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ECOLOGY AND MANAGEMENT OF TERMITES IN URBAN

ENVIRONMENT (ISLAMABAD)

ABDUL SATTAR

08-arid-772

Department of Entomology, Faculty of Crop and Food Sciences Pir

Mehr Ali Shah Arid Agriculture University, Rawalpindi Pakistan 2015

ECOLOGY AND MANAGEMENT OF TERMITES IN URBAN

ENVIRONMENT (ISLAMABAD)

by

2

ABDUL SATTAR (08-arid-772)

A thesis submitted in partial fulfillment of the requirements for the degree of

Doctor of Philosophy

in

Entomology

Department of Entomology, Faculty of Crop and Food Sciences Pir

Mehr Ali Shah Arid Agriculture University, Rawalpindi Pakistan 2015

CERTIFICATION I hereby undertake that this research is an original one and no part of this thesis falls under

plagiarism. If found otherwise, at any stage, I will be responsible for the consequences.

Student’s Name: ABDUL SATTAR Signature: ______________________

Registration No.: 08-arid-772 Date: __________________________

Certified that the contents and form of the thesis entitled “Ecology and

Management of Termites in Urban Environment (Islamabad)” submitted by Mr.

Abdul Sattar have been found satisfactory for the requirements of the degree.

3

Supervisor: __________________________

(Dr. Muhammad Naeem)

Co-Supervisor: _______________________

(Dr. Ehsan-ul-Haq)

Member:_____________________________

(Dr. Ata-ul-Mohsin)

Member:_____________________________

(Dr. Fayyaz-ul-Hassan)

Chairman: ___________________

Dean: _______________________

Director, Advanced Studies: ____________________________

4

IN THE NAME OF ALLAH THE MOST MERCIFUL THE MOST GRACIOUS

DEDICATION

5

I dedicate this humble effort to my parents, whose

unprecedented love and affection can never be

compensated

Abdul Sattar

CONTENTS

Page

List of Tables xi

List of Figures xiii

Acknowledgments xvi

Abstract xviii

1 INTRODUCTION 1

2 REVIEW OF LITERATURE 6

2.1. ECOLOGICAL STUDIES 6

2.1.1. Population dynamics 7

2.1.2. Foraging ecology 9

2.1.3. Species identification 10

2.1.4. Caste composition in termite colony 11

2.2. EVALUATION OF DYE MARKERS 12

2.3. PLANTS AND THEIR PRODUCTS 14

2.4. PHAGOSTIMULANTS 15 i) Yeast 16

6

ii) Urea 16

iii) Plant Extracts 17

iv) Glucose 17 v) Chemicals 18

2.5. BAITS 19 i) Metabolic inhibitors 20

ii) Fungi (bioagent) 21

iii) Insect Growth Regulators (IGRs) 21

3 IMPACT OF ENVIRONMENTAL FACTORS ON THE 22 POPULATION

DYNAMICS, DENSITY AND FORAGING ACTIVITIES OF O. LOKANANDI

AND M. OBESI IN

ISLAMABAD

3.1. ABSTRACT 22

3.2. INTRODUCTION 23

3.3. MATERIALS AND METHODS 25

3.3.1 Ecological Studies 25

3.3.2. Survey 26

3.3.3. Population Dynamics of Subterranean Termites 26

3.3.4. Identification of termites 26

3.3.5. Ecology of foraging termites 27

3.3.6. Statistical Analysis 27

3.4. RESULTS 27

3.4.1. Ecological studies of termites in urban environment

(Islamabad)

27

3.4.1.1. Survey 27

3.4.1.2. Population dynamics 28

3.4.1.3. Species of termites in the experimental areas 29

3.4.1.4. Yield (g) and number of termites in 1.0 gm sample 29

3.4.1.5. Foraging Ecology of Subterranean Termites. 33

3.4.1.6. Caste composition of foraging groups of subterranean

termites.

36

3.5. DISCUSSION 37

3.5.1. Population dynamics 37

3.5.2. Foraging Ecology 41

4 EFFECT OF DYE-MARKERS I.E. NILE BLUE-A AND SUDAN

RED-7B ON MICROTERMES OBESI AND ODONTOTERMES

LOKANANDI

44

4.1. ABSTRACT 44



4.3.1. Biological Stains 46

4.3.2. Experimental Termites 47

4.3.3. Preparation of Dye Attractive Bait 47

4.4. RESULTS 49

7

4.4.1. Toxicity of Biological dyes 49

4.4.2. Nile blue-A 49

4.4.3. Sudan red-7B 53

4.5. DISCUSSION 59

5 INVESTIGATION OF DYE MARKERS WHICH REMAIN

VISIBLE IN BODY OF TERMITES AT DIFFERENT RELATIVE

HUMIDITIES

62

5.1. ABSTRACT 62



5.3.1. Experimental Termites 64

5.3.2. Visibility of dye markers in the body of termites under at 65

different relative humidity

5.3.3. Retention test 66

5.4. RESULTS 66

5.4.1. Visibility of dye markers i.e., Nile blue-A and Sudan

66 red-7B in the body of termites at different relative

humidities

5.4.1.1. Nile blue-A 66

5.4.1.2. Sudan red-7B 69

5.4.2. Retention of dye 69

5.5. DISCUSSION 70

6 SCREENING OF PLANT EXTRACTS TO FIND OUT 82

PROPER CONCENTRATION FOR DEVELOPMENT OF

SLOW-ACTING TOXICANT BAITS TO MANAGE

TