced fecundity, altered host development, reduced egg viability and changes

100

in sex ratio. The environmental factors, such as temperature, on baculovirus host-

interaction should also be investigated.

5. Larval weight has also been noted to influence host susceptibility to baculovirus

infection (Allen & Ignoffo, 1969; Klein & Podoler, 1978). Therefore, future studies should

take this into consideration especially when working with larger larvae or higher instars.

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Appendix 1 PROBAN (VAN ARK, 1995) OUPUT OF PROBIT ANALYSIS OF SURFACE DOSE-RESPONSE BIOASSAY DATA (LC) ON AN AGAR DIET, WITH CRYPTOGRAN AGAINST LABORATORY REARED 2ND, 3RD AND 4TH INSTAR FCM LARVAE

****************************************************************************** rep 1. Dose-response bioassays with 2nd instar FCM larvae (See section: 2.3.2) ****************************************************************************** GENERAL INPUT INFORMATION ========================= DOSES TRANSFORMED TO LOG10 NATURAL MORTALITY = 4% NON-RESPONSE = 0% DOSE TRANSFORMED NUMBER NUMBER %RESPONSE CORRECTED EMPIRICAL DOSE(X) EXPOSED RESPON. %RESPONSE PROBITS *************** 6.1271 25 21 84.00 83.33 5.967 268000.00000000 5.4281 25 17 68.00 66.67 5.431 53600.00000000 4.7292 25 14 56.00 54.17 5.105 10700.00000000 4.0294 25 11 44.00 41.67 4.790 2140.00000000 3.3304 25 7 28.00 25.00 4.326 TEST FOR FIT OF LINE ==================== CHI-SQUARED FOR DEVIATIONS = .194 D. F. = 3 APPROXIMATE PROBABILITY = .9730 TEST LEVEL = 0,05 TABLED CHI-SQUARED VALUE = 7.815 DEVIATIONS FROM LINE ARE HOMOGENEOUS FITTED PROBIT LINE ================== REGRESSION EQUATION IS Y = a + bX WHERE Y IS EMPIRICAL PROBITS AND X IS DOSE INTERCEPT (a) = 2.4926 REGRESSION COEFFICIENT (b) = .5559 STANDARD ERROR OF b = .1309 MEAN FOR EMPIRICAL PROBITS (Y) = 5.1132 MEAN FOR DOSE (X) = 4.7139

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DOSE (X) EXPECTED EMP.PROBIT (Y) 6.12710 5.899 5.42813 5.510 4.72916 5.122 4.02938 4.733 3.33041 4.344 EXPECTED QUANTITIES FOR LINE ============================ G FOR FIDUCIAL LIMITS = .2131 EXPECTED DOSE STANDARD FIDUCIAL LIMITS % RESPONSE ERROR UPPER LOWER 50.0 4.510 .2277 4.961 3.949 90.0 6.816 .5427 8.712 6.057 95.0 7.469 .6860 9.904 6.526 99.0 8.695 .9636 12.159 7.386 99.9 10.069 1.2806 14.698 8.339 QUANTITIES TRANSFORMED BACK TO ORIGINAL ======================================= EXPECTED DOSE STANDARD FIDUCIAL LIMITS % RESPONSE ERROR UPPER LOWER 50.0 .3238167E+05 .1695776E+05 .9146373E+05 .8893884E+04 90.0 .6540600E+07 .8163971E+07 .5150532E+09 .1141324E+07 95.0 .2945197E+08 .4646691E+08 .8021963E+10 .3356050E+07 99.0 .4952268E+09 .1097516E+10 .1441442E+13 .2434323E+08 99.9 .1171910E+11 .3451694E+11 .4987913E+15 .2185136E+09

116

****************************************************************************** rep 2. Dose-response bioassays with 2nd instar FCM larvae (See section: 2.3.2) ****************************************************************************** GENERAL INPUT INFORMATION ========================= DOSES TRANSFORMED TO LOG10 NATURAL MORTALITY = 0% NON-RESPONSE = 0% DOSE TRANSFORMED NUMBER NUMBER %RESPONSE CORRECTED EMPIRICAL DOSE(X) EXPOSED RESPON. %RESPONSE PROBITS *************** 6.1271 25 21 84.00 84.00 5.995 268000.00000000 5.4281 25 17 68.00 68.00 5.468 53600.00000000 4.7292 25 16 64.00 64.00 5.359 10700.00000000 4.0294 25 12 48.00 48.00 4.950 2140.00000000 3.3304 25 4 16.00 16.00 4.005 TEST FOR FIT OF LINE ==================== CHI-SQUARED FOR DEVIATIONS = 2.528 D. F. = 3 APPROXIMATE PROBABILITY = .4730 TEST LEVEL = 0,05 TABLED CHI-SQUARED VALUE = 7.815 DEVIATIONS FROM LINE ARE HOMOGENEOUS FITTED PROBIT LINE ================== REGRESSION EQUATION IS Y = a + bX WHERE Y IS EMPIRICAL PROBITS AND X IS DOSE INTERCEPT (a) = 2.2071 REGRESSION COEFFICIENT (b) = .6278 STANDARD ERROR OF b = .1284 MEAN FOR EMPIRICAL PROBITS (Y) = 5.1295 MEAN FOR DOSE (X) = 4.6553 DOSE (X) EXPECTED EMP.PROBIT (Y) 6.12710 6.053 5.42813 5.615 4.72916 5.176 4.02938 4.737 3.33041 4.298

117

EXPECTED QUANTITIES FOR LINE ============================ G FOR FIDUCIAL LIMITS = .1608 EXPECTED DOSE STANDARD FIDUCIAL LIMITS % RESPONSE ERROR UPPER LOWER 50.0 4.449 .1964 4.831 3.988 90.0 6.491 .4216 7.810 5.874 95.0 7.069 .5298 8.756 6.308 99.0 8.155 .7412 10.547 7.104 99.9 9.372 .9838 12.566 7.985 QUANTITIES TRANSFORMED BACK TO ORIGINAL ======================================= EXPECTED DOSE STANDARD FIDUCIAL LIMITS % RESPONSE ERROR UPPER LOWER 50.0 .2811897E+05 .1269884E+05 .6783127E+05 .9716358E+04 90.0 .3094195E+07 .3000426E+07 .6462016E+08 .7482189E+06 95.0 .1172931E+08 .1429254E+08 .5702701E+09 .2030232E+07 99.0 .1427980E+09 .2434311E+09 .3523577E+11 .1269141E+08 99.9 .2352830E+10 .5323728E+10 .3680643E+13 .9652465E+08

118

****************************************************************************** rep 3. Dose-response bioassays with 2nd instar FCM larvae (See section: 2.3.2) ****************************************************************************** GENERAL INPUT INFORMATION ========================= DOSES TRANSFORMED TO LOG10 NATURAL MORTALITY = 4% NON-RESPONSE = 0% DOSE TRANSFORMED NUMBER NUMBER %RESPONSE CORRECTED EMPIRICAL DOSE(X) EXPOSED RESPON. %RESPONSE PROBITS *************** 6.1271 25 20 80.00 79.17 5.812 268000.00000000 5.4281 25 19 76.00 75.00 5.674 53600.00000000 4.7292 25 12 48.00 45.83 4.895 10700.00000000 4.0294 25 6 24.00 20.83 4.188 2140.00000000 3.3304 25 4 16.00 12.50 3.850 TEST FOR FIT OF LINE ==================== CHI-SQUARED FOR DEVIATIONS = 1.339 D. F. = 3 APPROXIMATE PROBABILITY = .7240 TEST LEVEL = 0,05 TABLED CHI-SQUARED VALUE = 7.815 DEVIATIONS FROM LINE ARE HOMOGENEOUS FITTED PROBIT LINE ================== REGRESSION EQUATION IS Y = a + bX WHERE Y IS EMPIRICAL PROBITS AND X IS DOSE INTERCEPT (a) = 1.1762 REGRESSION COEFFICIENT (b) = .7844 STANDARD ERROR OF b = .1482 MEAN FOR EMPIRICAL PROBITS (Y) = 4.9810 MEAN FOR DOSE (X) = 4.8507 DOSE (X) EXPECTED EMP.PROBIT (Y) 6.12710 5.982 5.42813 5.434 4.72916 4.886 4.02938 4.337 3.33041 3.789

119

EXPECTED QUANTITIES FOR LINE ============================ G FOR FIDUCIAL LIMITS = .1372 EXPECTED DOSE STANDARD FIDUCIAL LIMITS % RESPONSE ERROR UPPER LOWER 50.0 4.875 .1679 5.233 4.525 90.0 6.509 .3554 7.567 5.977 95.0 6.972 .4346 8.286 6.332 99.0 7.841 .5894 9.648 6.985 99.9 8.815 .7676 11.183 7.707 QUANTITIES TRANSFORMED BACK TO ORIGINAL ======================================= EXPECTED DOSE STANDARD FIDUCIAL LIMITS % RESPONSE ERROR UPPER LOWER 50.0 .7497616E+05 .2894753E+05 .1710020E+06 .3346134E+05 90.0 .3227040E+07 .2638137E+07 .3693448E+08 .9494056E+06 95.0 .9374902E+07 .9370226E+07 .1933200E+09 .2148912E+07 99.0 .6928947E+08 .9393237E+08 .4441636E+10 .9651606E+07 99.9 .6524476E+09 .1151922E+10 .1523499E+12 .5090410E+08

120

*************************** * COMPARISON OF LINES * *************************** ABRIDGED ANALYSIS OF VARIANCE ============================= DEVIATIONS FROM REGRESSION INTERCEPT REGR.COEF. D.F. SUM OF RESIDUAL (a) (b) SQUARES VARIANCES LINE: rep 1 2.493 .556 3 .193738E+00 .065 LINE: rep 2 2.207 .628 3 .252767E+01 .843 LINE: rep 3 1.176 .784 3 .133857E+01 .446 ---------------------------------------------------------------------- 9 .405997E+01 .451 ---------------------------------------------------------------------- COMBINED .646 11 .542505E+01 .493 ---------------------------------------------------------------------- DIFFERENCE BETWEEN SLOPES 2 .136508E+01 .683 ---------------------------------------------------------------------- TOTAL 2.074 .635 13 .799337E+01 .615 ---------------------------------------------------------------------- DIFFERENCE BETWEEN ADJ. MEANS 2 .256832E+01 1.284 ---------------------------------------------------------------------- BARTLETTS TEST FOR HOMOGENEITY OF RESIDUAL VARIANCES ==================================================== CHI-SQUARED VALUE = 2.112 D. F. = 2 APPROXIMATE PROBABILITY = .3490 TEST LEVEL = .0100 TABLED CHI-SQUARED VALUE = 9.210 RESIDUAL VARIANCES ARE HOMOGENEOUS - SLOPES OF LINES MAY BE COMPARED COMPARISON OF SLOPES (PARALLELISM) ================================== SOURCE D.F. SUM OF SQ. MEAN SQ. F-VALUE --------------------------------------------------------- PARALLELISM 2 .136508E+01 .683 1.51 HETEROGENEITY 9 .405997E+01 .451 -------------------------------- TOTAL 11 .5425E+01 CHI-SQUARED TEST FOR PARALLELISM -------------------------------- CHI-SQUARED VALUE = 1.365 D. F. = 2 APPROXIMATE PROBABILITY = .5100 TEST LEVEL = 0,05 TABLED CHI-SQUARED VALUE = 5.991

121

LINES ARE PARALLEL AND ELEVATIONS MAY BE COMPARED REGRESSION COEFFICIENTS ----------------------- rep 1 .5559 rep 2 .6278 rep 3 .7844 COMPARISON OF ELEVATIONS (ADJUSTED MEANS) ========================================= F-VALUE FOR ELEVATIONS = 2.604 D.F. = 2 & 11 APPROXIMATE PROBABILITY = .1190 TEST LEVEL = 0,05 TABLED F-VALUE = 3.98 ELEVATIONS ARE NOT SIGNIFICANTLY DIFFERENT MEANS OF EMPIRICAL PROBITS -------------------------- rep 3 4.9810 rep 1 5.1132 rep 2 5.1295 INTERCEPTS OF LINES ------------------- rep 3 1.176 rep 2 2.207 rep 1 2.493

122

****************************************************************************** rep 1. Dose-response bioassays with 3nd instar FCM larvae (See section: 2.3.3) ****************************************************************************** GENERAL INPUT INFORMATION ========================= DOSES TRANSFORMED TO LOG10 NATURAL MORTALITY = 0% NON-RESPONSE = 0% DOSE TRANSFORMED NUMBER NUMBER %RESPONSE CORRECTED EMPIRICAL DOSE(X) EXPOSED RESPON. %RESPONSE PROBITS *************** 6.0000 25 17 68.00 68.00 5.468 200000.00000000 5.3010 25 12 48.00 48.00 4.950 40000.00000000 4.6021 25 7 28.00 28.00 4.417 8000.00000000 3.9031 25 5 20.00 20.00 4.158 1600.00000000 3.2041 25 2 8.00 8.00 3.595 TEST FOR FIT OF LINE ==================== CHI-SQUARED FOR DEVIATIONS = .283 D. F. = 3 APPROXIMATE PROBABILITY = .9580 TEST LEVEL = 0,05 TABLED CHI-SQUARED VALUE = 7.815 DEVIATIONS FROM LINE ARE HOMOGENEOUS FITTED PROBIT LINE ================== REGRESSION EQUATION IS Y = a + bX WHERE Y IS EMPIRICAL PROBITS AND X IS DOSE INTERCEPT (a) = 1.5229 REGRESSION COEFFICIENT (b) = .6501 STANDARD ERROR OF b = .1351 MEAN FOR EMPIRICAL PROBITS (Y) = 4.6488 MEAN FOR DOSE (X) = 4.8084 DOSE (X) EXPECTED EMP.PROBIT (Y) 6.00000 5.424 5.30103 4.969 4.60206 4.515 3.90309 4.060 3.20412 3.606 EXPECTED QUANTITIES FOR LINE

123

============================ G FOR FIDUCIAL LIMITS = .1659 EXPECTED DOSE STANDARD FIDUCIAL LIMITS % RESPONSE ERROR UPPER LOWER 50.0 5.349 .2217 5.944 4.968 90.0 7.320 .5559 9.113 6.526 95.0 7.879 .6662 10.044 6.935 99.0 8.927 .8771 11.799 7.693 99.9 10.102 1.1167 13.773 8.537 QUANTITIES TRANSFORMED BACK TO ORIGINAL ======================================= EXPECTED DOSE STANDARD FIDUCIAL LIMITS % RESPONSE ERROR UPPER LOWER 50.0 .2231163E+06 .1137682E+06 .8785830E+06 .9294406E+05 90.0 .2088962E+08 .2670881E+08 .1297398E+10 .3358656E+07 95.0 .7564328E+08 .1158983E+09 .1107525E+11 .8608594E+07 99.0 .8451286E+09 .1704848E+10 .6298875E+12 .4936227E+08 99.9 .1264676E+11 .3248169E+11 .5928430E+14 .3446652E+09

124

****************************************************************************** rep 2. Dose-response bioassays with 3nd instar FCM larvae (See section: 2.3.3) ****************************************************************************** GENERAL INPUT INFORMATION ========================= DOSES TRANSFORMED TO LOG10 NATURAL MORTALITY = 0% NON-RESPONSE = 0% DOSE TRANSFORMED NUMBER NUMBER %RESPONSE CORRECTED EMPIRICAL DOSE(X) EXPOSED RESPON. %RESPONSE PROBITS *************** 6.0000 25 18 72.00 72.00 5.583 200000.00000000 5.3010 25 14 56.00 56.00 5.151 40000.00000000 4.6021 25 9 36.00 36.00 4.641 8000.00000000 3.9031 25 3 12.00 12.00 3.825 1600.00000000 3.2041 25 1 4.00 4.00 3.249 TEST FOR FIT OF LINE ==================== CHI-SQUARED FOR DEVIATIONS = .599 D. F. = 3 APPROXIMATE PROBABILITY = .8950 TEST LEVEL = 0,05 TABLED CHI-SQUARED VALUE = 7.815 DEVIATIONS FROM LINE ARE HOMOGENEOUS FITTED PROBIT LINE ================== REGRESSION EQUATION IS Y = a + bX WHERE Y IS EMPIRICAL PROBITS AND X IS DOSE INTERCEPT (a) = .6502 REGRESSION COEFFICIENT (b) = .8387 STANDARD ERROR OF b = .1476 MEAN FOR EMPIRICAL PROBITS (Y) = 4.7205 MEAN FOR DOSE (X) = 4.8531 DOSE (X) EXPECTED EMP.PROBIT (Y) 6.00000 5.682 5.30103 5.096 4.60206 4.510 3.90309 3.924 3.20412 3.338

125

EXPECTED QUANTITIES FOR LINE ============================ G FOR FIDUCIAL LIMITS = .1189 EXPECTED DOSE STANDARD FIDUCIAL LIMITS % RESPONSE ERROR UPPER LOWER 50.0 5.186 .1652 5.579 4.884 90.0 6.714 .3620 7.762 6.169 95.0 7.148 .4322 8.411 6.503 99.0 7.960 .5680 9.637 7.121 99.9 8.871 .7235 11.018 7.808 QUANTITIES TRANSFORMED BACK TO ORIGINAL ======================================= EXPECTED DOSE STANDARD FIDUCIAL LIMITS % RESPONSE ERROR UPPER LOWER 50.0 .1535805E+06 .5833773E+05 .3793829E+06 .7647830E+05 90.0 .5180910E+07 .4314040E+07 .5783304E+08 .1475697E+07 95.0 .1404669E+08 .1396286E+08 .2578026E+09 .3185158E+07 99.0 .9120470E+08 .1191474E+09 .4337252E+10 .1322405E+08 99.9 .7427228E+09 .1235936E+10 .1042509E+12 .6425975E+08

126

****************************************************************************** rep 3. Dose-response bioassays with 3nd instar FCM larvae (See section: 2.3.3) ****************************************************************************** GENERAL INPUT INFORMATION ========================= DOSES TRANSFORMED TO LOG10 NATURAL MORTALITY = 0% NON-RESPONSE = 0% DOSE TRANSFORMED NUMBER NUMBER %RESPONSE CORRECTED EMPIRICAL DOSE(X) EXPOSED RESPON. %RESPONSE PROBITS *************** 6.0000 25 20 80.00 80.00 5.842 200000.00000000 5.3010 25 15 60.00 60.00 5.253 40000.00000000 4.6021 25 8 32.00 32.00 4.532 8000.00000000 3.9031 25 4 16.00 16.00 4.005 1600.00000000 3.2041 25 2 8.00 8.00 3.595 TEST FOR FIT OF LINE ==================== CHI-SQUARED FOR DEVIATIONS = .328 D. F. = 3 APPROXIMATE PROBABILITY = .9500 TEST LEVEL = 0,05 TABLED CHI-SQUARED VALUE = 7.815 DEVIATIONS FROM LINE ARE HOMOGENEOUS FITTED PROBIT LINE ================== REGRESSION EQUATION IS Y = a + bX WHERE Y IS EMPIRICAL PROBITS AND X IS DOSE INTERCEPT (a) = .7690 REGRESSION COEFFICIENT (b) = .8394 STANDARD ERROR OF b = .1444 MEAN FOR EMPIRICAL PROBITS (Y) = 4.7887 MEAN FOR DOSE (X) = 4.7890 DOSE (X) EXPECTED EMP.PROBIT (Y) 6.00000 5.805 5.30103 5.218 4.60206 4.632 3.90309 4.045 3.20412 3.458

127

EXPECTED QUANTITIES FOR LINE ============================ G FOR FIDUCIAL LIMITS = .1137 EXPECTED DOSE STANDARD FIDUCIAL LIMITS % RESPONSE ERROR UPPER LOWER 50.0 5.041 .1584 5.404 4.742 90.0 6.568 .3418 7.543 6.048 95.0 7.000 .4098 8.183 6.385 99.0 7.812 .5419 9.393 7.007 99.9 8.722 .6935 10.756 7.697 QUANTITIES TRANSFORMED BACK TO ORIGINAL ======================================= EXPECTED DOSE STANDARD FIDUCIAL LIMITS % RESPONSE ERROR UPPER LOWER 50.0 .1098334E+06 .4002169E+05 .2537556E+06 .5515759E+05 90.0 .3694956E+07 .2904645E+07 .3490696E+08 .1118068E+07 95.0 .1001010E+08 .9434321E+07 .1524145E+09 .2426734E+07 99.0 .6490024E+08 .8088837E+08 .2470865E+10 .1016311E+08 99.9 .5276470E+09 .8416440E+09 .5703453E+11 .4981400E+08

128

*************************** * COMPARISON OF LINES * *************************** ABRIDGED ANALYSIS OF VARIANCE ============================= DEVIATIONS FROM REGRESSION INTERCEPT REGR.COEF. D.F. SUM OF RESIDUAL (a) (b) SQUARES VARIANCES LINE: rep 1 1.523 .650 3 .283150E+00 .094 LINE: rep 2 .650 .839 3 .598633E+00 .200 LINE: rep 3 .769 .839 3 .328355E+00 .109 ---------------------------------------------------------------------- 9 .121014E+01 .134 ---------------------------------------------------------------------- COMBINED .769 11 .244523E+01 .222 ---------------------------------------------------------------------- DIFFERENCE BETWEEN SLOPES 2 .123509E+01 .618 ---------------------------------------------------------------------- TOTAL 1.018 .768 13 .325647E+01 .250 ---------------------------------------------------------------------- DIFFERENCE BETWEEN ADJ. MEANS 2 .811238E+00 .406 ---------------------------------------------------------------------- BARTLETTS TEST FOR HOMOGENEITY OF RESIDUAL VARIANCES ==================================================== CHI-SQUARED VALUE = .262 D. F. = 2 APPROXIMATE PROBABILITY = .8740 TEST LEVEL = .0100 TABLED CHI-SQUARED VALUE = 9.210 RESIDUAL VARIANCES ARE HOMOGENEOUS - SLOPES OF LINES MAY BE COMPARED COMPARISON OF SLOPES (PARALLELISM) ================================== SOURCE D.F. SUM OF SQ. MEAN SQ. F-VALUE --------------------------------------------------------- PARALLELISM 2 .123509E+01 .618 4.59 HETEROGENEITY 9 .121014E+01 .134 -------------------------------- TOTAL 11 .2445E+01 CHI-SQUARED TEST FOR PARALLELISM -------------------------------- CHI-SQUARED VALUE = 1.235 D. F. = 2 APPROXIMATE PROBABILITY = .5450 TEST LEVEL = 0,05 TABLED CHI-SQUARED VALUE = 5.991 LINES ARE PARALLEL AND ELEVATIONS MAY BE COMPARED

129

REGRESSION COEFFICIENTS ----------------------- rep 1 .6501 rep 2 .8387 rep 3 .8394 COMPARISON OF ELEVATIONS (ADJUSTED MEANS) ========================================= F-VALUE FOR ELEVATIONS = 1.825 D.F. = 2 & 11 APPROXIMATE PROBABILITY = .2070 TEST LEVEL = 0,05 TABLED F-VALUE = 3.98 ELEVATIONS ARE NOT SIGNIFICANTLY DIFFERENT MEANS OF EMPIRICAL PROBITS -------------------------- rep 1 4.6488 rep 2 4.7205 rep 3 4.7887 INTERCEPTS OF LINES ------------------- rep 2 .650 rep 3 .769 rep 1 1.523

130

****************************************************************************** rep 1. Dose-response bioassays with 4th instar FCM larvae (See section: 2.3.4) ****************************************************************************** GENERAL INPUT INFORMATION ========================= DOSES TRANSFORMED TO LOG10 NATURAL MORTALITY = 0% NON-RESPONSE = 0% DOSE TRANSFORMED NUMBER NUMBER %RESPONSE CORRECTED EMPIRICAL DOSE(X) EXPOSED RESPON. %RESPONSE PROBITS *************** 7.5237 25 19 76.00 76.00 5.706 *************** 6.8248 25 14 56.00 56.00 5.151 *************** 6.1271 25 10 40.00 40.00 4.747 267000.00000000 5.4265 25 7 28.00 28.00 4.417 53400.00000000 4.7275 25 3 12.00 12.00 3.825 TEST FOR FIT OF LINE ==================== CHI-SQUARED FOR DEVIATIONS = .256 D. F. = 3 APPROXIMATE PROBABILITY = .9630 TEST LEVEL = 0,05 TABLED CHI-SQUARED VALUE = 7.815 DEVIATIONS FROM LINE ARE HOMOGENEOUS FITTED PROBIT LINE ================== REGRESSION EQUATION IS Y = a + bX WHERE Y IS EMPIRICAL PROBITS AND X IS DOSE INTERCEPT (a) = .8734 REGRESSION COEFFICIENT (b) = .6362 STANDARD ERROR OF b = .1295 MEAN FOR EMPIRICAL PROBITS (Y) = 4.8302 MEAN FOR DOSE (X) = 6.2194 DOSE (X) EXPECTED EMP.PROBIT (Y) 7.52375 5.660 6.82478 5.215 6.12710 4.771 5.42651 4.326 4.72754 3.881 EXPECTED QUANTITIES FOR LINE ============================

131

G FOR FIDUCIAL LIMITS = .1591 EXPECTED DOSE STANDARD FIDUCIAL LIMITS % RESPONSE ERROR UPPER LOWER 50.0 6.486 .1976 6.962 6.111 90.0 8.501 .5017 10.088 7.777 95.0 9.072 .6108 11.024 8.199 99.0 10.143 .8208 12.790 8.980 99.9 11.343 1.0601 14.778 9.849 QUANTITIES TRANSFORMED BACK TO ORIGINAL ======================================= EXPECTED DOSE STANDARD FIDUCIAL LIMITS % RESPONSE ERROR UPPER LOWER 50.0 .3063851E+07 .1392289E+07 .9163973E+07 .1292564E+07 90.0 .3167441E+09 .3654734E+09 .1225782E+11 .5977365E+08 95.0 .1179641E+10 .1657287E+10 .1057770E+12 .1580392E+09 99.0 .1389271E+11 .2622772E+11 .6172005E+13 .9554572E+09 99.9 .2205455E+12 .5377280E+12 .5997014E+15 .7056958E+10

132

****************************************************************************** rep 2. Dose-response bioassays with 4th instar FCM larvae (See section: 2.3.4) ****************************************************************************** GENERAL INPUT INFORMATION ========================= DOSES TRANSFORMED TO LOG10 NATURAL MORTALITY = 0% NON-RESPONSE = 0% DOSE TRANSFORMED NUMBER NUMBER %RESPONSE CORRECTED EMPIRICAL DOSE(X) EXPOSED RESPON. %RESPONSE PROBITS *************** 7.5237 25 21 84.00 84.00 5.995 *************** 6.8248 25 16 64.00 64.00 5.359 *************** 6.1271 25 11 44.00 44.00 4.849 267000.00000000 5.4265 25 8 32.00 32.00 4.532 53400.00000000 4.7275 25 2 8.00 8.00 3.595 TEST FOR FIT OF LINE ==================== CHI-SQUARED FOR DEVIATIONS = .988 D. F. = 3 APPROXIMATE PROBABILITY = .8060 TEST LEVEL = 0,05 TABLED CHI-SQUARED VALUE = 7.815 DEVIATIONS FROM LINE ARE HOMOGENEOUS FITTED PROBIT LINE ================== REGRESSION EQUATION IS Y = a + bX WHERE Y IS EMPIRICAL PROBITS AND X IS DOSE INTERCEPT (a) = .1243 REGRESSION COEFFICIENT (b) = .7767 STANDARD ERROR OF b = .1364 MEAN FOR EMPIRICAL PROBITS (Y) = 4.9253 MEAN FOR DOSE (X) = 6.1815 DOSE (X) EXPECTED EMP.PROBIT (Y) 7.52375 5.968 6.82478 5.425 6.12710 4.883 5.42651 4.339 4.72754 3.796

133

EXPECTED QUANTITIES FOR LINE ============================ G FOR FIDUCIAL LIMITS = .1185 EXPECTED DOSE STANDARD FIDUCIAL LIMITS % RESPONSE ERROR UPPER LOWER 50.0 6.278 .1606 6.626 5.955 90.0 7.928 .3458 8.922 7.404 95.0 8.396 .4204 9.620 7.767 99.0 9.273 .5660 10.941 8.436 99.9 10.257 .7334 12.431 9.178 QUANTITIES TRANSFORMED BACK TO ORIGINAL ======================================= EXPECTED DOSE STANDARD FIDUCIAL LIMITS % RESPONSE ERROR UPPER LOWER 50.0 .1895544E+07 .7000000E+06 .4227758E+07 .9020502E+06 90.0 .8469744E+08 .6736895E+08 .8354543E+09 .2532309E+08 95.0 .2486797E+09 .2404714E+09 .4170954E+10 .5841948E+08 99.0 .1874924E+10 .2440926E+10 .8737807E+11 .2729295E+09 99.9 .1805306E+11 .3045287E+11 .2696417E+13 .1507800E+10

134

****************************************************************************** rep 3. Dose-response bioassays with 4th instar FCM larvae (See section: 2.3.4) ****************************************************************************** GENERAL INPUT INFORMATION ========================= DOSES TRANSFORMED TO LOG10 NATURAL MORTALITY = 0% NON-RESPONSE = 0% DOSE TRANSFORMED NUMBER NUMBER %RESPONSE CORRECTED EMPIRICAL DOSE(X) EXPOSED RESPON. %RESPONSE PROBITS *************** 7.5237 25 20 80.00 80.00 5.842 *************** 6.8248 25 17 68.00 68.00 5.468 *************** 6.1271 25 13 52.00 52.00 5.050 267000.00000000 5.4265 25 7 28.00 28.00 4.417 53400.00000000 4.7275 25 3 12.00 12.00 3.825 TEST FOR FIT OF LINE ==================== CHI-SQUARED FOR DEVIATIONS = .443 D. F. = 3 APPROXIMATE PROBABILITY = .9280 TEST LEVEL = 0,05 TABLED CHI-SQUARED VALUE = 7.815 DEVIATIONS FROM LINE ARE HOMOGENEOUS FITTED PROBIT LINE ================== REGRESSION EQUATION IS Y = a + bX WHERE Y IS EMPIRICAL PROBITS AND X IS DOSE INTERCEPT (a) = .4927 REGRESSION COEFFICIENT (b) = .7248 STANDARD ERROR OF b = .1331 MEAN FOR EMPIRICAL PROBITS (Y) = 4.9539 MEAN FOR DOSE (X) = 6.1548 DOSE (X) EXPECTED EMP.PROBIT (Y) 7.52375 5.946 6.82478 5.439 6.12710 4.934 5.42651 4.426 4.72754 3.919

135

EXPECTED QUANTITIES FOR LINE ============================ G FOR FIDUCIAL LIMITS = .1295 EXPECTED DOSE STANDARD FIDUCIAL LIMITS % RESPONSE ERROR UPPER LOWER 50.0 6.218 .1695 6.584 5.872 90.0 7.987 .3765 9.096 7.423 95.0 8.488 .4606 9.863 7.807 99.0 9.428 .6244 11.314 8.516 99.9 10.482 .8124 12.950 9.302 QUANTITIES TRANSFORMED BACK TO ORIGINAL ======================================= EXPECTED DOSE STANDARD FIDUCIAL LIMITS % RESPONSE ERROR UPPER LOWER 50.0 .1653655E+07 .6446131E+06 .3837840E+07 .7442844E+06 90.0 .9695620E+08 .8395333E+08 .1246616E+10 .2646220E+08 95.0 .3074637E+09 .3256981E+09 .7293757E+10 .6412478E+08 99.0 .2678383E+10 .3846281E+10 .2061391E+12 .3279309E+09 99.9 .3032295E+11 .5665619E+11 .8907438E+13 .2002959E+10

136

*************************** * COMPARISON OF LINES * *************************** ABRIDGED ANALYSIS OF VARIANCE ============================= DEVIATIONS FROM REGRESSION INTERCEPT REGR.COEF. D.F. SUM OF RESIDUAL (a) (b) SQUARES VARIANCES LINE: rep 1 .873 .636 3 .256350E+00 .085 LINE: rep 2 .124 .777 3 .988294E+00 .329 LINE: rep 3 .493 .725 3 .443467E+00 .148 ---------------------------------------------------------------------- 9 .168811E+01 .188 ---------------------------------------------------------------------- COMBINED .710 11 .226405E+01 .206 ---------------------------------------------------------------------- DIFFERENCE BETWEEN SLOPES 2 .575942E+00 .288 ---------------------------------------------------------------------- TOTAL .524 .708 13 .329784E+01 .254 ---------------------------------------------------------------------- DIFFERENCE BETWEEN ADJ. MEANS 2 .103379E+01 .517 ---------------------------------------------------------------------- BARTLETTS TEST FOR HOMOGENEITY OF RESIDUAL VARIANCES ==================================================== CHI-SQUARED VALUE = .732 D. F. = 2 APPROXIMATE PROBABILITY = .6990 TEST LEVEL = .0100 TABLED CHI-SQUARED VALUE = 9.210 RESIDUAL VARIANCES ARE HOMOGENEOUS - SLOPES OF LINES MAY BE COMPARED COMPARISON OF SLOPES (PARALLELISM) ================================== SOURCE D.F. SUM OF SQ. MEAN SQ. F-VALUE --------------------------------------------------------- PARALLELISM 2 .575942E+00 .288 1.54 HETEROGENEITY 9 .168811E+01 .188 -------------------------------- TOTAL 11 .2264E+01 CHI-SQUARED TEST FOR PARALLELISM -------------------------------- CHI-SQUARED VALUE = .576 D. F. = 2 APPROXIMATE PROBABILITY = .7540 TEST LEVEL = 0,05 TABLED CHI-SQUARED VALUE = 5.991

137

LINES ARE PARALLEL AND ELEVATIONS MAY BE COMPARED REGRESSION COEFFICIENTS ----------------------- rep 1 .6362 rep 3 .7248 rep 2 .7767 COMPARISON OF ELEVATIONS (ADJUSTED MEANS) ========================================= F-VALUE FOR ELEVATIONS = 2.511 D.F. = 2 & 11 APPROXIMATE PROBABILITY = .1260 TEST LEVEL = 0,05 TABLED F-VALUE = 3.98 ELEVATIONS ARE NOT SIGNIFICANTLY DIFFERENT MEANS OF EMPIRICAL PROBITS -------------------------- rep 1 4.8302 rep 2 4.9253 rep 3 4.9539 INTERCEPTS OF LINES ------------------- rep 2 .124 rep 3 .493 rep 1 .873

138

Appendix 2 SPSS 11.0 OUTPUT DATA ANALYSIS OF BIOASSAYS WITH LABORATORY REARED AND FIELD COLLECTED FCM LARVAE FROM THE EASTERN AND WESTERN CAPE PROVINCE ON A NON-AGAR DIET 2.1 EASTERN CAPE 2.1.1 Lone Tree Farm (Addo) 2.1.1.1 Comparative analysis of bioassays with Cryptogran (at 1.661 x 108 OBs/ml) against field collected 2nd, 3rd and 4th instar FCM larvae from Lone Tree (Addo, Eastern Cape)

Case Processing Summary

237 100.0% 0 .0% 237 100.0%Quantal response *FCM Larval Instars

N Percent N Percent N PercentValid Missing Total

Cases

Quantal response * FCM Larval Instars Crosstabulation

64 103 68 23565.4 102.1 67.4 235.0

97.0% 100.0% 100.0% 99.2%

2 0 0 2.6 .9 .6 2.0

3.0% .0% .0% .8%

66 103 68 23766.0 103.0 68.0 237.0

100.0% 100.0% 100.0% 100.0%

CountExpected Count% within FCMLarval InstarsCountExpected Count% within FCMLarval InstarsCountExpected Count% within FCMLarval Instars

Dead

Alive

Quantal response

Total

2nd FCMInstar

3rd FCMInstar

4th FCMInstar

FCM Larval Instars

Total

139

Chi-Square Tests

5.226a 2 .0735.158 2 .076

3.613 1 .057

237

Pearson Chi-SquareLikelihood RatioLinear-by-LinearAssociationN of Valid Cases

Value dfAsymp. Sig.

(2-sided)

3 cells (50.0%) have expected count less than 5. Theminimum expected count is .56.

a.

Directional Measures

.019 .013 1.423 .076c

.223 .032 1.423 .076c

.010 .007 1.423 .076c

SymmetricRESPONSE DependentINSTAR Dependent

Uncertainty CoefficientNominal by NominalValue

Asymp.Std. Errora Approx. Tb Approx. Sig.

Not assuming the null hypothesis.a.

Using the asymptotic standard error assuming the null hypothesis.b.

Likelihood ratio chi-square probability.c.

Symmetric Measures

.148 .073

.148 .073-.124 .043 -1.911 .057c

-.123 .043 -1.906 .058c

237

PhiCramer's V

Nominal byNominal

Pearson's RInterval by IntervalSpearman CorrelationOrdinal by Ordinal

N of Valid Cases

ValueAsymp.

Std. Errora Approx. Tb Approx. Sig.

Not assuming the null hypothesis.a.

Using the asymptotic standard error assuming the null hypothesis.b.

Based on normal approximation.c.

140

2.1.1.2 Comparative analysis of bioassays with Cryptogran (at 1.661 x 108 OBs/ml) against field collected 2nd, 3rd, 4th and 5th instar FCM larvae from Lone Tree (Addo, Eastern Cape)

Case Processing Summary

322 100.0% 0 .0% 322 100.0%Quantal response *FCM Larval Instars

N Percent N Percent N PercentValid Missing Total

Cases

Quantal response * FCM Larval Instars Crosstabulation

64 103 68 37 27255.8 87.0 57.4 71.8 272.0

97.0% 100.0% 100.0% 43.5% 84.5%

2 0 0 48 5010.2 16.0 10.6 13.2 50.0

3.0% .0% .0% 56.5% 15.5%

66 103 68 85 32266.0 103.0 68.0 85.0 322.0

100.0% 100.0% 100.0% 100.0% 100.0%

CountExpected Count% within FCMLarval InstarsCountExpected Count% within FCMLarval InstarsCountExpected Count% within FCMLarval Instars

Dead

Alive

Quantal response

Total

2nd FCMInstar

3rd FCMInstar

4th FCMInstar

5th FCMInstar

FCM Larval Instars

Total

Chi-Square Tests

147.921a 3 .000143.720 3 .000

90.062 1 .000

322

Pearson Chi-SquareLikelihood RatioLinear-by-LinearAssociationN of Valid Cases

Value dfAsymp. Sig.

(2-sided)

0 cells (.0%) have expected count less than 5. Theminimum expected count is 10.25.

a.

141

Directional Measures

.248 .030 7.506 .000c

.517 .046 7.506 .000c

.163 .022 7.506 .000c

SymmetricRESPONSE DependentINSTAR Dependent

Uncertainty CoefficientNominal by NominalValue

Asymp.Std. Errora Approx. Tb Approx. Sig.

Not assuming the null hypothesis.a.

Using the asymptotic standard error assuming the null hypothesis.b.

Likelihood ratio chi-square probability.c.

Symmetric Measures

.678 .000

.678 .000

.530 .041 11.171 .000c

.519 .042 10.872 .000c

322

PhiCramer's V

Nominal byNominal

Pearson's RInterval by IntervalSpearman CorrelationOrdinal by Ordinal

N of Valid Cases

ValueAsymp.

Std. Errora Approx. Tb Approx. Sig.

Not assuming the null hypothesis.a.

Using the asymptotic standard error assuming the null hypothesis.b.

Based on normal approximation.c.

2.1.2 Tregaron Farm (Kirkwood) 2.1.2.1 Comparative analysis of bioassays with Cryptogran (at 1.661 x 108 OBs/ml) against field collected 2nd, 3rd and 4th instar FCM larvae from Tregaron Tree (Kirkwood, Eastern Cape)

Case Processing Summary

232 100.0% 0 .0% 232 100.0%Quantal response *FCM Larval Instars

N Percent N Percent N PercentValid Missing Total

Cases

142

Quantal response * FCM Larval Instars Crosstabulation

41 107 81 22941.5 107.6 80.0 229.0

97.6% 98.2% 100.0% 98.7%

1 2 0 3.5 1.4 1.0 3.0

2.4% 1.8% .0% 1.3%

42 109 81 23242.0 109.0 81.0 232.0

100.0% 100.0% 100.0% 100.0%

CountExpected Count% within FCMLarval InstarsCountExpected Count% within FCMLarval InstarsCountExpected Count% within FCMLarval Instars

Dead

Alive

Quantal response

Total

2nd FCMInstar

3rd FCMInstar

4th FCMInstar

FCM Larval Instars

Total

Chi-Square Tests

1.701a 2 .4272.643 2 .267

1.516 1 .218

232

Pearson Chi-SquareLikelihood RatioLinear-by-LinearAssociationN of Valid Cases

Value dfAsymp. Sig.

(2-sided)

3 cells (50.0%) have expected count less than 5. Theminimum expected count is .54.

a.

Directional Measures

.010 .006 1.662 .267c

.082 .018 1.662 .267c

.006 .003 1.662 .267c

SymmetricRESPONSE DependentINSTAR Dependent

Uncertainty CoefficientNominal by NominalValue

Asymp.Std. Errora Approx. Tb Approx. Sig.

Not assuming the null hypothesis.a.

Using the asymptotic standard error assuming the null hypothesis.b.

Likelihood ratio chi-square probability.c.

143

Symmetric Measures

.086 .427

.086 .427-.081 .049 -1.233 .219c

-.083 .045 -1.262 .208c

232

PhiCramer's V

Nominal byNominal

Pearson's RInterval by IntervalSpearman CorrelationOrdinal by Ordinal

N of Valid Cases

ValueAsymp.

Std. Errora Approx. Tb Approx. Sig.

Not assuming the null hypothesis.a.

Using the asymptotic standard error assuming the null hypothesis.b.

Based on normal approximation.c.

2.1.2.2 Comparative analysis of bioassays with Cryptogran (at 1.661 x 108 OBs/ml) against field collected 2nd, 3rd, 4th and 5th instar FCM larvae from Tregaron Farm (Kirkwood, Eastern Cape)

Case Processing Summary

297 100.0% 0 .0% 297 100.0%Quantal response *FCM Larval Instars

N Percent N Percent N PercentValid Missing Total

Cases

Quantal response * FCM Larval Instars Crosstabulation

41 107 81 42 27138.3 99.5 73.9 59.3 271.0

97.6% 98.2% 100.0% 64.6% 91.2%

1 2 0 23 263.7 9.5 7.1 5.7 26.0

2.4% 1.8% .0% 35.4% 8.8%

42 109 81 65 29742.0 109.0 81.0 65.0 297.0

100.0% 100.0% 100.0% 100.0% 100.0%

CountExpected Count% within FCMLarval InstarsCountExpected Count% within FCMLarval InstarsCountExpected Count% within FCMLarval Instars

Dead

Alive

Quantal response

Total

2nd FCMInstar

3rd FCMInstar

4th FCMInstar

5th FCMInstar

FCM Larval Instars

Total

144

Chi-Square Tests

74.149a 3 .00062.427 3 .000

39.686 1 .000

297

Pearson Chi-SquareLikelihood RatioLinear-by-LinearAssociationN of Valid Cases

Value dfAsymp. Sig.

(2-sided)

1 cells (12.5%) have expected count less than 5. Theminimum expected count is 3.68.

a.

Directional Measures

.129 .029 4.224 .000c

.354 .062 4.224 .000c

.079 .019 4.224 .000c

SymmetricRESPONSE DependentINSTAR Dependent

Uncertainty CoefficientNominal by NominalValue

Asymp.Std. Errora Approx. Tb Approx. Sig.

Not assuming the null hypothesis.a.

Using the asymptotic standard error assuming the null hypothesis.b.

Likelihood ratio chi-square probability.c.

Symmetric Measures

.500 .000

.500 .000

.366 .054 6.758 .000c

.354 .054 6.492 .000c

297

PhiCramer's V

Nominal byNominal

Pearson's RInterval by IntervalSpearman CorrelationOrdinal by Ordinal

N of Valid Cases

ValueAsymp.

Std. Errora Approx. Tb Approx. Sig.

Not assuming the null hypothesis.a.

Using the asymptotic standard error assuming the null hypothesis.b.

Based on normal approximation.c.

145

2.1.3 Comparative analysis of bioassays with Cryptogran (at 1.661 x 108 OBs/ml) against field collected 5th instar FCM larvae from both Lone Tree and Tregaron Farm (Eastern Cape)

Case Processing Summary

150 100.0% 0 .0% 150 100.0%Quantal response *FCM Larval Instars

N Percent N Percent N PercentValid Missing Total

Cases

Quantal response * FCM Larval Instars Crosstabulation

37 42 7944.8 34.2 79.0

43.5% 64.6% 52.7%

48 23 7140.2 30.8 71.0

56.5% 35.4% 47.3%

85 65 15085.0 65.0 150.0

100.0% 100.0% 100.0%

CountExpected Count% within FCMLarval InstarsCountExpected Count% within FCMLarval InstarsCountExpected Count% within FCMLarval Instars

Dead

Alive

Quantal response

Total

5th InstarADDO

5th InstarKIRKWOOD

FCM Larval Instars

Total

Chi-Square Tests

6.569b 1 .0105.751 1 .0166.636 1 .010

.013 .008

6.526 1 .011

150

Pearson Chi-SquareContinuity Correctiona

Likelihood RatioFisher's Exact TestLinear-by-LinearAssociationN of Valid Cases

Value dfAsymp. Sig.

(2-sided)Exact Sig.(2-sided)

Exact Sig.(1-sided)

Computed only for a 2x2 tablea.

0 cells (.0%) have expected count less than 5. The minimum expected count is30.77.

b.

146

Directional Measures

.032 .025 1.305 .010c

.032 .025 1.305 .010c

.032 .025 1.305 .010c

SymmetricRESPONSE DependentINSTAR Dependent

Uncertainty CoefficientNominal by NominalValue

Asymp.Std. Errora Approx. Tb Approx. Sig.

Not assuming the null hypothesis.a.

Using the asymptotic standard error assuming the null hypothesis.b.

Likelihood ratio chi-square probability.c.

Symmetric Measures

-.209 .010.209 .010

-.209 .079 -2.604 .010c

-.209 .079 -2.604 .010c

150

PhiCramer's V

Nominal byNominal

Pearson's RInterval by IntervalSpearman CorrelationOrdinal by Ordinal

N of Valid Cases

ValueAsymp.

Std. Errora Approx. Tb Approx. Sig.

Not assuming the null hypothesis.a.

Using the asymptotic standard error assuming the null hypothesis.b.

Based on normal approximation.c.

2.2 WESTERN CAPE 2.2.1 Rondegat Farm (Clanwilliam) 2.2.1.1 Comparative analysis of bioassays with Cryptogran (at 1.661 x 108 OBs/ml) against field collected 2nd, 3rd and 4th instar FCM larvae from Rondegat Tree (Clanwilliam, Western Cape)

Case Processing Summary

187 100.0% 0 .0% 187 100.0%Quantal response* FCM Instars

N Percent N Percent N PercentValid Missing Total

Cases

147

RESPONSE * INSTAR Crosstabulation

21 38 31 41 35 20 18620.9 37.8 30.8 40.8 34.8 20.9 186.0

100.0% 100.0% 100.0% 100.0% 100.0% 95.2% 99.5%0 0 0 0 0 1 1.1 .2 .2 .2 .2 .1 1.0

.0% .0% .0% .0% .0% 4.8% .5%21 38 31 41 35 21 187

21.0 38.0 31.0 41.0 35.0 21.0 187.0100.0% 100.0% 100.0% 100.0% 100.0% 100.0% 100.0%

CountExpected Count% within INSTARCountExpected Count% within INSTARCountExpected Count% within INSTAR

Dead

Alive

RESPONSE

Total

2nd FCMInstar Van Zyl

3rd FCMInstar Van Zyl

4th FCM InstarVan Zyl

2nd FCMInstar G. Grib

3rd FCMInstar G. Grib

4th FCMInstar G. Grib

INSTAR

Total

Chi-Square Tests

7.947a 5 .1594.416 5 .491

2.622 1 .105

187

Pearson Chi-SquareLikelihood RatioLinear-by-LinearAssociationN of Valid Cases

Value dfAsymp. Sig.

(2-sided)

6 cells (50.0%) have expected count less than 5. Theminimum expected count is .11.

a.

Directional Measures

.013 .013 1.008 .491c

.355 .063 1.008 .491c

.007 .007 1.008 .491c

SymmetricRESPONSE DependentINSTAR Dependent

Uncertainty CoefficientNominal by NominalValue

Asymp.Std. Errora Approx. Tb Approx. Sig.

Not assuming the null hypothesis.a.

Using the asymptotic standard error assuming the null hypothesis.b.

Likelihood ratio chi-square probability.c.

148

Symmetric Measures

.206 .159

.206 .159

.119 .059 1.626 .106c

.115 .057 1.570 .118c

187

PhiCramer's V

Nominal byNominal

Pearson's RInterval by IntervalSpearman CorrelationOrdinal by Ordinal

N of Valid Cases

ValueAsymp.

Std. Errora Approx. Tb Approx. Sig.

Not assuming the null hypothesis.a.

Using the asymptotic standard error assuming the null hypothesis.b.

Based on normal approximation.c.

2.2.1.2 Comparative analysis of bioassays with Cryptogran (at 1.661 x 108 OBs/ml) against field collected 2nd, 3rd, 4th and 5th instar FCM larvae from Rondegat Farm (Clanwilliam, Western Cape)

Case Processing Summary

183 100.0% 0 .0% 183 100.0%Quantal response *FCM Larval Instars * Orchards used

N Percent N Percent N PercentValid Missing Total

Cases

Quantal response * FCM Larval Instars * Orchards used Crosstabulation

21 38 31 65 15517.8 32.2 26.3 78.8 155.0

100.0% 100.0% 100.0% 69.9% 84.7%

0 0 0 28 283.2 5.8 4.7 14.2 28.0

.0% .0% .0% 30.1% 15.3%

21 38 31 93 18321.0 38.0 31.0 93.0 183.0

100.0% 100.0% 100.0% 100.0% 100.0%

CountExpected Count% within FCMLarval InstarsCountExpected Count% within FCMLarval InstarsCountExpected Count% within FCMLarval Instars

Dead

Alive

Quantal response

Total

Orchards usedVan Zyl

2nd FCMInstar

3rd FCMInstar

4th FCMInstar

5th FCMInstar

FCM Larval Instars

Total

149

Chi-Square Tests

31.992a 3 .00042.817 3 .000

24.254 1 .000

183

Pearson Chi-SquareLikelihood RatioLinear-by-LinearAssociationN of Valid Cases

Orchards usedVan Zyl

Value dfAsymp. Sig.

(2-sided)

2 cells (25.0%) have expected count less than 5. The minimum expectedcount is 3.21.

a.

Directional Measures

.142 .022 5.490 .000c

.273 .031 5.490 .000c

.096 .017 5.490 .000c

SymmetricRESPONSE DependentINSTAR Dependent

Uncertainty CoefficientNominal by NominalValue

Asymp.Std. Errora Approx. Tb Approx. Sig.

Not assuming the null hypothesis.a.

Using the asymptotic standard error assuming the null hypothesis.b.

Likelihood ratio chi-square probability.c.

Symmetric Measures

.418 .000

.418 .000

.365 .035 5.275 .000c

.392 .037 5.731 .000c

183

PhiCramer's V

Nominal byNominal

Pearson's RInterval by IntervalSpearman CorrelationOrdinal by Ordinal

N of Valid Cases

ValueAsymp.

Std. Errora Approx. Tb Approx. Sig.

Not assuming the null hypothesis.a.

Using the asymptotic standard error assuming the null hypothesis.b.

Based on normal approximation.c.

150

2.2.2 Jansekraal Farm (Citrusdal) 2.2.2.1 Comparative analysis of bioassays with Cryptogran (at 1.661 x 108 OBs/ml) against field collected 2nd, 3rd, 4th and 5th instar FCM larvae from Jansekraal Farm (Citrusdal, Western Cape)

Case Processing Summary

164 100.0% 0 .0% 164 100.0%Quantal response *FCM Larval Instars * Orchards used

N Percent N Percent N PercentValid Missing Total

Cases

Quantal response * FCM Larval Instars * Orchards used Crosstabulation

41 35 20 47 14335.8 30.5 18.3 58.4 143.0

100.0% 100.0% 95.2% 70.1% 87.2%

0 0 1 20 215.3 4.5 2.7 8.6 21.0

.0% .0% 4.8% 29.9% 12.8%

41 35 21 67 16441.0 35.0 21.0 67.0 164.0

100.0% 100.0% 100.0% 100.0% 100.0%

CountExpected Count% within FCMLarval InstarsCountExpected Count% within FCMLarval InstarsCountExpected Count% within FCMLarval Instars

Dead

Alive

Quantal response

Total

Orchards usedGeorge Grib

2nd FCMInstar

3rd FCMInstar

4th FCMInstar

5th FCMInstar

FCM Larval Instars

Total

Chi-Square Tests

29.813a 3 .00035.786 3 .000

24.746 1 .000

164

Pearson Chi-SquareLikelihood RatioLinear-by-LinearAssociationN of Valid Cases

Orchards usedGeorge Grib

Value dfAsymp. Sig.

(2-sided)

2 cells (25.0%) have expected count less than 5. The minimum expectedcount is 2.69.

a.

151

Directional Measures

.129 .027 4.319 .000c

.285 .044 4.319 .000c

.084 .020 4.319 .000c

SymmetricRESPONSE DependentINSTAR Dependent

Uncertainty CoefficientNominal by NominalValue

Asymp.Std. Errora Approx. Tb Approx. Sig.

Not assuming the null hypothesis.a.

Using the asymptotic standard error assuming the null hypothesis.b.

Likelihood ratio chi-square probability.c.

Symmetric Measures

.426 .000

.426 .000

.390 .044 5.385 .000c

.395 .044 5.472 .000c

164

PhiCramer's V

Nominal byNominal

Pearson's RInterval by IntervalSpearman CorrelationOrdinal by Ordinal

N of Valid Cases

ValueAsymp.

Std. Errora Approx. Tb Approx. Sig.

Not assuming the null hypothesis.a.

Using the asymptotic standard error assuming the null hypothesis.b.

Based on normal approximation.c.

2.2.3 Comparative analysis of bioassays with Cryptogran (at 1.661 x 108 OBs/ml) against field collected 2nd, 3rd, 4th and 5th instar FCM larvae from both Rondegat and Jansekraal Farm (Western Cape)

Case Processing Summary

160 100.0% 0 .0% 160 100.0%Quantal response *FCM Larval Instars

N Percent N Percent N PercentValid Missing Total

Cases

152

Quantal response * FCM Larval Instars Crosstabulation

65 47 11265.1 46.9 112.0

69.9% 70.1% 70.0%

28 20 4827.9 20.1 48.0

30.1% 29.9% 30.0%

93 67 16093.0 67.0 160.0

100.0% 100.0% 100.0%

CountExpected Count% within FCMLarval InstarsCountExpected Count% within FCMLarval InstarsCountExpected Count% within FCMLarval Instars

Dead

Alive

Quantal response

Total

5th FCM -Van Zyl

5th FCMInstar -

George Grib

FCM Larval Instars

Total

Chi-Square Tests

.001b 1 .972

.000 1 1.000

.001 1 .9721.000 .557

.001 1 .972

160

Pearson Chi-SquareContinuity Correctiona

Likelihood RatioFisher's Exact TestLinear-by-LinearAssociationN of Valid Cases

Value dfAsymp. Sig.

(2-sided)Exact Sig.(2-sided)

Exact Sig.(1-sided)

Computed only for a 2x2 tablea.

0 cells (.0%) have expected count less than 5. The minimum expected count is20.10.

b.

Directional Measures

.000 .000 .017 .972c

.000 .000 .017 .972c

.000 .000 .017 .972c

SymmetricRESPONSE DependentINSTAR Dependent

Uncertainty CoefficientNominal by NominalValue

Asymp.Std. Errora Approx. Tb Approx. Sig.

Not assuming the null hypothesis.a.

Using the asymptotic standard error assuming the null hypothesis.b.

Likelihood ratio chi-square probability.c.

153

Symmetric Measures

-.003 .972.003 .972

-.003 .079 -.035 .972c

-.003 .079 -.035 .972c

160

PhiCramer's V

Nominal byNominal

Pearson's RInterval by IntervalSpearman CorrelationOrdinal by Ordinal

N of Valid Cases

ValueAsymp.

Std. Errora Approx. Tb Approx. Sig.

Not assuming the null hypothesis.a.

Using the asymptotic standard error assuming the null hypothesis.b.

Based on normal approximation.c.

2.3 COMPARATIVE ANALYSIS OF BIOASSAYS WITH CRYPTOGRAN (AT 1.661 X 108 OBs/ML) AGAINST FIELD COLLECTED 5TH INSTAR FCM LARVAE FROM BOTH THE EASTERN CAPE AND WESTERN CAPE PROVINCES

Case Processing Summary

310 100.0% 0 .0% 310 100.0%Quantal Response *5th FCM Larval Instar

N Percent N Percent N PercentValid Missing Total

Cases

Quantal Response * 5th FCM Larval Instar Crosstabulation

37 42 65 47 19152.4 40.0 57.3 41.3 191.0

43.5% 64.6% 69.9% 70.1% 61.6%

48 23 28 20 11932.6 25.0 35.7 25.7 119.0

56.5% 35.4% 30.1% 29.9% 38.4%

85 65 93 67 31085.0 65.0 93.0 67.0 310.0

100.0% 100.0% 100.0% 100.0% 100.0%

CountExpected Count% within 5th FCMLarval InstarCountExpected Count% within 5th FCMLarval InstarCountExpected Count% within 5th FCMLarval Instar

Dead

Alive

Quantal Response

Total

5th FCMInstar fromLone TreeFarm (E/C)

5th FCMInstar fromTregaron

Farm (E/C)

5th FCM Instarfrom Van Zyl

(W/C)

5th FCMInstar from

G. Grib (W/C)

5th FCM Larval Instar

Total

154

Chi-Square Tests

16.760a 3 .00116.519 3 .001

13.175 1 .000

310

Pearson Chi-SquareLikelihood RatioLinear-by-LinearAssociationN of Valid Cases

Value dfAsymp. Sig.

(2-sided)

0 cells (.0%) have expected count less than 5. Theminimum expected count is 24.95.

a.

Directional Measures

.026 .013 2.040 .001c

.040 .020 2.040 .001c

.019 .010 2.040 .001c

SymmetricRESPONSE DependentINSTAR Dependent

Uncertainty CoefficientNominal by NominalValue

Asymp.Std. Errora Approx. Tb Approx. Sig.

Not assuming the null hypothesis.a.

Using the asymptotic standard error assuming the null hypothesis.b.

Likelihood ratio chi-square probability.c.

Symmetric Measures

.233 .001

.233 .001-.206 .056 -3.704 .000c

-.205 .056 -3.674 .000c

310

PhiCramer's V

Nominal byNominal

Pearson's RInterval by IntervalSpearman CorrelationOrdinal by Ordinal

N of Valid Cases

ValueAsymp.

Std. Errora Approx. Tb Approx. Sig.

Not assuming the null hypothesis.a.

Using the asymptotic standard error assuming the null hypothesis.b.

Based on normal approximation.c.

155

2.4 COMPARATIVE ANALYSIS OF BIOASSAYS WITH CRYPTOGRAN (AT 1.661 X 108 OBs/ML) AGAINST FIELD COLLECTED 5TH INSTAR FCM LARVAE FROM TREGARON (KIRKWOOD, EASTERN CAPE), CLANWILLIAM (WESTERN CAPE) AND CITRUSDAL (WESTERN CAPE).

Case Processing Summary

225 100.0% 0 .0% 225 100.0%Quantal Response *5th FCM Larval Instar

N Percent N Percent N PercentValid Missing Total

Cases

Quantal Response * 5th FCM Larval Instar Crosstabulation

42 65 47 15444.5 63.7 45.9 154.0

64.6% 69.9% 70.1% 68.4%

23 28 20 7120.5 29.3 21.1 71.0

35.4% 30.1% 29.9% 31.6%

65 93 67 22565.0 93.0 67.0 225.0

100.0% 100.0% 100.0% 100.0%

CountExpected Count% within 5th FCMLarval InstarCountExpected Count% within 5th FCMLarval InstarCountExpected Count% within 5th FCMLarval Instar

Dead

Alive

Quantal Response

Total

5th FCMInstar fromTregaron

Farm (E/C)

5th FCM Instarfrom Van Zyl

(W/C)

5th FCMInstar from

G. Grib (W/C)

5th FCM Larval Instar

Total

Chi-Square Tests

.622a 2 .733

.615 2 .735

.460 1 .497

225

Pearson Chi-SquareLikelihood RatioLinear-by-LinearAssociationN of Valid Cases

Value dfAsymp. Sig.

(2-sided)

0 cells (.0%) have expected count less than 5. Theminimum expected count is 20.51.

a.

156

Directional Measures

.002 .004 .390 .735c

.002 .006 .390 .735c

.001 .003 .390 .735c

SymmetricRESPONSE DependentINSTAR Dependent

Uncertainty CoefficientNominal by NominalValue

Asymp.Std. Errora Approx. Tb Approx. Sig.

Not assuming the null hypothesis.a.

Using the asymptotic standard error assuming the null hypothesis.b.

Likelihood ratio chi-square probability.c.

Symmetric Measures

.053 .733

.053 .733-.045 .067 -.678 .499c

-.045 .067 -.676 .500c

225

PhiCramer's V

Nominal byNominal

Pearson's RInterval by IntervalSpearman CorrelationOrdinal by Ordinal

N of Valid Cases

ValueAsymp.

Std. Errora Approx. Tb Approx. Sig.

Not assuming the null hypothesis.a.

Using the asymptotic standard error assuming the null hypothesis.b.

Based on normal approximation.c.

2.5 COMPARATIVE ANALYSIS OF BIOASSAYS WITH CRYPTOGRAN (AT 1.661 X 108 OBs/ML) AGAINST FIELD COLLECTED AND LABORATORY REARED 5TH INSTAR FCM LARVAE

2.5.1 Comparative analysis of bioassays with Cryptogran (at 1.661 x 108 OBs/ml) against field collected 5th instar FCM larvae from Lone Tree Farm (Addo, Eastern Cape) and the laboratory colony

Case Processing Summary

235 100.0% 0 .0% 235 100.0%Quantal response* 5th FCM Instar

N Percent N Percent N PercentValid Missing Total

Cases

157

Quantal response * 5th FCM Instar Crosstabulation

37 104 14151.0 90.0 141.0

43.5% 69.3% 60.0%48 46 94

34.0 60.0 94.056.5% 30.7% 40.0%

85 150 23585.0 150.0 235.0

100.0% 100.0% 100.0%

CountExpected Count% within 5th FCM InstarCountExpected Count% within 5th FCM InstarCountExpected Count% within 5th FCM Instar

Dead

Alive

Quantal response

Total

5th instarfrom Addo

(fieldcollected)

5th instar(laboratory

reared)

5th FCM Instar

Total

Chi-Square Tests

15.052b 1 .00013.996 1 .00014.986 1 .000

.000 .000

14.988 1 .000

235

Pearson Chi-SquareContinuity Correctiona

Likelihood RatioFisher's Exact TestLinear-by-LinearAssociationN of Valid Cases

Value dfAsymp. Sig.

(2-sided)Exact Sig.(2-sided)

Exact Sig.(1-sided)

Computed only for a 2x2 tablea.

0 cells (.0%) have expected count less than 5. The minimum expected count is34.00.

b.

Directional Measures

.048 .025 1.954 .000c

.047 .024 1.954 .000c

.049 .025 1.954 .000c

SymmetricRESPONSE DependentINSTAR Dependent

Uncertainty CoefficientNominal by NominalValue

Asymp.Std. Errora Approx. Tb Approx. Sig.

Not assuming the null hypothesis.a.

Using the asymptotic standard error assuming the null hypothesis.b.

Likelihood ratio chi-square probability.c.

158

Symmetric Measures

-.253 .000.253 .000

-.253 .064 -3.993 .000c

-.253 .064 -3.993 .000c

235

PhiCramer's V

Nominal byNominal

Pearson's RInterval by IntervalSpearman CorrelationOrdinal by Ordinal

N of Valid Cases

ValueAsymp.

Std. Errora Approx. Tb Approx. Sig.

Not assuming the null hypothesis.a.

Using the asymptotic standard error assuming the null hypothesis.b.

Based on normal approximation.c.

2.5.2 Comparative analysis of bioassays with Cryptogran (at 1.661 x 108 OBs/ml) against field collected 5th instar FCM larvae from Tregaron Farm (Kirkwood, Eastern Cape) and a laboratory colony.

Case Processing Summary

215 100.0% 0 .0% 215 100.0%Quantal response* 5th FCM Instar

N Percent N Percent N PercentValid Missing Total

Cases

Quantal response * 5th FCM Instar Crosstabulation

42 104 14644.1 101.9 146.0

64.6% 69.3% 67.9%23 46 69

20.9 48.1 69.035.4% 30.7% 32.1%

65 150 21565.0 150.0 215.0

100.0% 100.0% 100.0%

CountExpected Count% within 5th FCM InstarCountExpected Count% within 5th FCM InstarCountExpected Count% within 5th FCM Instar

Dead

Alive

Quantal response

Total

5th instarfrom

Kirkwood(field

collected)

5th instar(laboratory

reared)

5th FCM Instar

Total

159

Chi-Square Tests

.463b 1 .496

.272 1 .602

.459 1 .498.527 .299

.461 1 .497

215

Pearson Chi-SquareContinuity Correctiona

Likelihood RatioFisher's Exact TestLinear-by-LinearAssociationN of Valid Cases

Value dfAsymp. Sig.

(2-sided)Exact Sig.(2-sided)

Exact Sig.(1-sided)

Computed only for a 2x2 tablea.

0 cells (.0%) have expected count less than 5. The minimum expected count is20.86.

b.

Directional Measures

.002 .005 .337 .498c

.002 .005 .337 .498c

.002 .005 .337 .498c

SymmetricRESPONSE DependentINSTAR Dependent

Uncertainty CoefficientNominal by NominalValue

Asymp.Std. Errora Approx. Tb Approx. Sig.

Not assuming the null hypothesis.a.

Using the asymptotic standard error assuming the null hypothesis.b.

Likelihood ratio chi-square probability.c.

Symmetric Measures

-.046 .496.046 .496

-.046 .069 -.678 .498c

-.046 .069 -.678 .498c

215

PhiCramer's V

Nominal byNominal

Pearson's RInterval by IntervalSpearman CorrelationOrdinal by Ordinal

N of Valid Cases

ValueAsymp.

Std. Errora Approx. Tb Approx. Sig.

Not assuming the null hypothesis.a.

Using the asymptotic standard error assuming the null hypothesis.b.

Based on normal approximation.c.

160

2.5.3 Comparative analysis of bioassays with Cryptogran (at 1.661 x 108 OBs/ml) against field collected 5th instar FCM larvae from Rondegat Farm (Clanwilliam) and the laboratory colony

Case Processing Summary

243 100.0% 0 .0% 243 100.0%Quantal response* 5th FCM Instar

N Percent N Percent N PercentValid Missing Total

Cases

Quantal response * 5th FCM Instar Crosstabulation

65 104 16964.7 104.3 169.0

69.9% 69.3% 69.5%28 46 74

28.3 45.7 74.030.1% 30.7% 30.5%

93 150 24393.0 150.0 243.0

100.0% 100.0% 100.0%

CountExpected Count% within 5th FCM InstarCountExpected Count% within 5th FCM InstarCountExpected Count% within 5th FCM Instar

Dead

Alive

Quantal response

Total

5th instarfrom VanZyl (fieldcollected)

5th instar(laboratory

reared)

5th FCM Instar

Total

Chi-Square Tests

.008b 1 .927

.000 1 1.000

.008 1 .9271.000 .522

.008 1 .927

243

Pearson Chi-SquareContinuity Correctiona

Likelihood RatioFisher's Exact TestLinear-by-LinearAssociationN of Valid Cases

Value dfAsymp. Sig.

(2-sided)Exact Sig.(2-sided)

Exact Sig.(1-sided)

Computed only for a 2x2 tablea.

0 cells (.0%) have expected count less than 5. The minimum expected count is28.32.

b.

161

Directional Measures

.000 .001 .046 .927c

.000 .001 .046 .927c

.000 .001 .046 .927c

SymmetricRESPONSE DependentINSTAR Dependent

Uncertainty CoefficientNominal by NominalValue

Asymp.Std. Errora Approx. Tb Approx. Sig.

Not assuming the null hypothesis.a.

Using the asymptotic standard error assuming the null hypothesis.b.

Likelihood ratio chi-square probability.c.

Symmetric Measures

.006 .927

.006 .927

.006 .064 .092 .927c

.006 .064 .092 .927c

243

PhiCramer's V

Nominal byNominal

Pearson's RInterval by IntervalSpearman CorrelationOrdinal by Ordinal

N of Valid Cases

ValueAsymp.

Std. Errora Approx. Tb Approx. Sig.

Not assuming the null hypothesis.a.

Using the asymptotic standard error assuming the null hypothesis.b.

Based on normal approximation.c.

2.5.4 Comparative analysis of bioassays with Cryptogran (at 1.661 x 108 OBs/ml) against field collected 5th instar FCM larvae from Jansekraal Farm (Citrusdal) and the laboratory colony

Case Processing Summary

217 100.0% 0 .0% 217 100.0%Quantal response* 5th FCM Instar

N Percent N Percent N PercentValid Missing Total

Cases

162

Quantal response * 5th FCM Instar Crosstabulation

47 104 15146.6 104.4 151.0

70.1% 69.3% 69.6%20 46 66

20.4 45.6 66.029.9% 30.7% 30.4%

67 150 21767.0 150.0 217.0

100.0% 100.0% 100.0%

CountExpected Count% within 5th FCM InstarCountExpected Count% within 5th FCM InstarCountExpected Count% within 5th FCM Instar

Dead

Alive

Quantal response

Total

5th instarfrom George

Grib (fieldcollected)

5th instar(laboratory

reared)

5th FCM Instar

Total

Chi-Square Tests

.015b 1 .904

.000 1 1.000

.015 1 .9041.000 .519

.015 1 .904

217

Pearson Chi-SquareContinuity Correctiona

Likelihood RatioFisher's Exact TestLinear-by-LinearAssociationN of Valid Cases

Value dfAsymp. Sig.

(2-sided)Exact Sig.(2-sided)

Exact Sig.(1-sided)

Computed only for a 2x2 tablea.

0 cells (.0%) have expected count less than 5. The minimum expected count is20.38.

b.

Directional Measures

.000 .001 .060 .904c

.000 .001 .060 .904c

.000 .001 .060 .904c

SymmetricRESPONSE DependentINSTAR Dependent

Uncertainty CoefficientNominal by NominalValue

Asymp.Std. Errora Approx. Tb Approx. Sig.

Not assuming the null hypothesis.a.

Using the asymptotic standard error assuming the null hypothesis.b.

Likelihood ratio chi-square probability.c.

163

Symmetric Measures

.008 .904

.008 .904

.008 .068 .120 .904c

.008 .068 .120 .904c

217

PhiCramer's V

Nominal byNominal

Pearson's RInterval by IntervalSpearman CorrelationOrdinal by Ordinal

N of Valid Cases

ValueAsymp.

Std. Errora Approx. Tb Approx. Sig.

Not assuming the null hypothesis.a.

Using the asymptotic standard error assuming the null hypothesis.b.

Based on normal approximation.c.

164

Appendix 3 PROBAN (VAN ARK, 1995) OUPUT OF PROBIT ANALYSIS OF SURFACE DOSE-RESPONSE BIOASSAY DATA (LC) ON A NON-AGAR DIET, WITH CRYPTOGRAN AGAINST LABORATORY REARED 4TH INSTAR FCM LARVAE

****************************************************************************** Rep 1. Dose-response bioassays with 4th instar FCM larvae ****************************************************************************** GENERAL INPUT INFORMATION ========================= DOSES TRANSFORMED TO LOG10 NATURAL MORTALITY = 0% NON-RESPONSE = 0% DOSE TRANSFORMED NUMBER NUMBER %RESPONSE CORRECTED EMPIRICAL DOSE(X) EXPOSED RESPON. %RESPONSE PROBITS *************** 6.6990 25 21 84.00 84.00 5.995 *************** 6.0000 25 20 80.00 80.00 5.842 200000.00000000 5.3010 25 16 64.00 64.00 5.359 40000.00000000 4.6021 25 13 52.00 52.00 5.050 8000.00000000 3.9031 25 9 36.00 36.00 4.641 TEST FOR FIT OF LINE ==================== CHI-SQUARED FOR DEVIATIONS = .263 D. F. = 3 APPROXIMATE PROBABILITY = .9620 TEST LEVEL = 0,05 TABLED CHI-SQUARED VALUE = 7.815 DEVIATIONS FROM LINE ARE HOMOGENEOUS FITTED PROBIT LINE ================== REGRESSION EQUATION IS Y = a + bX WHERE Y IS EMPIRICAL PROBITS AND X IS DOSE INTERCEPT (a) = 2.7062 REGRESSION COEFFICIENT (b) = .5040 STANDARD ERROR OF b = .1258 MEAN FOR EMPIRICAL PROBITS (Y) = 5.3114 MEAN FOR DOSE (X) = 5.1690

165

DOSE (X) EXPECTED EMP.PROBIT (Y) 6.69897 6.082 6.00000 5.730 5.30103 5.378 4.60206 5.026 3.90309 4.673 EXPECTED QUANTITIES FOR LINE ============================ G FOR FIDUCIAL LIMITS = .2392 EXPECTED DOSE STANDARD FIDUCIAL LIMITS % RESPONSE ERROR UPPER LOWER 50.0 4.551 .2832 5.022 3.692 90.0 7.094 .5359 9.047 6.351 95.0 7.815 .7017 10.430 6.864 99.0 9.167 1.0255 13.049 7.799 99.9 10.682 1.3962 16.001 8.832 QUANTITIES TRANSFORMED BACK TO ORIGINAL ======================================= EXPECTED DOSE STANDARD FIDUCIAL LIMITS % RESPONSE ERROR UPPER LOWER 50.0 .3557916E+05 .2317565E+05 .1052773E+06 .4915167E+04 90.0 .1241734E+08 .1530598E+08 .1114923E+10 .2246435E+07 95.0 .6529120E+08 .1053756E+09 .2689900E+11 .7311156E+07 99.0 .1468339E+10 .3463446E+10 .1119661E+14 .6292778E+08 99.9 .4813783E+11 .1545867E+12 .1002075E+17 .6785057E+09

166

****************************************************************************** Rep 2. Dose-response bioassays with 4th instar FCM larvae ****************************************************************************** GENERAL INPUT INFORMATION ========================= DOSES TRANSFORMED TO LOG10 NATURAL MORTALITY = 0% NON-RESPONSE = 0% DOSE TRANSFORMED NUMBER NUMBER %RESPONSE CORRECTED EMPIRICAL DOSE(X) EXPOSED RESPON. %RESPONSE PROBITS *************** 6.6990 25 22 88.00 88.00 6.175 *************** 6.0000 25 21 84.00 84.00 5.995 200000.00000000 5.3010 25 18 72.00 72.00 5.583 40000.00000000 4.6021 25 12 48.00 48.00 4.950 8000.00000000 3.9031 25 9 36.00 36.00 4.641 TEST FOR FIT OF LINE ==================== CHI-SQUARED FOR DEVIATIONS = .638 D. F. = 3 APPROXIMATE PROBABILITY = .8870 TEST LEVEL = 0,05 TABLED CHI-SQUARED VALUE = 7.815 DEVIATIONS FROM LINE ARE HOMOGENEOUS FITTED PROBIT LINE ================== REGRESSION EQUATION IS Y = a + bX WHERE Y IS EMPIRICAL PROBITS AND X IS DOSE INTERCEPT (a) = 2.2836 REGRESSION COEFFICIENT (b) = .6017 STANDARD ERROR OF b = .1323 MEAN FOR EMPIRICAL PROBITS (Y) = 5.3596 MEAN FOR DOSE (X) = 5.1125 DOSE (X) EXPECTED EMP.PROBIT (Y) 6.69897 6.314 6.00000 5.894 5.30103 5.473 4.60206 5.053 3.90309 4.632

167

EXPECTED QUANTITIES FOR LINE ============================ G FOR FIDUCIAL LIMITS = .1857 EXPECTED DOSE STANDARD FIDUCIAL LIMITS % RESPONSE ERROR UPPER LOWER 50.0 4.515 .2433 4.924 3.833 90.0 6.645 .3943 7.919 6.069 95.0 7.249 .5124 8.951 6.521 99.0 8.381 .7474 10.913 7.340 99.9 9.651 1.0187 13.129 8.243 QUANTITIES TRANSFORMED BACK TO ORIGINAL ======================================= EXPECTED DOSE STANDARD FIDUCIAL LIMITS % RESPONSE ERROR UPPER LOWER 50.0 .3271462E+05 .1830910E+05 .8398966E+05 .6800540E+04 90.0 .4414008E+07 .4002972E+07 .8306697E+08 .1173171E+07 95.0 .1772792E+08 .2089355E+08 .8932824E+09 .3318485E+07 99.0 .2405380E+09 .4134663E+09 .8189054E+11 .2190046E+08 99.9 .4475364E+10 .1048615E+11 .1346199E+14 .1750381E+09

168

****************************************************************************** Rep 3. Dose-response bioassays with 4th instar FCM larvae ****************************************************************************** GENERAL INPUT INFORMATION ========================= DOSES TRANSFORMED TO LOG10 NATURAL MORTALITY = 0% NON-RESPONSE = 0% DOSE TRANSFORMED NUMBER NUMBER %RESPONSE CORRECTED EMPIRICAL DOSE(X) EXPOSED RESPON. %RESPONSE PROBITS *************** 6.6990 25 23 92.00 92.00 6.405 *************** 6.0000 25 21 84.00 84.00 5.995 200000.00000000 5.3010 25 17 68.00 68.00 5.468 40000.00000000 4.6021 25 10 40.00 40.00 4.747 8000.00000000 3.9031 25 7 28.00 28.00 4.417 TEST FOR FIT OF LINE ==================== CHI-SQUARED FOR DEVIATIONS = .461 D. F. = 3 APPROXIMATE PROBABILITY = .9240 TEST LEVEL = 0,05 TABLED CHI-SQUARED VALUE = 7.815 DEVIATIONS FROM LINE ARE HOMOGENEOUS FITTED PROBIT LINE ================== REGRESSION EQUATION IS Y = a + bX WHERE Y IS EMPIRICAL PROBITS AND X IS DOSE INTERCEPT (a) = 1.4111 REGRESSION COEFFICIENT (b) = .7536 STANDARD ERROR OF b = .1392 MEAN FOR EMPIRICAL PROBITS (Y) = 5.2574 MEAN FOR DOSE (X) = 5.1041 DOSE (X) EXPECTED EMP.PROBIT (Y) 6.69897 6.459 6.00000 5.933 5.30103 5.406 4.60206 4.879 3.90309 4.352

169

EXPECTED QUANTITIES FOR LINE ============================ G FOR FIDUCIAL LIMITS = .1312 EXPECTED DOSE STANDARD FIDUCIAL LIMITS % RESPONSE ERROR UPPER LOWER 50.0 4.762 .1783 5.089 4.332 90.0 6.463 .3015 7.335 6.002 95.0 6.945 .3789 8.067 6.379 99.0 7.849 .5340 9.461 7.067 99.9 8.863 .7143 11.036 7.825 QUANTITIES TRANSFORMED BACK TO ORIGINAL ======================================= EXPECTED DOSE STANDARD FIDUCIAL LIMITS % RESPONSE ERROR UPPER LOWER 50.0 .5787496E+05 .2374045E+05 .1228772E+06 .2149658E+05 90.0 .2905208E+07 .2014449E+07 .2160936E+08 .1004720E+07 95.0 .8816114E+07 .7682919E+07 .1166905E+09 .2395567E+07 99.0 .7071261E+08 .8684976E+08 .2889744E+10 .1166885E+08 99.9 .7297842E+09 .1199019E+10 .1087326E+12 .6683128E+08

170

*************************** * COMPARISON OF LINES * *************************** ABRIDGED ANALYSIS OF VARIANCE ============================= DEVIATIONS FROM REGRESSION INTERCEPT REGR.COEF. D.F. SUM OF RESIDUAL (a) (b) SQUARES VARIANCES LINE: rep 1 2.706 .504 3 .263390E+00 .088 LINE: rep 2 2.284 .602 3 .638172E+00 .213 LINE: rep 3 1.411 .754 3 .460849E+00 .154 ---------------------------------------------------------------------- 9 .136241E+01 .151 ---------------------------------------------------------------------- COMBINED .611 11 .313955E+01 .285 ---------------------------------------------------------------------- DIFFERENCE BETWEEN SLOPES 2 .177714E+01 .889 ---------------------------------------------------------------------- TOTAL 2.176 .611 13 .350039E+01 .269 ---------------------------------------------------------------------- DIFFERENCE BETWEEN ADJ. MEANS 2 .360844E+00 .180 ---------------------------------------------------------------------- BARTLETTS TEST FOR HOMOGENEITY OF RESIDUAL VARIANCES ==================================================== CHI-SQUARED VALUE = .302 D. F. = 2 APPROXIMATE PROBABILITY = .8580 TEST LEVEL = .0100 TABLED CHI-SQUARED VALUE = 9.210 RESIDUAL VARIANCES ARE HOMOGENEOUS - SLOPES OF LINES MAY BE COMPARED COMPARISON OF SLOPES (PARALLELISM) ================================== SOURCE D.F. SUM OF SQ. MEAN SQ. F-VALUE --------------------------------------------------------- PARALLELISM 2 .177714E+01 .889 5.87 HETEROGENEITY 9 .136241E+01 .151 -------------------------------- TOTAL 11 .3140E+01 CHI-SQUARED TEST FOR PARALLELISM -------------------------------- CHI-SQUARED VALUE = 1.777 D. F. = 2 APPROXIMATE PROBABILITY = .4140 TEST LEVEL = 0,05 TABLED CHI-SQUARED VALUE = 5.991

171

LINES ARE PARALLEL AND ELEVATIONS MAY BE COMPARED REGRESSION COEFFICIENTS ----------------------- rep 1 .5040 rep 2 .6017 rep 3 .7536 COMPARISON OF ELEVATIONS (ADJUSTED MEANS) ========================================= F-VALUE FOR ELEVATIONS = .632 D.F. = 2 & 11 APPROXIMATE PROBABILITY = .5500 TEST LEVEL = 0,05 TABLED F-VALUE = 3.98 ELEVATIONS ARE NOT SIGNIFICANTLY DIFFERENT MEANS OF EMPIRICAL PROBITS -------------------------- rep 3 5.2574 rep 1 5.3114 rep 2 5.3596 INTERCEPTS OF LINES ------------------- rep 3 1.411 rep 2 2.284 rep 1 2.706

172

Appendix 4 PROBAN (VAN ARK, 1995) OUPUT OF PROBIT ANALYSIS OF SURFACE DOSE-RESPONSE BIOASSAY DATA (LC) ON AN AGAR DIET, WITH CRYPTOGRAN AND CRYPTEX AGAINST AN OLD FCM COLONY 4.1 DOSE-RESPONSE BIOASSAYS WITH CRYPTEX AGAINST 1st INSTAR FCM LARVAE FROM THE OLD COLONY ****************************************************************************** rep 1. Dose-response bioassays with 1st instar FCM larvae (See section: 4.3.2.1) ****************************************************************************** GENERAL INPUT INFORMATION ========================= DOSES TRANSFORMED TO LOG10 NATURAL MORTALITY = 5% NON-RESPONSE = 0% DOSE TRANSFORMED NUMBER NUMBER %RESPONSE CORRECTED EMPIRICAL DOSE(X) EXPOSED RESPON. %RESPONSE PROBITS 100000.00000000 5.0000 30 23 76.67 75.44 5.688 20000.00000000 4.3010 30 15 50.00 47.37 4.934 4000.00000000 3.6021 30 13 43.33 40.35 4.756 800.00000000 2.9031 30 7 23.33 19.30 4.133 160.00000000 2.2041 30 4 13.33 8.77 3.645 TEST FOR FIT OF LINE ==================== CHI-SQUARED FOR DEVIATIONS = .956 D. F. = 3 APPROXIMATE PROBABILITY = .8140 TEST LEVEL = 0,05 TABLED CHI-SQUARED VALUE = 7.815 DEVIATIONS FROM LINE ARE HOMOGENEOUS FITTED PROBIT LINE ================== REGRESSION EQUATION IS Y = a + bX WHERE Y IS EMPIRICAL PROBITS AND X IS DOSE INTERCEPT (a) = 2.1422 REGRESSION COEFFICIENT (b) = .6911 STANDARD ERROR OF b = .1383 MEAN FOR EMPIRICAL PROBITS (Y) = 4.8099

173

MEAN FOR DOSE (X) = 3.8600 DOSE (X) EXPECTED EMP.PROBIT (Y) 5.00000 5.598 4.30103 5.115 3.60206 4.632 2.90309 4.149 2.20412 3.665 EXPECTED QUANTITIES FOR LINE ============================ G FOR FIDUCIAL LIMITS = .1538 EXPECTED DOSE STANDARD FIDUCIAL LIMITS % RESPONSE ERROR UPPER LOWER 50.0 4.135 .1857 4.584 3.786 90.0 5.989 .4615 7.433 5.320 95.0 6.515 .5601 8.285 5.710 99.0 7.501 .7499 9.892 6.433 99.9 8.606 .9662 11.701 7.237 QUANTITIES TRANSFORMED BACK TO ORIGINAL ======================================= EXPECTED DOSE STANDARD FIDUCIAL LIMITS % RESPONSE ERROR UPPER LOWER 50.0 .1364701E+05 .5827868E+04 .3835363E+05 .6113390E+04 90.0 .9758868E+06 .1035948E+07 .2712654E+08 .2087630E+06 95.0 .3273877E+07 .4217638E+07 .1928066E+09 .5133082E+06 99.0 .3169480E+08 .5466388E+08 .7804815E+10 .2713104E+07 99.9 .4039096E+09 .8975832E+09 .5027501E+12 .1725628E+08

174

****************************************************************************** rep 2. Dose-response bioassays with 1st instar FCM larvae (See section: 4.3.2.1) ****************************************************************************** GENERAL INPUT INFORMATION ========================= DOSES TRANSFORMED TO LOG10 NATURAL MORTALITY = 13% NON-RESPONSE = 0% DOSE TRANSFORMED NUMBER NUMBER %RESPONSE CORRECTED EMPIRICAL DOSE(X) EXPOSED RESPON. %RESPONSE PROBITS 100000.00000000 5.0000 30 24 80.00 77.01 5.739 20000.00000000 4.3010 30 16 53.33 46.36 4.909 4000.00000000 3.6021 30 13 43.33 34.87 4.611 800.00000000 2.9031 30 10 33.33 23.37 4.273 160.00000000 2.2041 30 8 26.67 15.71 3.994 TEST FOR FIT OF LINE ==================== CHI-SQUARED FOR DEVIATIONS = 1.343 D. F. = 3 APPROXIMATE PROBABILITY = .7230 TEST LEVEL = 0,05 TABLED CHI-SQUARED VALUE = 7.815 DEVIATIONS FROM LINE ARE HOMOGENEOUS FITTED PROBIT LINE ================== REGRESSION EQUATION IS Y = a + bX WHERE Y IS EMPIRICAL PROBITS AND X IS DOSE INTERCEPT (a) = 2.4603 REGRESSION COEFFICIENT (b) = .6156 STANDARD ERROR OF b = .1500 MEAN FOR EMPIRICAL PROBITS (Y) = 4.8580 MEAN FOR DOSE (X) = 3.8946 DOSE (X) EXPECTED EMP.PROBIT (Y) 5.00000 5.539 4.30103 5.108 3.60206 4.678 2.90309 4.248 2.20412 3.817

175

EXPECTED QUANTITIES FOR LINE ============================ G FOR FIDUCIAL LIMITS = .2281 EXPECTED DOSE STANDARD FIDUCIAL LIMITS % RESPONSE ERROR UPPER LOWER 50.0 4.125 .2228 4.695 3.692 90.0 6.207 .6033 8.400 5.381 95.0 6.797 .7394 9.514 5.796 99.0 7.904 1.0005 11.616 6.562 99.9 9.145 1.2974 13.980 7.412 QUANTITIES TRANSFORMED BACK TO ORIGINAL ======================================= EXPECTED DOSE STANDARD FIDUCIAL LIMITS % RESPONSE ERROR UPPER LOWER 50.0 .1334230E+05 .6837922E+04 .4956098E+05 .4916217E+04 90.0 .1610440E+07 .2234719E+07 .2510991E+09 .2403365E+06 95.0 .6266936E+07 .1065790E+08 .3266739E+10 .6245641E+06 99.0 .8014190E+08 .1844185E+09 .4128518E+12 .3645196E+07 99.9 .1395306E+10 .4163615E+10 .9556881E+14 .2583691E+08

176

****************************************************************************** rep 3. Dose-response bioassays with 1st instar FCM larvae (See section: 4.3.2.1) ****************************************************************************** GENERAL INPUT INFORMATION ========================= DOSES TRANSFORMED TO LOG10 NATURAL MORTALITY = 13% NON-RESPONSE = 0% DOSE TRANSFORMED NUMBER NUMBER %RESPONSE CORRECTED EMPIRICAL DOSE(X) EXPOSED RESPON. %RESPONSE PROBITS 100000.00000000 5.0000 30 23 76.67 73.18 5.618 20000.00000000 4.3010 30 15 50.00 42.53 4.812 4000.00000000 3.6021 30 12 40.00 31.03 4.505 800.00000000 2.9031 30 10 33.33 23.37 4.273 160.00000000 2.2041 30 6 20.00 8.05 3.598 TEST FOR FIT OF LINE ==================== CHI-SQUARED FOR DEVIATIONS = 1.199 D. F. = 3 APPROXIMATE PROBABILITY = .7570 TEST LEVEL = 0,05 TABLED CHI-SQUARED VALUE = 7.815 DEVIATIONS FROM LINE ARE HOMOGENEOUS FITTED PROBIT LINE ================== REGRESSION EQUATION IS Y = a + bX WHERE Y IS EMPIRICAL PROBITS AND X IS DOSE INTERCEPT (a) = 2.1861 REGRESSION COEFFICIENT (b) = .6567 STANDARD ERROR OF b = .1624 MEAN FOR EMPIRICAL PROBITS (Y) = 4.8057 MEAN FOR DOSE (X) = 3.9886 DOSE (X) EXPECTED EMP.PROBIT (Y) 5.00000 5.470 4.30103 5.011 3.60206 4.552 2.90309 4.093 2.20412 3.634

177

EXPECTED QUANTITIES FOR LINE ============================ G FOR FIDUCIAL LIMITS = .2348 EXPECTED DOSE STANDARD FIDUCIAL LIMITS % RESPONSE ERROR UPPER LOWER 50.0 4.285 .2225 4.882 3.869 90.0 6.236 .5940 8.424 5.427 95.0 6.789 .7235 9.483 5.814 99.0 7.827 .9718 11.480 6.529 99.9 8.990 1.2541 13.726 7.323 QUANTITIES TRANSFORMED BACK TO ORIGINAL ======================================= EXPECTED DOSE STANDARD FIDUCIAL LIMITS % RESPONSE ERROR UPPER LOWER 50.0 .1925411E+05 .9854223E+04 .7622664E+05 .7388220E+04 90.0 .1721693E+07 .2352216E+07 .2657602E+09 .2670238E+06 95.0 .6153688E+07 .1024064E+08 .3042740E+10 .6510322E+06 99.0 .6709213E+08 .1499673E+09 .3019232E+12 .3378669E+07 99.9 .9768484E+09 .2817742E+10 .5322459E+14 .2101987E+08

178

****************************************************************************** rep 4. Dose-response bioassays with 1st instar FCM larvae (See section: 4.3.2.1) ****************************************************************************** GENERAL INPUT INFORMATION ========================= DOSES TRANSFORMED TO LOG10 NATURAL MORTALITY = 17% NON-RESPONSE = 0% DOSE TRANSFORMED NUMBER NUMBER %RESPONSE CORRECTED EMPIRICAL DOSE(X) EXPOSED RESPON. %RESPONSE PROBITS 100000.00000000 5.0000 30 25 83.33 79.92 5.839 20000.00000000 4.3010 30 19 63.33 55.82 5.146 4000.00000000 3.6021 30 15 50.00 39.76 4.740 800.00000000 2.9031 30 11 36.67 23.69 4.284 160.00000000 2.2041 30 9 30.00 15.66 3.991 TEST FOR FIT OF LINE ==================== CHI-SQUARED FOR DEVIATIONS = .469 D. F. = 3 APPROXIMATE PROBABILITY = .9230 TEST LEVEL = 0,05 TABLED CHI-SQUARED VALUE = 7.815 DEVIATIONS FROM LINE ARE HOMOGENEOUS FITTED PROBIT LINE ================== REGRESSION EQUATION IS Y = a + bX WHERE Y IS EMPIRICAL PROBITS AND X IS DOSE INTERCEPT (a) = 2.3529 REGRESSION COEFFICIENT (b) = .6734 STANDARD ERROR OF b = .1591 MEAN FOR EMPIRICAL PROBITS (Y) = 4.9716 MEAN FOR DOSE (X) = 3.8888 DOSE (X) EXPECTED EMP.PROBIT (Y) 5.00000 5.720 4.30103 5.249 3.60206 4.778 2.90309 4.308 2.20412 3.837

179

EXPECTED QUANTITIES FOR LINE ============================ G FOR FIDUCIAL LIMITS = .2145 EXPECTED DOSE STANDARD FIDUCIAL LIMITS % RESPONSE ERROR UPPER LOWER 50.0 3.931 .2061 4.398 3.487 90.0 5.834 .5037 7.600 5.131 95.0 6.374 .6222 8.587 5.518 99.0 7.386 .8515 10.452 6.229 99.9 8.520 1.1135 12.553 7.016 QUANTITIES TRANSFORMED BACK TO ORIGINAL ======================================= EXPECTED DOSE STANDARD FIDUCIAL LIMITS % RESPONSE ERROR UPPER LOWER 50.0 .8531357E+04 .4043172E+04 .2502519E+05 .3067083E+04 90.0 .6826911E+06 .7908599E+06 .3976938E+08 .1353365E+06 95.0 .2364466E+07 .3383758E+07 .3859726E+09 .3296710E+06 99.0 .2430123E+08 .4759512E+08 .2831284E+11 .1694714E+07 99.9 .3311598E+09 .8481476E+09 .3573360E+13 .1038453E+08

180

*************************** * COMPARISON OF LINES * *************************** ABRIDGED ANALYSIS OF VARIANCE ============================= DEVIATIONS FROM REGRESSION INTERCEPT REGR.COEF. D.F. SUM OF RESIDUAL (a) (b) SQUARES VARIANCES LINE: rep 1 2.142 .691 3 .956200E+00 .319 LINE: rep 2 2.460 .616 3 .134252E+01 .448 LINE: rep 3 2.186 .657 3 .119867E+01 .400 LINE: rep 4 2.353 .673 3 .469053E+00 .156 ---------------------------------------------------------------------- 12 .396644E+01 .331 ---------------------------------------------------------------------- COMBINED .660 15 .411196E+01 .274 ---------------------------------------------------------------------- DIFFERENCE BETWEEN SLOPES 3 .145521E+00 .049 ---------------------------------------------------------------------- TOTAL 2.291 .657 18 .554750E+01 .308 ---------------------------------------------------------------------- DIFFERENCE BETWEEN ADJ. MEANS 3 .143553E+01 .479 ---------------------------------------------------------------------- BARTLETTS TEST FOR HOMOGENEITY OF RESIDUAL VARIANCES ==================================================== CHI-SQUARED VALUE = .329 D. F. = 3 APPROXIMATE PROBABILITY = .9500 TEST LEVEL = .0100 TABLED CHI-SQUARED VALUE = 11.345 RESIDUAL VARIANCES ARE HOMOGENEOUS - SLOPES OF LINES MAY BE COMPARED COMPARISON OF SLOPES (PARALLELISM) ================================== SOURCE D.F. SUM OF SQ. MEAN SQ. F-VALUE --------------------------------------------------------- PARALLELISM 3 .145521E+00 .049 .15 HETEROGENEITY 12 .396644E+01 .331 -------------------------------- TOTAL 15 .4112E+01 CHI-SQUARED TEST FOR PARALLELISM -------------------------------- CHI-SQUARED VALUE = .146 D. F. = 3 APPROXIMATE PROBABILITY = .9800 TEST LEVEL = 0,05 TABLED CHI-SQUARED VALUE = 7.815

181

LINES ARE PARALLEL AND ELEVATIONS MAY BE COMPARED REGRESSION COEFFICIENTS ----------------------- rep 2 .6156 rep 3 .6567 rep 4 .6734 rep 1 .6911 COMPARISON OF ELEVATIONS (ADJUSTED MEANS) ========================================= F-VALUE FOR ELEVATIONS = 1.746 D.F. = 3 & 15 APPROXIMATE PROBABILITY = .2010 TEST LEVEL = 0,05 TABLED F-VALUE = 3.29 ELEVATIONS ARE NOT SIGNIFICANTLY DIFFERENT MEANS OF EMPIRICAL PROBITS -------------------------- rep 3 4.8057 rep 1 4.8099 rep 2 4.8580 rep 4 4.9716 INTERCEPTS OF LINES ------------------- rep 1 2.142 rep 3 2.186 rep 4 2.353 rep 2 2.460

182

4.2 DOSE-RESPONSE BIOASSAYS WITH CRYPTOGRAN AGAINST 1st INSTAR FCM LARVAE FROM THE OLD COLONY ****************************************************************************** rep 1. Dose-response bioassays with 1st instar FCM larvae (See section: 4.3.2.2) ****************************************************************************** GENERAL INPUT INFORMATION ========================= DOSES TRANSFORMED TO LOG10 NATURAL MORTALITY = 5% NON-RESPONSE = 0% DOSE TRANSFORMED NUMBER NUMBER %RESPONSE CORRECTED EMPIRICAL DOSE(X) EXPOSED RESPON. %RESPONSE PROBITS 100000.00000000 5.0000 30 29 96.67 96.49 6.811 20000.00000000 4.3010 30 23 76.67 75.44 5.688 4000.00000000 3.6021 30 16 53.33 50.88 5.022 800.00000000 2.9031 30 11 36.67 33.33 4.569 160.00000000 2.2041 30 8 26.67 22.81 4.255 TEST FOR FIT OF LINE ==================== CHI-SQUARED FOR DEVIATIONS = 2.413 D. F. = 3 APPROXIMATE PROBABILITY = .4940 TEST LEVEL = 0,05 TABLED CHI-SQUARED VALUE = 7.815 DEVIATIONS FROM LINE ARE HOMOGENEOUS FITTED PROBIT LINE ================== REGRESSION EQUATION IS Y = a + bX WHERE Y IS EMPIRICAL PROBITS AND X IS DOSE INTERCEPT (a) = 2.1949 REGRESSION COEFFICIENT (b) = .8358 STANDARD ERROR OF b = .1386 MEAN FOR EMPIRICAL PROBITS (Y) = 5.1649 MEAN FOR DOSE (X) = 3.5537 DOSE (X) EXPECTED EMP.PROBIT (Y) 5.00000 6.374 4.30103 5.789 3.60206 5.205 2.90309 4.621

183

2.20412 4.037 EXPECTED QUANTITIES FOR LINE ============================ G FOR FIDUCIAL LIMITS = .1057 EXPECTED DOSE STANDARD FIDUCIAL LIMITS % RESPONSE ERROR UPPER LOWER 50.0 3.356 .1504 3.646 3.020 90.0 4.890 .2659 5.621 4.475 95.0 5.325 .3284 6.246 4.822 99.0 6.140 .4535 7.434 5.457 99.9 7.054 .5989 8.776 6.159 QUANTITIES TRANSFORMED BACK TO ORIGINAL ======================================= EXPECTED DOSE STANDARD FIDUCIAL LIMITS % RESPONSE ERROR UPPER LOWER 50.0 .2271951E+04 .7859964E+03 .4422866E+04 .1048217E+04 90.0 .7760031E+05 .4745366E+05 .4179128E+06 .2982479E+05 95.0 .2111352E+06 .1594848E+06 .1762302E+07 .6633738E+05 99.0 .1379978E+07 .1439254E+07 .2716114E+08 .2866086E+06 99.9 .1132130E+08 .1559573E+08 .5973448E+09 .1442703E+07

184

****************************************************************************** rep 2. Dose-response bioassays with 1st instar FCM larvae (See section: 4.3.2.2) ****************************************************************************** GENERAL INPUT INFORMATION ========================= DOSES TRANSFORMED TO LOG10 NATURAL MORTALITY = 17% NON-RESPONSE = 0% DOSE TRANSFORMED NUMBER NUMBER %RESPONSE CORRECTED EMPIRICAL DOSE(X) EXPOSED RESPON. %RESPONSE PROBITS 100000.00000000 5.0000 30 26 86.67 83.94 5.992 20000.00000000 4.3010 30 17 56.67 47.79 4.945 4000.00000000 3.6021 30 14 46.67 35.74 4.635 800.00000000 2.9031 30 12 40.00 27.71 4.408 160.00000000 2.2041 30 9 30.00 15.66 3.991 TEST FOR FIT OF LINE ==================== CHI-SQUARED FOR DEVIATIONS = 2.480 D. F. = 3 APPROXIMATE PROBABILITY = .4820 TEST LEVEL = 0,05 TABLED CHI-SQUARED VALUE = 7.815 DEVIATIONS FROM LINE ARE HOMOGENEOUS FITTED PROBIT LINE ================== REGRESSION EQUATION IS Y = a + bX WHERE Y IS EMPIRICAL PROBITS AND X IS DOSE INTERCEPT (a) = 2.3220 REGRESSION COEFFICIENT (b) = .6744 STANDARD ERROR OF b = .1606 MEAN FOR EMPIRICAL PROBITS (Y) = 4.9566 MEAN FOR DOSE (X) = 3.9066 DOSE (X) EXPECTED EMP.PROBIT (Y) 5.00000 5.694 4.30103 5.223 3.60206 4.751 2.90309 4.280 2.20412 3.808

185

EXPECTED QUANTITIES FOR LINE ============================ G FOR FIDUCIAL LIMITS = .2180 EXPECTED DOSE STANDARD FIDUCIAL LIMITS % RESPONSE ERROR UPPER LOWER 50.0 3.971 .2072 4.448 3.529 90.0 5.871 .5116 7.678 5.160 95.0 6.410 .6311 8.671 5.545 99.0 7.420 .8621 10.547 6.253 99.9 8.553 1.1259 12.660 7.037 QUANTITIES TRANSFORMED BACK TO ORIGINAL ======================================= EXPECTED DOSE STANDARD FIDUCIAL LIMITS % RESPONSE ERROR UPPER LOWER 50.0 .9352659E+04 .4456680E+04 .2808136E+05 .3383235E+04 90.0 .7434701E+06 .8747863E+06 .4765047E+08 .1444500E+06 95.0 .2570138E+07 .3730609E+07 .4686852E+09 .3504248E+06 99.0 .2632221E+08 .5219335E+08 .3522865E+11 .1788799E+07 99.9 .3572866E+09 .9252370E+09 .4567640E+13 .1087921E+08

186

****************************************************************************** rep 3. Dose-response bioassays with 1st instar FCM larvae (See section: 4.3.2.2) ****************************************************************************** GENERAL INPUT INFORMATION ========================= DOSES TRANSFORMED TO LOG10 NATURAL MORTALITY = 13% NON-RESPONSE = 0% DOSE TRANSFORMED NUMBER NUMBER %RESPONSE CORRECTED EMPIRICAL DOSE(X) EXPOSED RESPON. %RESPONSE PROBITS 100000.00000000 5.0000 30 28 93.33 92.34 6.428 20000.00000000 4.3010 30 22 73.33 69.35 5.506 4000.00000000 3.6021 30 18 60.00 54.02 5.101 800.00000000 2.9031 30 10 33.33 23.37 4.273 160.00000000 2.2041 30 5 16.67 4.21 3.273 TEST FOR FIT OF LINE ==================== CHI-SQUARED FOR DEVIATIONS = .834 D. F. = 3 APPROXIMATE PROBABILITY = .8420 TEST LEVEL = 0,05 TABLED CHI-SQUARED VALUE = 7.815 DEVIATIONS FROM LINE ARE HOMOGENEOUS FITTED PROBIT LINE ================== REGRESSION EQUATION IS Y = a + bX WHERE Y IS EMPIRICAL PROBITS AND X IS DOSE INTERCEPT (a) = 1.3590 REGRESSION COEFFICIENT (b) = .9987 STANDARD ERROR OF b = .1803 MEAN FOR EMPIRICAL PROBITS (Y) = 5.1671 MEAN FOR DOSE (X) = 3.8132 DOSE (X) EXPECTED EMP.PROBIT (Y) 5.00000 6.352 4.30103 5.654 3.60206 4.956 2.90309 4.258 2.20412 3.560

187

EXPECTED QUANTITIES FOR LINE ============================ G FOR FIDUCIAL LIMITS = .1253 EXPECTED DOSE STANDARD FIDUCIAL LIMITS % RESPONSE ERROR UPPER LOWER 50.0 3.646 .1464 3.930 3.314 90.0 4.929 .2472 5.631 4.547 95.0 5.293 .3032 6.175 4.835 99.0 5.975 .4158 7.210 5.360 99.9 6.740 .5476 8.381 5.938 QUANTITIES TRANSFORMED BACK TO ORIGINAL ======================================= EXPECTED DOSE STANDARD FIDUCIAL LIMITS % RESPONSE ERROR UPPER LOWER 50.0 .4424809E+04 .1489631E+04 .8503718E+04 .2061855E+04 90.0 .8496233E+05 .4831057E+05 .4277568E+06 .3522517E+05 95.0 .1963439E+06 .1369060E+06 .1494960E+07 .6842023E+05 99.0 .9447969E+06 .9036499E+06 .1620879E+08 .2291437E+06 99.9 .5498840E+07 .6925548E+07 .2404815E+09 .8663676E+06

188

****************************************************************************** rep 4. Dose-response bioassays with 1st instar FCM larvae (See section: 4.3.2.2) ****************************************************************************** GENERAL INPUT INFORMATION ========================= DOSES TRANSFORMED TO LOG10 NATURAL MORTALITY = 7% NON-RESPONSE = 0% DOSE TRANSFORMED NUMBER NUMBER %RESPONSE CORRECTED EMPIRICAL DOSE(X) EXPOSED RESPON. %RESPONSE PROBITS 100000.00000000 5.0000 30 28 93.33 92.83 6.463 20000.00000000 4.3010 30 21 70.00 67.74 5.460 4000.00000000 3.6021 30 19 63.33 60.57 5.268 800.00000000 2.9031 30 14 46.67 42.65 4.815 160.00000000 2.2041 30 9 30.00 24.73 4.317 TEST FOR FIT OF LINE ==================== CHI-SQUARED FOR DEVIATIONS = 1.703 D. F. = 3 APPROXIMATE PROBABILITY = .6400 TEST LEVEL = 0,05 TABLED CHI-SQUARED VALUE = 7.815 DEVIATIONS FROM LINE ARE HOMOGENEOUS FITTED PROBIT LINE ================== REGRESSION EQUATION IS Y = a + bX WHERE Y IS EMPIRICAL PROBITS AND X IS DOSE INTERCEPT (a) = 2.8155 REGRESSION COEFFICIENT (b) = .6733 STANDARD ERROR OF b = .1304 MEAN FOR EMPIRICAL PROBITS (Y) = 5.2065 MEAN FOR DOSE (X) = 3.5512 DOSE (X) EXPECTED EMP.PROBIT (Y) 5.00000 6.182 4.30103 5.711 3.60206 5.241 2.90309 4.770 2.20412 4.300

189

EXPECTED QUANTITIES FOR LINE ============================ G FOR FIDUCIAL LIMITS = .1441 EXPECTED DOSE STANDARD FIDUCIAL LIMITS % RESPONSE ERROR UPPER LOWER 50.0 3.245 .1877 3.594 2.792 90.0 5.148 .3569 6.219 4.614 95.0 5.688 .4505 7.067 5.027 99.0 6.700 .6353 8.676 5.783 99.9 7.834 .8484 10.492 6.619 QUANTITIES TRANSFORMED BACK TO ORIGINAL ======================================= EXPECTED DOSE STANDARD FIDUCIAL LIMITS % RESPONSE ERROR UPPER LOWER 50.0 .1756077E+04 .7582109E+03 .3926060E+04 .6192524E+03 90.0 .1406260E+06 .1154289E+06 .1657186E+07 .4115868E+05 95.0 .4871509E+06 .5047334E+06 .1167521E+08 .1065271E+06 99.0 .5008724E+07 .7318301E+07 .4745833E+09 .6071935E+06 99.9 .6828500E+08 .1332521E+09 .3105880E+11 .4156230E+07 *************************** * COMPARISON OF LINES * *************************** ABRIDGED ANALYSIS OF VARIANCE ============================= DEVIATIONS FROM REGRESSION INTERCEPT REGR.COEF. D.F. SUM OF RESIDUAL (a) (b) SQUARES VARIANCES LINE: rep 1 2.195 .836 3 .241329E+01 .804 LINE: rep 2 2.322 .674 3 .247979E+01 .827 LINE: rep 3 1.359 .999 3 .833745E+00 .278 LINE: rep 4 2.816 .673 3 .170329E+01 .568 ---------------------------------------------------------------------- 12 .743011E+01 .619 ---------------------------------------------------------------------- COMBINED .776 15 .101609E+02 .677 ---------------------------------------------------------------------- DIFFERENCE BETWEEN SLOPES 3 .273075E+01 .910 ---------------------------------------------------------------------- TOTAL 2.417 .737 18 .197144E+02 1.095 ---------------------------------------------------------------------- DIFFERENCE BETWEEN ADJ. MEANS 3 .955355E+01 3.185 ----------------------------------------------------------------------

190

BARTLETTS TEST FOR HOMOGENEITY OF RESIDUAL VARIANCES ==================================================== CHI-SQUARED VALUE = .379 D. F. = 3 APPROXIMATE PROBABILITY = .9400 TEST LEVEL = .0100 TABLED CHI-SQUARED VALUE = 11.345 RESIDUAL VARIANCES ARE HOMOGENEOUS - SLOPES OF LINES MAY BE COMPARED COMPARISON OF SLOPES (PARALLELISM) ================================== SOURCE D.F. SUM OF SQ. MEAN SQ. F-VALUE --------------------------------------------------------- PARALLELISM 3 .273075E+01 .910 1.47 HETEROGENEITY 12 .743011E+01 .619 -------------------------------- TOTAL 15 .1016E+02 CHI-SQUARED TEST FOR PARALLELISM -------------------------------- CHI-SQUARED VALUE = 2.731 D. F. = 3 APPROXIMATE PROBABILITY = .4370 TEST LEVEL = 0,05 TABLED CHI-SQUARED VALUE = 7.815 LINES ARE PARALLEL AND ELEVATIONS MAY BE COMPARED REGRESSION COEFFICIENTS ----------------------- rep 4 .6733 rep 2 .6744 rep 1 .8358 rep 3 .9987 COMPARISON OF ELEVATIONS (ADJUSTED MEANS) ========================================= F-VALUE FOR ELEVATIONS = 4.701 D.F. = 3 & 15 APPROXIMATE PROBABILITY = .0170 TEST LEVEL = 0,05 TABLED F-VALUE = 3.29 ELEVATIONS ARE SIGNIFICANTLY DIFFERENT MEANS OF EMPIRICAL PROBITS -------------------------- rep 2 4.9566 rep 1 5.1649 rep 3 5.1671 rep 4 5.2065

191

INTERCEPTS OF LINES ------------------- rep 3 1.359 rep 1 2.195 rep 2 2.322 rep 4 2.816 MULTIPLE COMPARISON OF ELEVATIONS WITH BONFERRONI METHOD -------------------------------------------------------- NUMBER OF COMPARISONS = 6 TEST LEVEL = (0,05/ 6) = .00833 * = SIGNIFICANTLY DIFFERENT LINE: rep 1 rep 3 rep 4 rep 2 rep 1 rep 3 RELATIVE POTENCY COMPARISONS ============================ LD-50 LD-50 LINE REGRESSION EQUATION TRANSFORMED ORIGINAL ---- ------------------- ----------- -------- rep 1 Y = 2.4077 + .7759X 3.3412 .2193691E+04 rep 2 Y = 1.9256 + .7759X 3.9625 .9173134E+04 rep 3 Y = 2.2085 + .7759X 3.5978 .3961395E+04 rep 4 Y = 2.4512 + .7759X 3.2851 .1927954E+04 LINE: rep 1 rep 2 rep 3 rep 4 rep 1 1.000 .239 .554 1.138 rep 2 4.182 1.000 2.316 4.758 rep 3 1.806 .432 1.000 2.055 rep 4 .879 .210 .487 1.000

192

Appendix 5 PROBAN (VAN ARK, 1995) OUPUT OF PROBIT ANALYSIS OF SURFACE DOSE-RESPONSE BIOASSAY DATA (LC) ON AN AGAR DIET, WITH CRYPTOGRAN AND CRYPTEX AGAINST THE ADDO FCM COLONY 5.1 DOSE-RESPONSE BIOASSAYS WITH CRYPTEX AGAINST 1st INSTAR FCM LARVAE FROM THE ADDO ****************************************************************************** rep 1. Dose-response bioassays with 1st instar FCM larvae (See section: 4.3.2.3) ****************************************************************************** GENERAL INPUT INFORMATION ========================= DOSES TRANSFORMED TO LOG10 NATURAL MORTALITY = 10% NON-RESPONSE = 0% DOSE TRANSFORMED NUMBER NUMBER %RESPONSE CORRECTED EMPIRICAL DOSE(X) EXPOSED RESPON. %RESPONSE PROBITS 100000.00000000 5.0000 30 27 90.00 88.89 6.221 20000.00000000 4.3010 30 23 76.67 74.07 5.645 4000.00000000 3.6021 30 22 73.33 70.37 5.535 800.00000000 2.9031 30 11 36.67 29.63 4.465 160.00000000 2.2041 30 5 16.67 7.41 3.554 TEST FOR FIT OF LINE ==================== CHI-SQUARED FOR DEVIATIONS = 3.245 D. F. = 3 APPROXIMATE PROBABILITY = .3560 TEST LEVEL = 0,05 TABLED CHI-SQUARED VALUE = 7.815 DEVIATIONS FROM LINE ARE HOMOGENEOUS FITTED PROBIT LINE ================== REGRESSION EQUATION IS Y = a + bX WHERE Y IS EMPIRICAL PROBITS AND X IS DOSE INTERCEPT (a) = 1.9592 REGRESSION COEFFICIENT (b) = .8836 STANDARD ERROR OF b = .1543 MEAN FOR EMPIRICAL PROBITS (Y) = 5.1811

193

MEAN FOR DOSE (X) = 3.6464 DOSE (X) EXPECTED EMP.PROBIT (Y) 5.00000 6.377 4.30103 5.760 3.60206 5.142 2.90309 4.524 2.20412 3.907 EXPECTED QUANTITIES FOR LINE ============================ G FOR FIDUCIAL LIMITS = .1171 EXPECTED DOSE STANDARD FIDUCIAL LIMITS % RESPONSE ERROR UPPER LOWER 50.0 3.441 .1539 3.736 3.092 90.0 4.892 .2640 5.632 4.482 95.0 5.303 .3257 6.237 4.809 99.0 6.074 .4496 7.388 5.405 99.9 6.939 .5940 8.689 6.062 QUANTITIES TRANSFORMED BACK TO ORIGINAL ======================================= EXPECTED DOSE STANDARD FIDUCIAL LIMITS % RESPONSE ERROR UPPER LOWER 50.0 .2763095E+04 .9779551E+03 .5449363E+04 .1236079E+04 90.0 .7794898E+05 .4732923E+05 .4285316E+06 .3034653E+05 95.0 .2008932E+06 .1504856E+06 .1725023E+07 .6437345E+05 99.0 .1186099E+07 .1226425E+07 .2442165E+08 .2539022E+06 99.9 .8682473E+07 .1186287E+08 .4890325E+09 .1152265E+07

194

****************************************************************************** rep 2.Dose-response bioassays with 1st instar FCM larvae (See section: 4.3.2.3) ****************************************************************************** GENERAL INPUT INFORMATION ========================= DOSES TRANSFORMED TO LOG10 NATURAL MORTALITY = 13% NON-RESPONSE = 0% DOSE TRANSFORMED NUMBER NUMBER %RESPONSE CORRECTED EMPIRICAL DOSE(X) EXPOSED RESPON. %RESPONSE PROBITS 100000.00000000 5.0000 30 28 93.33 92.34 6.428 20000.00000000 4.3010 30 17 56.67 50.19 5.005 4000.00000000 3.6021 30 13 43.33 34.87 4.611 800.00000000 2.9031 30 10 33.33 23.37 4.273 160.00000000 2.2041 30 9 30.00 19.54 4.142 TEST FOR FIT OF LINE ==================== CHI-SQUARED FOR DEVIATIONS = 5.626 D. F. = 3 APPROXIMATE PROBABILITY = .1290 TEST LEVEL = 0,05 TABLED CHI-SQUARED VALUE = 7.815 DEVIATIONS FROM LINE ARE HOMOGENEOUS FITTED PROBIT LINE ================== REGRESSION EQUATION IS Y = a + bX WHERE Y IS EMPIRICAL PROBITS AND X IS DOSE INTERCEPT (a) = 1.9754 REGRESSION COEFFICIENT (b) = .7825 STANDARD ERROR OF b = .1600 MEAN FOR EMPIRICAL PROBITS (Y) = 5.0097 MEAN FOR DOSE (X) = 3.8778 DOSE (X) EXPECTED EMP.PROBIT (Y) 5.00000 5.888 4.30103 5.341 3.60206 4.794 2.90309 4.247 2.20412 3.700

195

EXPECTED QUANTITIES FOR LINE ============================ G FOR FIDUCIAL LIMITS = .1605 EXPECTED DOSE STANDARD FIDUCIAL LIMITS % RESPONSE ERROR UPPER LOWER 50.0 3.865 .1737 4.235 3.491 90.0 5.503 .3749 6.674 4.954 95.0 5.968 .4611 7.431 5.303 99.0 6.838 .6296 8.865 5.943 99.9 7.815 .8233 10.483 6.652 QUANTITIES TRANSFORMED BACK TO ORIGINAL ======================================= EXPECTED DOSE STANDARD FIDUCIAL LIMITS % RESPONSE ERROR UPPER LOWER 50.0 .7334391E+04 .2930758E+04 .1716544E+05 .3099778E+04 90.0 .3185822E+06 .2747318E+06 .4723024E+07 .8991885E+05 95.0 .9279214E+06 .9841853E+06 .2700515E+08 .2007999E+06 99.0 .6891694E+07 .9980042E+07 .7335873E+09 .8778415E+06 99.9 .6524906E+08 .1235514E+09 .3038427E+11 .4488129E+07

196

****************************************************************************** rep 3. Dose-response bioassays with 1st instar FCM larvae (See section: 4.3.2.3) ****************************************************************************** GENERAL INPUT INFORMATION ========================= DOSES TRANSFORMED TO LOG10 NATURAL MORTALITY = 13% NON-RESPONSE = 0% DOSE TRANSFORMED NUMBER NUMBER %RESPONSE CORRECTED EMPIRICAL DOSE(X) EXPOSED RESPON. %RESPONSE PROBITS 100000.00000000 5.0000 30 20 66.67 61.69 5.297 20000.00000000 4.3010 30 17 56.67 50.19 5.005 4000.00000000 3.6021 30 15 50.00 42.53 4.812 800.00000000 2.9031 30 12 40.00 31.03 4.505 160.00000000 2.2041 30 8 26.67 15.71 3.994 TEST FOR FIT OF LINE ==================== CHI-SQUARED FOR DEVIATIONS = .327 D. F. = 3 APPROXIMATE PROBABILITY = .9500 TEST LEVEL = 0,05 TABLED CHI-SQUARED VALUE = 7.815 DEVIATIONS FROM LINE ARE HOMOGENEOUS FITTED PROBIT LINE ================== REGRESSION EQUATION IS Y = a + bX WHERE Y IS EMPIRICAL PROBITS AND X IS DOSE INTERCEPT (a) = 3.1899 REGRESSION COEFFICIENT (b) = .4280 STANDARD ERROR OF b = .1346 MEAN FOR EMPIRICAL PROBITS (Y) = 4.8128 MEAN FOR DOSE (X) = 3.7921 DOSE (X) EXPECTED EMP.PROBIT (Y) 5.00000 5.330 4.30103 5.031 3.60206 4.731 2.90309 4.432 2.20412 4.133

197

EXPECTED QUANTITIES FOR LINE ============================ G FOR FIDUCIAL LIMITS = .3799 G IS RATHER LARGE: ACCURACY OF EXPERIMENTAL PROCEDURES MAY BE SUSPECT EXPECTED DOSE STANDARD FIDUCIAL LIMITS % RESPONSE ERROR UPPER LOWER 50.0 4.230 .3263 5.353 3.642 90.0 7.224 1.1191 12.817 5.837 95.0 8.073 1.3784 15.014 6.377 99.0 9.665 1.8705 19.147 7.379 99.9 11.450 2.4264 23.789 8.494 QUANTITIES TRANSFORMED BACK TO ORIGINAL ======================================= EXPECTED DOSE STANDARD FIDUCIAL LIMITS % RESPONSE ERROR UPPER LOWER 50.0 .1696555E+05 .1273365E+05 .2253584E+06 .4387948E+04 90.0 .1675719E+08 .4313242E+08 .6558304E+13 .6868367E+06 95.0 .1183317E+09 .3751423E+09 .1033238E+16 .2384397E+07 99.0 .4626857E+10 .1990525E+11 .1404081E+20 .2395612E+08 99.9 .2819960E+12 .1573710E+13 .6156438E+24 .3122148E+09

198

****************************************************************************** rep 4. Dose-response bioassays with 1st instar FCM larvae (See section: 4.3.2.3) ****************************************************************************** GENERAL INPUT INFORMATION ========================= DOSES TRANSFORMED TO LOG10 NATURAL MORTALITY = 8% NON-RESPONSE = 0% DOSE TRANSFORMED NUMBER NUMBER %RESPONSE CORRECTED EMPIRICAL DOSE(X) EXPOSED RESPON. %RESPONSE PROBITS 100000.00000000 5.0000 30 20 66.67 63.77 5.352 20000.00000000 4.3010 30 14 46.67 42.03 4.799 4000.00000000 3.6021 30 12 40.00 34.78 4.609 800.00000000 2.9031 30 10 33.33 27.54 4.403 160.00000000 2.2041 30 4 13.33 5.80 3.428 TEST FOR FIT OF LINE ==================== CHI-SQUARED FOR DEVIATIONS = 1.499 D. F. = 3 APPROXIMATE PROBABILITY = .6870 TEST LEVEL = 0,05 TABLED CHI-SQUARED VALUE = 7.815 DEVIATIONS FROM LINE ARE HOMOGENEOUS FITTED PROBIT LINE ================== REGRESSION EQUATION IS Y = a + bX WHERE Y IS EMPIRICAL PROBITS AND X IS DOSE INTERCEPT (a) = 2.5916 REGRESSION COEFFICIENT (b) = .5446 STANDARD ERROR OF b = .1393 MEAN FOR EMPIRICAL PROBITS (Y) = 4.7080 MEAN FOR DOSE (X) = 3.8860 DOSE (X) EXPECTED EMP.PROBIT (Y) 5.00000 5.315 4.30103 4.934 3.60206 4.553 2.90309 4.173 2.20412 3.792

199

EXPECTED QUANTITIES FOR LINE ============================ G FOR FIDUCIAL LIMITS = .2512 G IS RATHER LARGE: ACCURACY OF EXPERIMENTAL PROCEDURES MAY BE SUSPECT EXPECTED DOSE STANDARD FIDUCIAL LIMITS % RESPONSE ERROR UPPER LOWER 50.0 4.422 .2689 5.237 3.967 90.0 6.775 .7742 9.749 5.741 95.0 7.442 .9384 11.073 6.198 99.0 8.693 1.2510 13.567 7.046 99.9 10.096 1.6049 16.370 7.990 QUANTITIES TRANSFORMED BACK TO ORIGINAL ======================================= EXPECTED DOSE STANDARD FIDUCIAL LIMITS % RESPONSE ERROR UPPER LOWER 50.0 .2642931E+05 .1634583E+05 .1726096E+06 .9265744E+04 90.0 .5960221E+07 .1061381E+08 .5605044E+10 .5506338E+06 95.0 .2769017E+08 .5976630E+08 .1183777E+12 .1577407E+07 99.0 .4936980E+09 .1420521E+10 .3690898E+14 .1111606E+08 99.9 .1247605E+11 .4605195E+11 .2343266E+17 .9766628E+08

200

****************************************************************************** rep 5. Dose-response bioassays with 1st instar FCM larvae (See section: 4.3.2.3) ****************************************************************************** GENERAL INPUT INFORMATION ========================= DOSES TRANSFORMED TO LOG10 NATURAL MORTALITY = 5% NON-RESPONSE = 0% DOSE TRANSFORMED NUMBER NUMBER %RESPONSE CORRECTED EMPIRICAL DOSE(X) EXPOSED RESPON. %RESPONSE PROBITS 100000.00000000 5.0000 30 20 66.67 64.91 5.383 20000.00000000 4.3010 30 15 50.00 47.37 4.934 4000.00000000 3.6021 30 10 33.33 29.82 4.470 800.00000000 2.9031 30 6 20.00 15.79 3.997 160.00000000 2.2041 30 3 10.00 5.26 3.380 TEST FOR FIT OF LINE ==================== CHI-SQUARED FOR DEVIATIONS = .069 D. F. = 3 APPROXIMATE PROBABILITY = .9910 TEST LEVEL = 0,05 TABLED CHI-SQUARED VALUE = 7.815 DEVIATIONS FROM LINE ARE HOMOGENEOUS FITTED PROBIT LINE ================== REGRESSION EQUATION IS Y = a + bX WHERE Y IS EMPIRICAL PROBITS AND X IS DOSE INTERCEPT (a) = 1.9782 REGRESSION COEFFICIENT (b) = .6853 STANDARD ERROR OF b = .1473 MEAN FOR EMPIRICAL PROBITS (Y) = 4.6943 MEAN FOR DOSE (X) = 3.9635 DOSE (X) EXPECTED EMP.PROBIT (Y) 5.00000 5.405 4.30103 4.926 3.60206 4.447 2.90309 3.968 2.20412 3.489

201

EXPECTED QUANTITIES FOR LINE ============================ G FOR FIDUCIAL LIMITS = .1776 EXPECTED DOSE STANDARD FIDUCIAL LIMITS % RESPONSE ERROR UPPER LOWER 50.0 4.410 .2082 4.966 4.046 90.0 6.280 .5312 8.032 5.528 95.0 6.810 .6393 8.937 5.912 99.0 7.804 .8462 10.642 6.625 99.9 8.919 1.0814 12.560 7.419 QUANTITIES TRANSFORMED BACK TO ORIGINAL ======================================= EXPECTED DOSE STANDARD FIDUCIAL LIMITS % RESPONSE ERROR UPPER LOWER 50.0 .2567839E+05 .1229376E+05 .9246323E+05 .1111245E+05 90.0 .1904424E+07 .2326637E+07 .1076894E+09 .3370448E+06 95.0 .6455273E+07 .9491387E+07 .8644956E+09 .8172572E+06 99.0 .6371758E+08 .1240112E+09 .4382950E+11 .4221589E+07 99.9 .8298374E+09 .2063915E+10 .3626784E+13 .2621890E+08

202

****************************************************************************** rep 6. Dose-response bioassays with 1st instar FCM larvae (See section: 4.3.2.3) ****************************************************************************** GENERAL INPUT INFORMATION ========================= DOSES TRANSFORMED TO LOG10 NATURAL MORTALITY = 3% NON-RESPONSE = 0% DOSE TRANSFORMED NUMBER NUMBER %RESPONSE CORRECTED EMPIRICAL DOSE(X) EXPOSED RESPON. %RESPONSE PROBITS 100000.00000000 5.0000 30 22 73.33 72.51 5.598 20000.00000000 4.3010 30 15 50.00 48.45 4.961 4000.00000000 3.6021 30 12 40.00 38.14 4.698 800.00000000 2.9031 30 10 33.33 31.27 4.512 160.00000000 2.2041 30 8 26.67 24.40 4.306 TEST FOR FIT OF LINE ==================== CHI-SQUARED FOR DEVIATIONS = 1.295 D. F. = 3 APPROXIMATE PROBABILITY = .7340 TEST LEVEL = 0,05 TABLED CHI-SQUARED VALUE = 7.815 DEVIATIONS FROM LINE ARE HOMOGENEOUS FITTED PROBIT LINE ================== REGRESSION EQUATION IS Y = a + bX WHERE Y IS EMPIRICAL PROBITS AND X IS DOSE INTERCEPT (a) = 3.2346 REGRESSION COEFFICIENT (b) = .4365 STANDARD ERROR OF b = .1155 MEAN FOR EMPIRICAL PROBITS (Y) = 4.8441 MEAN FOR DOSE (X) = 3.6876 DOSE (X) EXPECTED EMP.PROBIT (Y) 5.00000 5.417 4.30103 5.112 3.60206 4.807 2.90309 4.502 2.20412 4.197

203

EXPECTED QUANTITIES FOR LINE ============================ G FOR FIDUCIAL LIMITS = .2688 G IS RATHER LARGE: ACCURACY OF EXPERIMENTAL PROCEDURES MAY BE SUSPECT EXPECTED DOSE STANDARD FIDUCIAL LIMITS % RESPONSE ERROR UPPER LOWER 50.0 4.045 .2706 4.810 3.542 90.0 6.981 .9073 10.598 5.785 95.0 7.813 1.1204 12.312 6.347 99.0 9.374 1.5255 15.539 7.391 99.9 11.125 1.9835 19.163 8.553 QUANTITIES TRANSFORMED BACK TO ORIGINAL ======================================= EXPECTED DOSE STANDARD FIDUCIAL LIMITS % RESPONSE ERROR UPPER LOWER 50.0 .1108373E+05 .6897826E+04 .6455088E+05 .3483532E+04 90.0 .9570962E+07 .1997247E+08 .3961645E+11 .6098868E+06 95.0 .6505948E+08 .1676503E+09 .2052497E+13 .2225743E+07 99.0 .2368454E+10 .8309954E+10 .3456808E+16 .2460977E+08 99.9 .1332397E+12 .6078378E+12 .1456610E+20 .3576460E+09

204

****************************************************************************** rep 7. Dose-response bioassays with 1st instar FCM larvae (See section: 4.3.2.3) ****************************************************************************** GENERAL INPUT INFORMATION ========================= DOSES TRANSFORMED TO LOG10 NATURAL MORTALITY = 3% NON-RESPONSE = 0% DOSE TRANSFORMED NUMBER NUMBER %RESPONSE CORRECTED EMPIRICAL DOSE(X) EXPOSED RESPON. %RESPONSE PROBITS 100000.00000000 5.0000 30 23 76.67 75.95 5.705 20000.00000000 4.3010 30 18 60.00 58.76 5.221 4000.00000000 3.6021 30 13 43.33 41.58 4.787 800.00000000 2.9031 30 12 40.00 38.14 4.698 160.00000000 2.2041 30 10 33.33 31.27 4.512 TEST FOR FIT OF LINE ==================== CHI-SQUARED FOR DEVIATIONS = 1.234 D. F. = 3 APPROXIMATE PROBABILITY = .7480 TEST LEVEL = 0,05 TABLED CHI-SQUARED VALUE = 7.815 DEVIATIONS FROM LINE ARE HOMOGENEOUS FITTED PROBIT LINE ================== REGRESSION EQUATION IS Y = a + bX WHERE Y IS EMPIRICAL PROBITS AND X IS DOSE INTERCEPT (a) = 3.4796 REGRESSION COEFFICIENT (b) = .4159 STANDARD ERROR OF b = .1131 MEAN FOR EMPIRICAL PROBITS (Y) = 4.9877 MEAN FOR DOSE (X) = 3.6256 DOSE (X) EXPECTED EMP.PROBIT (Y) 5.00000 5.559 4.30103 5.269 3.60206 4.978 2.90309 4.687 2.20412 4.396

205

EXPECTED QUANTITIES FOR LINE ============================ G FOR FIDUCIAL LIMITS = .2838 G IS RATHER LARGE: ACCURACY OF EXPERIMENTAL PROCEDURES MAY BE SUSPECT EXPECTED DOSE STANDARD FIDUCIAL LIMITS % RESPONSE ERROR UPPER LOWER 50.0 3.655 .2617 4.273 3.061 90.0 6.736 .8850 10.361 5.577 95.0 7.610 1.1140 12.213 6.164 99.0 9.248 1.5503 15.701 7.250 99.9 11.085 2.0441 19.621 8.459 QUANTITIES TRANSFORMED BACK TO ORIGINAL ======================================= EXPECTED DOSE STANDARD FIDUCIAL LIMITS % RESPONSE ERROR UPPER LOWER 50.0 .4521137E+04 .2720852E+04 .1875459E+05 .1150561E+04 90.0 .5450159E+07 .1109407E+08 .2294637E+11 .3777788E+06 95.0 .4072274E+08 .1043417E+09 .1633595E+13 .1458127E+07 99.0 .1770218E+10 .6312245E+10 .5026054E+16 .1779285E+08 99.9 .1214953E+12 .5711962E+12 .4175647E+20 .2878344E+09

206

*************************** * COMPARISON OF LINES * *************************** ABRIDGED ANALYSIS OF VARIANCE ============================= DEVIATIONS FROM REGRESSION INTERCEPT REGR.COEF. D.F. SUM OF RESIDUAL (a) (b) SQUARES VARIANCES LINE: rep 1 1.959 .884 3 .324480E+01 1.082 LINE: rep 2 1.975 .782 3 .562595E+01 1.875 LINE: rep 3 3.190 .428 3 .327372E+00 .109 LINE: rep 4 2.592 .545 3 .149887E+01 .500 LINE: rep 5 1.978 .685 3 .687376E-01 .023 LINE: rep 6 3.235 .436 3 .129454E+01 .432 LINE: rep 7 3.480 .416 3 .123441E+01 .411 ---------------------------------------------------------------------- 21 .132947E+02 .633 ---------------------------------------------------------------------- COMBINED .559 27 .240937E+02 .892 ---------------------------------------------------------------------- DIFFERENCE BETWEEN SLOPES 6 .107990E+02 1.800 ---------------------------------------------------------------------- TOTAL 2.877 .533 33 .442300E+02 1.340 ---------------------------------------------------------------------- DIFFERENCE BETWEEN ADJ. MEANS 6 .201363E+02 3.356 ---------------------------------------------------------------------- BARTLETTS TEST FOR HOMOGENEITY OF RESIDUAL VARIANCES ==================================================== CHI-SQUARED VALUE = 2.135 D. F. = 6 APPROXIMATE PROBABILITY = .9070 TEST LEVEL = .0100 TABLED CHI-SQUARED VALUE = 16.812 RESIDUAL VARIANCES ARE HOMOGENEOUS - SLOPES OF LINES MAY BE COMPARED COMPARISON OF SLOPES (PARALLELISM) ================================== SOURCE D.F. SUM OF SQ. MEAN SQ. F-VALUE --------------------------------------------------------- PARALLELISM 6 .107990E+02 1.800 2.84 HETEROGENEITY 21 .132947E+02 .633 -------------------------------- TOTAL 27 .2409E+02 CHI-SQUARED TEST FOR PARALLELISM -------------------------------- CHI-SQUARED VALUE = 10.799 D. F. = 6 APPROXIMATE PROBABILITY = .0940 TEST LEVEL = 0,05 TABLED CHI-SQUARED VALUE = 12.592

207

LINES ARE PARALLEL AND ELEVATIONS MAY BE COMPARED REGRESSION COEFFICIENTS ----------------------- rep 7 .4159 rep 3 .4280 rep 6 .4365 rep 4 .5446 rep 5 .6853 rep 2 .7825 rep 1 .8836 COMPARISON OF ELEVATIONS (ADJUSTED MEANS) ========================================= F-VALUE FOR ELEVATIONS = 3.761 D.F. = 6 & 27 APPROXIMATE PROBABILITY = .0080 TEST LEVEL = 0,05 TABLED F-VALUE = 2.46 ELEVATIONS ARE SIGNIFICANTLY DIFFERENT MEANS OF EMPIRICAL PROBITS -------------------------- rep 5 4.6943 rep 4 4.7080 rep 3 4.8128 rep 6 4.8441 rep 7 4.9877 rep 2 5.0097 rep 1 5.1811 INTERCEPTS OF LINES ------------------- rep 1 1.959 rep 2 1.975 rep 5 1.978 rep 4 2.592 rep 3 3.190 rep 6 3.235 rep 7 3.480 MULTIPLE COMPARISON OF ELEVATIONS WITH BONFERRONI METHOD -------------------------------------------------------- NUMBER OF COMPARISONS = 21 TEST LEVEL = (0,05/21) = .00238

208

* = SIGNIFICANTLY DIFFERENT LINE: rep 4 rep 3 rep 6 rep 7 rep 2 rep 1 rep 5 * rep 4 rep 3 rep 6 rep 7 rep 2 RELATIVE POTENCY COMPARISONS ============================ LD-50 LD-50 LINE REGRESSION EQUATION TRANSFORMED ORIGINAL ---- ------------------- ----------- -------- rep 1 Y = 3.1444 + .5586X 3.3221 .2099362E+04 rep 2 Y = 2.8438 + .5586X 3.8604 .7250899E+04 rep 3 Y = 2.6947 + .5586X 4.1273 .1340586E+05 rep 4 Y = 2.5375 + .5586X 4.4087 .2562826E+05 rep 5 Y = 2.4805 + .5586X 4.5108 .3241685E+05 rep 6 Y = 2.7844 + .5586X 3.9666 .9260420E+04 rep 7 Y = 2.9626 + .5586X 3.6477 .4442928E+04 LINE: rep 1 rep 2 rep 3 rep 4 rep 5 rep 6 rep 7 rep 1 1.000 .290 .157 .082 .065 .227 .473 rep 2 3.454 1.000 .541 .283 .224 .783 1.632 rep 3 6.386 1.849 1.000 .523 .414 1.448 3.017 rep 4 12.208 3.534 1.912 1.000 .791 2.768 5.768 rep 5 15.441 4.471 2.418 1.265 1.000 3.501 7.296 rep 6 4.411 1.277 .691 .361 .286 1.000 2.084 rep 7 2.116 .613 .331 .173 .137 .480 1.000

209

5.2 DOSE-RESPONSE BIOASSAYS WITH CRYPTOGRAN AGAINST 1st INSTAR FCM LARVAE FROM THE ADDO COLONY ****************************************************************************** rep 1. Dose-response bioassays with 1st instar FCM larvae (See section: 4.3.2.4) ****************************************************************************** GENERAL INPUT INFORMATION ========================= DOSES TRANSFORMED TO LOG10 NATURAL MORTALITY = 7% NON-RESPONSE = 0% DOSE TRANSFORMED NUMBER NUMBER %RESPONSE CORRECTED EMPIRICAL DOSE(X) EXPOSED RESPON. %RESPONSE PROBITS 100000.00000000 5.0000 30 25 83.33 82.08 5.918 20000.00000000 4.3010 30 16 53.33 49.82 4.996 4000.00000000 3.6021 30 14 46.67 42.65 4.815 800.00000000 2.9031 30 13 43.33 39.07 4.723 160.00000000 2.2041 30 5 16.67 10.39 3.740 TEST FOR FIT OF LINE ==================== CHI-SQUARED FOR DEVIATIONS = 3.833 D. F. = 3 APPROXIMATE PROBABILITY = .2790 TEST LEVEL = 0,05 TABLED CHI-SQUARED VALUE = 7.815 DEVIATIONS FROM LINE ARE HOMOGENEOUS FITTED PROBIT LINE ================== REGRESSION EQUATION IS Y = a + bX WHERE Y IS EMPIRICAL PROBITS AND X IS DOSE INTERCEPT (a) = 2.6256 REGRESSION COEFFICIENT (b) = .6167 STANDARD ERROR OF b = .1306 MEAN FOR EMPIRICAL PROBITS (Y) = 4.9362 MEAN FOR DOSE (X) = 3.7464

210

DOSE (X) EXPECTED EMP.PROBIT (Y) 5.00000 5.709 4.30103 5.278 3.60206 4.847 2.90309 4.416 2.20412 3.985 EXPECTED QUANTITIES FOR LINE ============================ G FOR FIDUCIAL LIMITS = .1724 EXPECTED DOSE STANDARD FIDUCIAL LIMITS % RESPONSE ERROR UPPER LOWER 50.0 3.850 .1959 4.294 3.449 90.0 5.928 .5014 7.554 5.210 95.0 6.517 .6183 8.546 5.642 99.0 7.622 .8437 10.418 6.440 99.9 8.861 1.1006 12.525 7.326 QUANTITIES TRANSFORMED BACK TO ORIGINAL ======================================= EXPECTED DOSE STANDARD FIDUCIAL LIMITS % RESPONSE ERROR UPPER LOWER 50.0 .7078438E+04 .3188932E+04 .1968120E+05 .2811542E+04 90.0 .8471958E+06 .9770384E+06 .3582914E+08 .1623239E+06 95.0 .3288937E+07 .4677236E+07 .3513733E+09 .4388835E+06 99.0 .4187069E+08 .8124682E+08 .2616765E+11 .2755796E+07 99.9 .7253268E+09 .1836099E+10 .3349622E+13 .2119162E+08

211

****************************************************************************** rep 2. Dose-response bioassays with 1st instar FCM larvae (See section: 4.3.2.4) ****************************************************************************** GENERAL INPUT INFORMATION ========================= DOSES TRANSFORMED TO LOG10 NATURAL MORTALITY = 17% NON-RESPONSE = 0% DOSE TRANSFORMED NUMBER NUMBER %RESPONSE CORRECTED EMPIRICAL DOSE(X) EXPOSED RESPON. %RESPONSE PROBITS 100000.00000000 5.0000 30 27 90.00 87.95 6.173 20000.00000000 4.3010 30 23 76.67 71.89 5.580 4000.00000000 3.6021 30 17 56.67 47.79 4.945 800.00000000 2.9031 30 14 46.67 35.74 4.635 160.00000000 2.2041 30 10 33.33 19.68 4.147 TEST FOR FIT OF LINE ==================== CHI-SQUARED FOR DEVIATIONS = .397 D. F. = 3 APPROXIMATE PROBABILITY = .9370 TEST LEVEL = 0,05 TABLED CHI-SQUARED VALUE = 7.815 DEVIATIONS FROM LINE ARE HOMOGENEOUS FITTED PROBIT LINE ================== REGRESSION EQUATION IS Y = a + bX WHERE Y IS EMPIRICAL PROBITS AND X IS DOSE INTERCEPT (a) = 2.4896 REGRESSION COEFFICIENT (b) = .7194 STANDARD ERROR OF b = .1520 MEAN FOR EMPIRICAL PROBITS (Y) = 5.1602 MEAN FOR DOSE (X) = 3.7124 DOSE (X) EXPECTED EMP.PROBIT (Y) 5.00000 6.086 4.30103 5.584 3.60206 5.081 2.90309 4.578 2.20412 4.075

212

EXPECTED QUANTITIES FOR LINE ============================ G FOR FIDUCIAL LIMITS = .1714 EXPECTED DOSE STANDARD FIDUCIAL LIMITS % RESPONSE ERROR UPPER LOWER 50.0 3.490 .1940 3.864 3.023 90.0 5.271 .3793 6.472 4.716 95.0 5.776 .4749 7.311 5.095 99.0 6.723 .6633 8.904 5.788 99.9 7.785 .8807 10.703 6.553 QUANTITIES TRANSFORMED BACK TO ORIGINAL ======================================= EXPECTED DOSE STANDARD FIDUCIAL LIMITS % RESPONSE ERROR UPPER LOWER 50.0 .3088093E+04 .1378235E+04 .7311091E+04 .1055058E+04 90.0 .1867287E+06 .1629002E+06 .2963192E+07 .5195390E+05 95.0 .5973454E+06 .6524196E+06 .2047498E+08 .1244872E+06 99.0 .5289602E+07 .8069914E+07 .8025734E+09 .6139698E+06 99.9 .6099611E+08 .1235537E+09 .5045905E+11 .3570975E+07

213

****************************************************************************** rep 3. Dose-response bioassays with 1st instar FCM larvae (See section: 4.3.2.4) ****************************************************************************** GENERAL INPUT INFORMATION ========================= DOSES TRANSFORMED TO LOG10 NATURAL MORTALITY = 20% NON-RESPONSE = 0% DOSE TRANSFORMED NUMBER NUMBER %RESPONSE CORRECTED EMPIRICAL DOSE(X) EXPOSED RESPON. %RESPONSE PROBITS 100000.00000000 5.0000 30 29 96.67 95.83 6.731 20000.00000000 4.3010 30 29 96.67 95.83 6.731 4000.00000000 3.6021 30 27 90.00 87.50 6.150 800.00000000 2.9031 30 23 76.67 70.83 5.548 160.00000000 2.2041 30 16 53.33 41.67 4.790 TEST FOR FIT OF LINE ==================== CHI-SQUARED FOR DEVIATIONS = 1.345 D. F. = 3 APPROXIMATE PROBABILITY = .7220 TEST LEVEL = 0,05 TABLED CHI-SQUARED VALUE = 7.815 DEVIATIONS FROM LINE ARE HOMOGENEOUS FITTED PROBIT LINE ================== REGRESSION EQUATION IS Y = a + bX WHERE Y IS EMPIRICAL PROBITS AND X IS DOSE INTERCEPT (a) = 3.1655 REGRESSION COEFFICIENT (b) = .7934 STANDARD ERROR OF b = .1820 MEAN FOR EMPIRICAL PROBITS (Y) = 5.6803 MEAN FOR DOSE (X) = 3.1695 DOSE (X) EXPECTED EMP.PROBIT (Y) 5.00000 7.133 4.30103 6.578 3.60206 6.023 2.90309 5.469 2.20412 4.914

214

EXPECTED QUANTITIES FOR LINE ============================ G FOR FIDUCIAL LIMITS = .2022 EXPECTED DOSE STANDARD FIDUCIAL LIMITS % RESPONSE ERROR UPPER LOWER 50.0 2.312 .2741 2.734 1.455 90.0 3.927 .2582 4.718 3.521 95.0 4.385 .3380 5.497 3.890 99.0 5.244 .5129 7.012 4.528 99.9 6.207 .7226 8.740 5.214 QUANTITIES TRANSFORMED BACK TO ORIGINAL ======================================= EXPECTED DOSE STANDARD FIDUCIAL LIMITS % RESPONSE ERROR UPPER LOWER 50.0 .2051808E+03 .1293637E+03 .5424911E+03 .2852182E+02 90.0 .8460654E+04 .5025180E+04 .5222900E+05 .3320258E+04 95.0 .2428229E+05 .1887867E+05 .3138856E+06 .7766821E+04 99.0 .1754260E+06 .2069295E+06 .1028703E+08 .3371058E+05 99.9 .1610189E+07 .2676053E+07 .5490666E+09 .1637389E+06

215

****************************************************************************** rep 4. Dose-response bioassays with 1st instar FCM larvae (See section: 4.3.2.4) ****************************************************************************** GENERAL INPUT INFORMATION ========================= DOSES TRANSFORMED TO LOG10 NATURAL MORTALITY = 8% NON-RESPONSE = 0% DOSE TRANSFORMED NUMBER NUMBER %RESPONSE CORRECTED EMPIRICAL DOSE(X) EXPOSED RESPON. %RESPONSE PROBITS 100000.00000000 5.0000 30 19 63.33 60.14 5.257 20000.00000000 4.3010 30 14 46.67 42.03 4.799 4000.00000000 3.6021 30 12 40.00 34.78 4.609 800.00000000 2.9031 30 10 33.33 27.54 4.403 160.00000000 2.2041 30 7 23.33 16.67 4.033 TEST FOR FIT OF LINE ==================== CHI-SQUARED FOR DEVIATIONS = .318 D. F. = 3 APPROXIMATE PROBABILITY = .9520 TEST LEVEL = 0,05 TABLED CHI-SQUARED VALUE = 7.815 DEVIATIONS FROM LINE ARE HOMOGENEOUS FITTED PROBIT LINE ================== REGRESSION EQUATION IS Y = a + bX WHERE Y IS EMPIRICAL PROBITS AND X IS DOSE INTERCEPT (a) = 3.1555 REGRESSION COEFFICIENT (b) = .4065 STANDARD ERROR OF b = .1280 MEAN FOR EMPIRICAL PROBITS (Y) = 4.6966 MEAN FOR DOSE (X) = 3.7907 DOSE (X) EXPECTED EMP.PROBIT (Y) 5.00000 5.188 4.30103 4.904 3.60206 4.620 2.90309 4.336 2.20412 4.052

216

EXPECTED QUANTITIES FOR LINE ============================ G FOR FIDUCIAL LIMITS = .3808 G IS RATHER LARGE: ACCURACY OF EXPERIMENTAL PROCEDURES MAY BE SUSPECT EXPECTED DOSE STANDARD FIDUCIAL LIMITS % RESPONSE ERROR UPPER LOWER 50.0 4.537 .3794 6.047 3.945 90.0 7.690 1.2631 14.043 6.132 95.0 8.583 1.5379 16.363 6.697 99.0 10.259 2.0582 20.726 7.748 99.9 12.138 2.6449 25.623 8.920 QUANTITIES TRANSFORMED BACK TO ORIGINAL ======================================= EXPECTED DOSE STANDARD FIDUCIAL LIMITS % RESPONSE ERROR UPPER LOWER 50.0 .3444230E+05 .3005749E+05 .1113433E+07 .8819092E+04 90.0 .4892678E+08 .1421395E+09 .1102955E+15 .1353765E+07 95.0 .3829872E+09 .1354734E+10 .2308556E+17 .4978426E+07 99.0 .1816683E+11 .8599862E+11 .5317086E+21 .5601771E+08 99.9 .1375009E+13 .8364622E+13 .4199039E+26 .8314882E+09

217

****************************************************************************** rep 5. Dose-response bioassays with 1st instar FCM larvae (See section: 4.3.2.4) ****************************************************************************** GENERAL INPUT INFORMATION ========================= DOSES TRANSFORMED TO LOG10 NATURAL MORTALITY = 5% NON-RESPONSE = 0% DOSE TRANSFORMED NUMBER NUMBER %RESPONSE CORRECTED EMPIRICAL DOSE(X) EXPOSED RESPON. %RESPONSE PROBITS 100000.00000000 5.0000 30 20 66.67 64.91 5.383 20000.00000000 4.3010 30 17 56.67 54.39 5.110 4000.00000000 3.6021 30 12 40.00 36.84 4.664 800.00000000 2.9031 30 9 30.00 26.32 4.366 160.00000000 2.2041 30 6 20.00 15.79 3.997 TEST FOR FIT OF LINE ==================== CHI-SQUARED FOR DEVIATIONS = .091 D. F. = 3 APPROXIMATE PROBABILITY = .9880 TEST LEVEL = 0,05 TABLED CHI-SQUARED VALUE = 7.815 DEVIATIONS FROM LINE ARE HOMOGENEOUS FITTED PROBIT LINE ================== REGRESSION EQUATION IS Y = a + bX WHERE Y IS EMPIRICAL PROBITS AND X IS DOSE INTERCEPT (a) = 2.8935 REGRESSION COEFFICIENT (b) = .5029 STANDARD ERROR OF b = .1241 MEAN FOR EMPIRICAL PROBITS (Y) = 4.7866 MEAN FOR DOSE (X) = 3.7645 DOSE (X) EXPECTED EMP.PROBIT (Y) 5.00000 5.408 4.30103 5.056 3.60206 4.705 2.90309 4.353 2.20412 4.002

218

EXPECTED QUANTITIES FOR LINE ============================ G FOR FIDUCIAL LIMITS = .2338 EXPECTED DOSE STANDARD FIDUCIAL LIMITS % RESPONSE ERROR UPPER LOWER 50.0 4.189 .2542 4.902 3.735 90.0 6.737 .7692 9.591 5.698 95.0 7.460 .9407 10.977 6.198 99.0 8.815 1.2674 13.586 7.127 99.9 10.334 1.6373 16.518 8.160 QUANTITIES TRANSFORMED BACK TO ORIGINAL ======================================= EXPECTED DOSE STANDARD FIDUCIAL LIMITS % RESPONSE ERROR UPPER LOWER 50.0 .1544534E+05 .9030854E+04 .7982005E+05 .5426490E+04 90.0 .5461709E+07 .9662493E+07 .3903871E+10 .4988028E+06 95.0 .2882506E+08 .6236543E+08 .9484961E+11 .1578639E+07 99.0 .6527873E+09 .1902857E+10 .3852993E+14 .1338668E+08 99.9 .2156895E+11 .8122339E+11 .3293707E+17 .1446100E+09

219

****************************************************************************** rep 6. Dose-response bioassays with 1st instar FCM larvae (See section: 4.3.2.4) ****************************************************************************** GENERAL INPUT INFORMATION ========================= DOSES TRANSFORMED TO LOG10 NATURAL MORTALITY = 3% NON-RESPONSE = 0% DOSE TRANSFORMED NUMBER NUMBER %RESPONSE CORRECTED EMPIRICAL DOSE(X) EXPOSED RESPON. %RESPONSE PROBITS 100000.00000000 5.0000 30 19 63.33 62.20 5.311 20000.00000000 4.3010 30 16 53.33 51.89 5.047 4000.00000000 3.6021 30 13 43.33 41.58 4.787 800.00000000 2.9031 30 11 36.67 34.71 4.607 160.00000000 2.2041 30 7 23.33 20.96 4.192 TEST FOR FIT OF LINE ==================== CHI-SQUARED FOR DEVIATIONS = .173 D. F. = 3 APPROXIMATE PROBABILITY = .9760 TEST LEVEL = 0,05 TABLED CHI-SQUARED VALUE = 7.815 DEVIATIONS FROM LINE ARE HOMOGENEOUS FITTED PROBIT LINE ================== REGRESSION EQUATION IS Y = a + bX WHERE Y IS EMPIRICAL PROBITS AND X IS DOSE INTERCEPT (a) = 3.4286 REGRESSION COEFFICIENT (b) = .3784 STANDARD ERROR OF b = .1137 MEAN FOR EMPIRICAL PROBITS (Y) = 4.8209 MEAN FOR DOSE (X) = 3.6793 DOSE (X) EXPECTED EMP.PROBIT (Y) 5.00000 5.321 4.30103 5.056 3.60206 4.792 2.90309 4.527 2.20412 4.263

220

EXPECTED QUANTITIES FOR LINE ============================ G FOR FIDUCIAL LIMITS = .3466 G IS RATHER LARGE: ACCURACY OF EXPERIMENTAL PROCEDURES MAY BE SUSPECT EXPECTED DOSE STANDARD FIDUCIAL LIMITS % RESPONSE ERROR UPPER LOWER 50.0 4.153 .3227 5.225 3.582 90.0 7.539 1.1951 13.135 6.039 95.0 8.499 1.4765 15.455 6.657 99.0 10.300 2.0096 19.818 7.805 99.9 12.319 2.6111 24.717 9.086 QUANTITIES TRANSFORMED BACK TO ORIGINAL ======================================= EXPECTED DOSE STANDARD FIDUCIAL LIMITS % RESPONSE ERROR UPPER LOWER 50.0 .1421045E+05 .1054799E+05 .1680628E+06 .3818231E+04 90.0 .3462350E+08 .9516886E+08 .1364123E+14 .1093302E+07 95.0 .3158128E+09 .1072482E+10 .2853160E+16 .4537638E+07 99.0 .1995728E+11 .9224595E+11 .6579682E+20 .6389630E+08 99.9 .2083476E+13 .1251241E+14 .5209556E+25 .1218169E+10

221

*************************** * COMPARISON OF LINES * *************************** ABRIDGED ANALYSIS OF VARIANCE ============================= DEVIATIONS FROM REGRESSION INTERCEPT REGR.COEF. D.F. SUM OF RESIDUAL (a) (b) SQUARES VARIANCES LINE: rep 1 2.626 .617 3 .383295E+01 1.278 LINE: rep 2 2.490 .719 3 .396525E+00 .132 LINE: rep 3 3.165 .793 3 .134459E+01 .448 LINE: rep 4 3.155 .407 3 .317843E+00 .106 LINE: rep 5 2.894 .503 3 .906790E-01 .030 LINE: rep 6 3.429 .378 3 .173485E+00 .058 ---------------------------------------------------------------------- 18 .615608E+01 .342 ---------------------------------------------------------------------- COMBINED .531 23 .130008E+02 .565 ---------------------------------------------------------------------- DIFFERENCE BETWEEN SLOPES 5 .684471E+01 1.369 ---------------------------------------------------------------------- TOTAL 3.278 .457 28 .681618E+02 2.434 ---------------------------------------------------------------------- DIFFERENCE BETWEEN ADJ. MEANS 5 .551610E+02 11.032 ---------------------------------------------------------------------- BARTLETTS TEST FOR HOMOGENEITY OF RESIDUAL VARIANCES ==================================================== CHI-SQUARED VALUE = 2.907 D. F. = 5 APPROXIMATE PROBABILITY = .7170 TEST LEVEL = .0100 TABLED CHI-SQUARED VALUE = 15.086 RESIDUAL VARIANCES ARE HOMOGENEOUS - SLOPES OF LINES MAY BE COMPARED COMPARISON OF SLOPES (PARALLELISM) ================================== SOURCE D.F. SUM OF SQ. MEAN SQ. F-VALUE --------------------------------------------------------- PARALLELISM 5 .684471E+01 1.369 4.00 HETEROGENEITY 18 .615608E+01 .342 -------------------------------- TOTAL 23 .1300E+02 CHI-SQUARED TEST FOR PARALLELISM -------------------------------- CHI-SQUARED VALUE = 6.845 D. F. = 5 APPROXIMATE PROBABILITY = .2310 TEST LEVEL = 0,05 TABLED CHI-SQUARED VALUE = 11.070

222

LINES ARE PARALLEL AND ELEVATIONS MAY BE COMPARED REGRESSION COEFFICIENTS ----------------------- rep 6 .3784 rep 4 .4065 rep 5 .5029 rep 1 .6167 rep 2 .7194 rep 3 .7934 COMPARISON OF ELEVATIONS (ADJUSTED MEANS) ========================================= F-VALUE FOR ELEVATIONS = 19.517 D.F. = 5 & 23 APPROXIMATE PROBABILITY = <.0001 TEST LEVEL = 0,05 TABLED F-VALUE = 2.64 ELEVATIONS ARE SIGNIFICANTLY DIFFERENT MEANS OF EMPIRICAL PROBITS -------------------------- rep 4 4.6966 rep 5 4.7866 rep 6 4.8209 rep 1 4.9362 rep 2 5.1602 rep 3 5.6803 INTERCEPTS OF LINES ------------------- rep 2 2.490 rep 1 2.626 rep 5 2.894 rep 4 3.155 rep 3 3.165 rep 6 3.429 MULTIPLE COMPARISON OF ELEVATIONS WITH BONFERRONI METHOD -------------------------------------------------------- NUMBER OF COMPARISONS = 15 TEST LEVEL = (0,05/15) = .00333 * = SIGNIFICANTLY DIFFERENT LINE: rep 5 rep 6 rep 1 rep 2 rep 3 rep 4 * rep 5 * rep 6 * rep 1 * rep 2 *

223

RELATIVE POTENCY COMPARISONS ============================ LD-50 LD-50 LINE REGRESSION EQUATION TRANSFORMED ORIGINAL ---- ------------------- ----------- -------- rep 1 Y = 2.9483 + .5306X 3.8667 .7357729E+04 rep 2 Y = 3.1904 + .5306X 3.4105 .2573168E+04 rep 3 Y = 3.9985 + .5306X 1.8874 .7716931E+02 rep 4 Y = 2.6852 + .5306X 4.3626 .2304577E+05 rep 5 Y = 2.7892 + .5306X 4.1666 .1467668E+05 rep 6 Y = 2.8687 + .5306X 4.0168 .1039558E+05

LINE: rep 1 rep 2 rep 3 rep 4 rep 5 rep 6 rep 1 1.000 2.859 95.345 .319 .501 .708 rep 2 .350 1.000 33.344 .112 .175 .248 rep 3 .010 .030 1.000 .003 .005 .007 rep 4 3.132 8.956298.639 1.000 1.570 2.217 rep 5 1.995 5.704190.188 .637 1.000 1.412 rep 6 1.413 4.040134.711 .451 .708 1.000

224

Appendix 6 COMBINED PROBAN (VAN ARK, 1995) OUPUT OF PROBIT ANALYSIS OF SURFACE DOSE-RESPONSE BIOASSAY DATA (LC) ON AN AGAR DIET, WITH CRYPTOGRAN AND CRYPTEX AGAINST THE OLD FCM COLONY AND THE ADDO COLONY ****************************************************************************** Combined replicates (4 reps) for the old colony treated with Cryptex ****************************************************************************** GENERAL INPUT INFORMATION ========================= DOSES TRANSFORMED TO LOG10 NATURAL MORTALITY = 13% NON-RESPONSE = 0% DOSE TRANSFORMED NUMBER NUMBER %RESPONSE CORRECTED EMPIRICAL DOSE(X) EXPOSED RESPON. %RESPONSE PROBITS 100000.00000000 5.0000 120 95 79.17 76.05 5.708 20000.00000000 4.3010 120 65 54.17 47.32 4.933 4000.00000000 3.6021 120 53 44.17 35.82 4.637 800.00000000 2.9031 120 38 31.67 21.46 4.209 160.00000000 2.2041 120 27 22.50 10.92 3.769 TEST FOR FIT OF LINE ==================== CHI-SQUARED FOR DEVIATIONS = 2.872 D. F. = 3 APPROXIMATE PROBABILITY = .4130 TEST LEVEL = 0,05 TABLED CHI-SQUARED VALUE = 7.815 DEVIATIONS FROM LINE ARE HOMOGENEOUS FITTED PROBIT LINE ================== REGRESSION EQUATION IS Y = a + bX WHERE Y IS EMPIRICAL PROBITS AND X IS DOSE INTERCEPT (a) = 2.2047 REGRESSION COEFFICIENT (b) = .6741 STANDARD ERROR OF b = .0793 MEAN FOR EMPIRICAL PROBITS (Y) = 4.8658 MEAN FOR DOSE (X) = 3.9473

225

DOSE (X) EXPECTED EMP.PROBIT (Y) 5.00000 5.575 4.30103 5.104 3.60206 4.633 2.90309 4.162 2.20412 3.691 EXPECTED QUANTITIES FOR LINE ============================ G FOR FIDUCIAL LIMITS = .0531 EXPECTED DOSE STANDARD FIDUCIAL LIMITS % RESPONSE ERROR UPPER LOWER 50.0 4.146 .1036 4.367 3.949 90.0 6.047 .2668 6.716 5.615 95.0 6.586 .3264 7.408 6.061 99.0 7.597 .4410 8.714 6.890 99.9 8.730 .5715 10.181 7.817 QUANTITIES TRANSFORMED BACK TO ORIGINAL ======================================= EXPECTED DOSE STANDARD FIDUCIAL LIMITS % RESPONSE ERROR UPPER LOWER 50.0 .1400798E+05 .3338193E+04 .2325657E+05 .8882763E+04 90.0 .1115441E+07 .6845778E+06 .5194917E+07 .4121629E+06 95.0 .3857895E+07 .2896167E+07 .2560952E+08 .1149608E+07 99.0 .3954679E+08 .4011137E+08 .5170737E+09 .7769384E+07 99.9 .5373391E+09 .7063618E+09 .1516473E+11 .6555151E+08

226

****************************************************************************** Combined replicates (4 reps) for the old colony treated with Cryptogran ****************************************************************************** GENERAL INPUT INFORMATION ========================= DOSES TRANSFORMED TO LOG10 NATURAL MORTALITY = 11% NON-RESPONSE = 0% DOSE TRANSFORMED NUMBER NUMBER %RESPONSE CORRECTED EMPIRICAL DOSE(X) EXPOSED RESPON. %RESPONSE PROBITS 100000.00000000 5.0000 120 111 92.50 91.57 6.377 20000.00000000 4.3010 120 83 69.17 65.36 5.395 4000.00000000 3.6021 120 67 55.83 50.37 5.009 800.00000000 2.9031 120 47 39.17 31.65 4.523 160.00000000 2.2041 120 31 25.83 16.67 4.033 TEST FOR FIT OF LINE ==================== CHI-SQUARED FOR DEVIATIONS = 4.387 D. F. = 3 APPROXIMATE PROBABILITY = .2210 TEST LEVEL = 0,05 TABLED CHI-SQUARED VALUE = 7.815 DEVIATIONS FROM LINE ARE HOMOGENEOUS FITTED PROBIT LINE ================== REGRESSION EQUATION IS Y = a + bX WHERE Y IS EMPIRICAL PROBITS AND X IS DOSE INTERCEPT (a) = 2.2288 REGRESSION COEFFICIENT (b) = .7790 STANDARD ERROR OF b = .0735 MEAN FOR EMPIRICAL PROBITS (Y) = 5.1173 MEAN FOR DOSE (X) = 3.7078 DOSE (X) EXPECTED EMP.PROBIT (Y) 5.00000 6.124 4.30103 5.579 3.60206 5.035 2.90309 4.490 2.20412 3.946 EXPECTED QUANTITIES FOR LINE

227

============================ G FOR FIDUCIAL LIMITS = .0342 EXPECTED DOSE STANDARD FIDUCIAL LIMITS % RESPONSE ERROR UPPER LOWER 50.0 3.557 .0844 3.720 3.384 90.0 5.202 .1637 5.586 4.925 95.0 5.669 .2028 6.148 5.328 99.0 6.543 .2801 7.211 6.076 99.9 7.524 .3694 8.408 6.910 QUANTITIES TRANSFORMED BACK TO ORIGINAL ======================================= EXPECTED DOSE STANDARD FIDUCIAL LIMITS % RESPONSE ERROR UPPER LOWER 50.0 .3607599E+04 .7000158E+03 .5250839E+04 .2418520E+04 90.0 .1593484E+06 .5998138E+05 .3853721E+06 .8405682E+05 95.0 .4663353E+06 .2174934E+06 .1407343E+07 .2126941E+06 99.0 .3494458E+07 .2250981E+07 .1626323E+08 .1191812E+07 99.9 .3341662E+08 .2839174E+08 .2558502E+09 .8128026E+07

228

****************************************************************************** Combined replicates (3 reps) for the Addo colony treated with Cryptex ****************************************************************************** GENERAL INPUT INFORMATION ========================= DOSES TRANSFORMED TO LOG10 NATURAL MORTALITY = 9% NON-RESPONSE = 0% DOSE TRANSFORMED NUMBER NUMBER %RESPONSE CORRECTED EMPIRICAL DOSE(X) EXPOSED RESPON. %RESPONSE PROBITS 100000.00000000 5.0000 90 75 83.33 81.68 5.903 20000.00000000 4.3010 90 55 61.11 57.26 5.183 4000.00000000 3.6021 90 45 50.00 45.05 4.876 800.00000000 2.9031 90 27 30.00 23.08 4.264 160.00000000 2.2041 90 17 18.89 10.87 3.767 TEST FOR FIT OF LINE ==================== CHI-SQUARED FOR DEVIATIONS = 1.317 D. F. = 3 APPROXIMATE PROBABILITY = .7290 TEST LEVEL = 0,05 TABLED CHI-SQUARED VALUE = 7.815 DEVIATIONS FROM LINE ARE HOMOGENEOUS FITTED PROBIT LINE ================== REGRESSION EQUATION IS Y = a + bX WHERE Y IS EMPIRICAL PROBITS AND X IS DOSE INTERCEPT (a) = 2.1381 REGRESSION COEFFICIENT (b) = .7375 STANDARD ERROR OF b = .0845 MEAN FOR EMPIRICAL PROBITS (Y) = 4.9627 MEAN FOR DOSE (X) = 3.8297 DOSE (X) EXPECTED EMP.PROBIT (Y) 5.00000 5.826 4.30103 5.310 3.60206 4.795 2.90309 4.279 2.20412 3.764 EXPECTED QUANTITIES FOR LINE

229

============================ G FOR FIDUCIAL LIMITS = .0505 EXPECTED DOSE STANDARD FIDUCIAL LIMITS % RESPONSE ERROR UPPER LOWER 50.0 3.880 .1004 4.085 3.681 90.0 5.618 .2282 6.182 5.244 95.0 6.111 .2800 6.808 5.656 99.0 7.034 .3808 7.990 6.420 99.9 8.070 .4963 9.319 7.272 QUANTITIES TRANSFORMED BACK TO ORIGINAL ======================================= EXPECTED DOSE STANDARD FIDUCIAL LIMITS % RESPONSE ERROR UPPER LOWER 50.0 .7591489E+04 .1752352E+04 .1215924E+05 .4798780E+04 90.0 .4149573E+06 .2177734E+06 .1519822E+07 .1755395E+06 95.0 .1289998E+07 .8307906E+06 .6426716E+07 .4526154E+06 99.0 .1082619E+08 .9481997E+07 .9762062E+08 .2631433E+07 99.9 .1175495E+09 .1341906E+09 .2085270E+10 .1871542E+08

230

****************************************************************************** Combined replicates (4 reps) for the Addo colony treated with Cryptogran ****************************************************************************** GENERAL INPUT INFORMATION ========================= DOSES TRANSFORMED TO LOG10 NATURAL MORTALITY = 13% NON-RESPONSE = 0% DOSE TRANSFORMED NUMBER NUMBER %RESPONSE CORRECTED EMPIRICAL DOSE(X) EXPOSED RESPON. %RESPONSE PROBITS 100000.00000000 5.0000 120 101 84.17 81.80 5.908 20000.00000000 4.3010 120 85 70.83 66.48 5.426 4000.00000000 3.6021 120 70 58.33 52.11 5.053 800.00000000 2.9031 120 59 49.17 41.57 4.787 160.00000000 2.2041 120 37 30.83 20.50 4.176 TEST FOR FIT OF LINE ==================== CHI-SQUARED FOR DEVIATIONS = 1.236 D. F. = 3 APPROXIMATE PROBABILITY = .7480 TEST LEVEL = 0,05 TABLED CHI-SQUARED VALUE = 7.815 DEVIATIONS FROM LINE ARE HOMOGENEOUS FITTED PROBIT LINE ================== REGRESSION EQUATION IS Y = a + bX WHERE Y IS EMPIRICAL PROBITS AND X IS DOSE INTERCEPT (a) = 2.9957 REGRESSION COEFFICIENT (b) = .5769 STANDARD ERROR OF b = .0671 MEAN FOR EMPIRICAL PROBITS (Y) = 5.1122 MEAN FOR DOSE (X) = 3.6689 DOSE (X) EXPECTED EMP.PROBIT (Y) 5.00000 5.880 4.30103 5.477 3.60206 5.074 2.90309 4.670 2.20412 4.267 EXPECTED QUANTITIES FOR LINE

231

============================ G FOR FIDUCIAL LIMITS = .0519 EXPECTED DOSE STANDARD FIDUCIAL LIMITS % RESPONSE ERROR UPPER LOWER 50.0 3.474 .1106 3.687 3.241 90.0 5.696 .2593 6.340 5.273 95.0 6.326 .3272 7.146 5.797 99.0 7.507 .4590 8.664 6.769 99.9 8.831 .6097 10.373 7.854 QUANTITIES TRANSFORMED BACK TO ORIGINAL ======================================= EXPECTED DOSE STANDARD FIDUCIAL LIMITS % RESPONSE ERROR UPPER LOWER 50.0 .2980857E+04 .7580593E+03 .4858636E+04 .1741282E+04 90.0 .4964894E+06 .2960711E+06 .2189940E+07 .1876307E+06 95.0 .2116735E+07 .1593056E+07 .1398387E+08 .6259887E+06 99.0 .3212394E+08 .3391542E+08 .4618411E+09 .5879476E+07 99.9 .6776405E+09 .9502318E+09 .2360070E+11 .7148504E+08

232

*************************** * COMPARISON OF LINES * *************************** ABRIDGED ANALYSIS OF VARIANCE ============================= DEVIATIONS FROM REGRESSION INTERCEPT REGR.COEF. D.F. SUM OF RESIDUAL (a) (b) SQUARES VARIANCES LINE: oldtex 2.205 .674 3 .287153E+01 .957 LINE: Oldgra 2.229 .779 3 .438674E+01 1.462 LINE: Adotex 2.138 .738 3 .131686E+01 .439 LINE: Adogra 2.996 .577 3 .123553E+01 .412 ---------------------------------------------------------------------- 12 .981067E+01 .818 ---------------------------------------------------------------------- COMBINED .684 15 .144514E+02 .963 ---------------------------------------------------------------------- DIFFERENCE BETWEEN SLOPES 3 .464070E+01 1.547 ---------------------------------------------------------------------- TOTAL 2.534 .659 18 .434777E+02 2.415 ---------------------------------------------------------------------- DIFFERENCE BETWEEN ADJ. MEANS 3 .290264E+02 9.675 ---------------------------------------------------------------------- BARTLETTS TEST FOR HOMOGENEITY OF RESIDUAL VARIANCES ==================================================== CHI-SQUARED VALUE = .640 D. F. = 3 APPROXIMATE PROBABILITY = .8860 TEST LEVEL = .0100 TABLED CHI-SQUARED VALUE = 11.345 RESIDUAL VARIANCES ARE HOMOGENEOUS - SLOPES OF LINES MAY BE COMPARED COMPARISON OF SLOPES (PARALLELISM) ================================== SOURCE D.F. SUM OF SQ. MEAN SQ. F-VALUE --------------------------------------------------------- PARALLELISM 3 .464070E+01 1.547 1.89 HETEROGENEITY 12 .981067E+01 .818 -------------------------------- TOTAL 15 .1445E+02 CHI-SQUARED TEST FOR PARALLELISM -------------------------------- CHI-SQUARED VALUE = 4.641 D. F. = 3 APPROXIMATE PROBABILITY = .1980 TEST LEVEL = 0,05 TABLED CHI-SQUARED VALUE = 7.815 LINES ARE PARALLEL AND ELEVATIONS MAY BE COMPARED

233

REGRESSION COEFFICIENTS ----------------------- Adogra .5769 oldtex .6741 Adotex .7375 Oldgra .7790 COMPARISON OF ELEVATIONS (ADJUSTED MEANS) ========================================= F-VALUE FOR ELEVATIONS = 10.043 D.F. = 3 & 15 APPROXIMATE PROBABILITY = .0010 TEST LEVEL = 0,05 TABLED F-VALUE = 3.29 ELEVATIONS ARE SIGNIFICANTLY DIFFERENT MEANS OF EMPIRICAL PROBITS -------------------------- oldtex 4.8658 Adotex 4.9627 Adogra 5.1122 Oldgra 5.1173 INTERCEPTS OF LINES ------------------- Adotex 2.138 oldtex 2.205 Oldgra 2.229 Adogra 2.996 MULTIPLE COMPARISON OF ELEVATIONS WITH BONFERRONI METHOD -------------------------------------------------------- NUMBER OF COMPARISONS = 6 TEST LEVEL = (0,05/ 6) = .00833 * = SIGNIFICANTLY DIFFERENT LINE: Adotex Adogra Oldgra oldtex * * Adotex Adogra RELATIVE POTENCY COMPARISONS ============================ LD-50 LD-50 LINE REGRESSION EQUATION TRANSFORMED ORIGINAL ---- ------------------- ----------- -------- oldtex Y = 2.1675 + .6836X 4.1436 .1391969E+05 Oldgra Y = 2.5827 + .6836X 3.5362 .3437064E+04 Adotex Y = 2.3448 + .6836X 3.8843 .7661668E+04 Adogra Y = 2.6043 + .6836X 3.5047 .3196721E+04

234

LINE: oldtex Oldgra Adotex Adogra oldtex 1.000 4.050 1.817 4.354 Oldgra .247 1.000 .449 1.075 Adotex .550 2.229 1.000 2.397 Adogra .230 .930 .417 1.000

235

Appendix 7

7.1 SURVIVAL, DEVELOPMENT, INCUBATION ROOM TEMPERATURE AND HUMIDITY DATA FOR FIELD COLLECTED FCM LARVAE FROM ADDO, CITRUSDAL, MARBLE HALL AND NELSPRUIT REGIONS Table 7.1 FCM larvae collected from navel oranges from a range of geographic regions from April 2007 to May 2008

Date of fruit collection

Area

Total

number of fruits

collected

Number of FCM larvae

parasitized

Total number of FCM

larvae isolated

1st 2nd 3rd 4th 5th 1st 2nd 3rd 4th 5th 03/04/2008 Addo 399 0 0 2 0 0 2 48 86 23 50 08/04/2008 Addo 867 0 1 5 5 0 1 46 96 66 134

Total 1266 0 1 7 5 0 3 94 182 89 184 24/04/2008 Citrusdal 172 - - - - - 15 33 23 5 3 17/04/2008 Citrusdal 408 - - - - - 0 3 23 11 46 21/06/2008 Citrusdal 83 - - - - - 0 2 4 4 16

Total 663 - - - - - 15 38 50 20 65 23/06/2008 Marble Hall 406 - - - - - 0 5 56 22 53 26/06/2008 Nelspruit 704 - - - - - 0 4 35 18 83

Table 7.2 Percentage survival of field collected FCM larvae emerging into Adults.

Instar Total larvae isolated

Total number of moths emerging

% Survival

Addo

1st 3 0 0.00 2nd 94 33 35.11 3rd 182 51 28.02 4th 89 58 65.17 5th 184 148 80.43

Citrusdal

1st 15 5 33.33 2nd 38 10 26.32 3rd 50 20 40.00 4th 20 13 65.00 5th 65 45 69.23

Marble Hall

1st 0 0 0.00 2nd 5 1 20.00 3rd 56 32 57.14 4th 22 12 54.55 5th 53 35 66.04

Nelspruit

1st 0 0 0.00 2nd 4 0 0.00 3rd 35 16 45.71 4th 18 7 38.89 5th 83 50 60.24

236

Table 7.3 Weekly temperature, humidity readings and FCM development data from the incubation room for the Addo colony (Eastern Cape).

Duration Temperature oC

(Average) Relative

(Average) Number of

moths Number of

Jars Weekly (7days) Minimum Maximum Humidity (%) emerging Progeny with eggs

2/5/2008 21 F1 2 4 - 10/05/2008 31 F1 5 11 - 17/05/2008 5 F1 4 18 - 24/05/2008 42 F1 4 25 - 31/05/2008 F1

1 - 7/05 - 06/2008 F1 08 -14 /06/ 2008 26.2 26.4 35.2 8 F2 15 - 21 /06/ 2008 25.71 26 40.7 44 F2 3 22 - 28 /06/ 2008 24.33 25.33 39.9 20 F2 2

29 -05 /06- 07/ 2008 24.75 25.25 37.2 36 F2 5 06 - 12 /07/ 2008 23 23.57 42.6 26 F2 10

13 - 19 /07/ 2008 23.5 24 41 F2

20 - 26 /07/ 2008 24 24.6 38.76 F2

27 - 02 /07-08 / 2008 24.57 25.57 45.29 F2

03 - 09 /08/ 2008 24.29 24.86 37.81 F3 1 10 -16 /08/ 2008 24.5 25 43.45 F3 5 17 - 23 /08/ 2008 25.25 26 39.28 F3 22 24 - 30 /08/ 2008 25.75 26.25 50.35 F3 10

31 - 06 /08 - 09/ 2008 23.67 24.33 31.3 F3 07 -13 /09/ 2008 25.25 26 38.53 F3 14 - 20 /09/ 2008 26.5 27 39.2 F3 21 - 27 /09/ 2008 26 26.33 30.87 F3

28 - 04 /09 - 10/ 2008 26 26.5 33.4 F4 8 05 - 11 /10/ 2008 25 25.67 34.33 F4 6 12 -18 /10/ 2008 25.67 26 40.6 F4 3 19 - 25 /10/ 2008 25.67 26 46.17 F4

26 - 01 /10 - 11/ 2008 25.33 25.67 45.53 F4 02 - 08 /11/ 2008 25 26.5 38 F4 09 - 15 /11/ 2008 26 26.5 45.8 F5 8 16 - 22 /11/ 2008 26.5 27 59.9 F5 6 23 - 29 /11/ 2008 24.5 25.5 47 F5

237

Table 7.4 Weekly temperature, humidity readings and FCM development data from the incubation room for the Citrusdal colony (Western Cape).

Duration Temperature oC

(Average) Relative

(Average) Number of

moths Number of Jars

Weekly (7days) Minimum Maximum Humidity (%) emerging Progeny with eggs

18 - 24/05/2008 30 F1 2 25 - 31/05/2008 43 F1 6

1 - 7/05 - 06/2008 F1 4 08 -14 /06/ 2008 25.6 25.8 37.56 F2 15 - 21 /06/ 2008 26 26.14 38.99 F2 2 22 - 28 /06/ 2008 25.25 25.75 38.16 1 F2 8

29 -05 /06- 07/ 2008 23.57 24 41.16 25 F2 2 06 - 12 /07/ 2008 24 25 48.7 67 F2 13 - 19 /07/ 2008 25 26.8 39.66 34 F3 3 20 - 26 /07/ 2008 26.71 27.71 51.03 16 F3 2

27 - 02 /07-08 / 2008 25.86 26.71 42.67 31 F3 4

03 - 09 /08/ 2008 26.5 27.5 46.95 F3 4 10 -16 /08/ 2008 27 28 44.08 F3 10 17 - 23 /08/ 2008 27 28 54.95 F3 4 24 - 30 /08/ 2008 26.5 27.5 40.75 F3 6

31 - 06 /08 - 09/ 2008 26.2 27 38.34 F3 8 07 -13 /09/ 2008 26.8 27.6 38.32 F3 5 14 - 20 /09/ 2008 25.4 26.2 38.82 F3 6 21 - 27 /09/ 2008 24.5 25.5 36.7 F4 7

28 - 04 /09 - 10/ 2008 25.67 26.33 44.17 F4 5 05 - 11 /10/ 2008 25.33 26.33 45.73 F4 6 12 -18 /10/ 2008 25.67 27.33 48.1 F4 10 19 - 25 /10/ 2008 26 27 41.15 F4 6

26 - 01 /10 - 11/ 2008 26.5 27.5 51.3 F4 7

02 - 08 /11/ 2008 25.67 26.67 55.57 F5

09 - 15 /11/ 2008 26.33 27.33 50.37 F5 3 19/11/2008 27 28 53.6 F5

Table 7.5 Weekly temperature, humidity readings and FCM development data from the incubation room for the Marble Hall colony (Mpumalanga Province).

Duration Temperature oC

(Average) Relative

(Average) Number of

moths Number of

Jars Weekly (7days) Minimum Maximum Humidity (%) emerging Progeny with eggs 27 - 02 /07/ 2008 24.75 25.75 44 18 F1 03 - 09 /08/ 2008 25 26 49.15 24 F1 1 10 -16 /08/ 2008 26 27 45.1 22 F1 4 17 - 23 /08/ 2008 26 27 53.3 8 F1 8

238

24 - 30 /08/ 2008 26.33 26.67 38.57 6 F1 5 31 - 06 /08 - 09/

2008 25.17 26.17 39.85 F1 07 -13 /09/ 2008 25.8 26.8 41.14 F1 14 - 20 /09/ 2008 27 27.8 39.18 12 F2 21 - 27 /09/ 2008 25.5 26.5 39.8 80 F2 3 28 - 04 /09 - 10/

2008 26.25 27 46 132 F2 6

05 - 11 /10/ 2008 26.33 27.33 47 69 F2 6 12 -18 /10/ 2008 26.75 27.75 49.9 17 F2 8 19 - 25 /10/ 2008 26 27 42.15 1 F2 26 - 01 /10 - 11/

2008 26.5 27.5 59.85 F3 3 02 - 08 /11/ 2008 26.67 27.33 52.5 F3 09 - 15 /11/ 2008 26.67 27.33 53.17 F3 12

19/11/2008 27 28 52.3 F3 3

Table 7.6 Weekly temperature, humidity readings and FCM development data from the incubation room for the Nelspruit colony (Mpumalanga Province).

Duration Temperature oC

(Average) Relative

(Average) Number of

moths Number of

Jars Weekly (7days) Minimum Maximum Humidity (%) emerging Progeny with eggs 13 - 19 /07/ 2008 23.75 24.75 50.35 19 F1 20 - 26 /07/ 2008 26.29 27.43 51.56 11 F1 2

27 - 02 /07 -08/ 2008 25.14 26 43.53 21 F1 4

03 - 09 /08/ 2008 25.5 26.5 47.8 1 F1 2

10 -16 /08/ 2008 26 27 42.3 1 F1 3 17 - 23 /08/ 2008 26 27 53.3 F1 24 - 30 /08/ 2008 25.67 26.67 38.2 6 F2 31 - 06 /08 - 09/

2008 25.1 26.17 39.97 42 F2 1 07 -13 /09/ 2008 25.8 26.8 41.46 54 F2 4 14 - 20 /09/ 2008 27 27.8 39.3 73 F2 3 21 - 27 /09/ 2008 25.67 26.67 36.2 5 F2 5 28 - 04 /09 - 10/

2008 26.25 27.25 44.93 F2 3 05 - 11 /10/ 2008 26 27 45.63 F2 2 12 -18 /10/ 2008 27 28 49.87 F2 19 - 25 /10/ 2008 26 27 42.1 F2 26 - 01 /10 - 11/

2008 26 27 52.8 F3 1 02 - 08 /11/ 2008 26.75 27.5 52.45 F3 5 09 - 15 /11/ 2008 26.67 27.33 52.37 F3 6

19/11/2008 27 28 52.6 F3 1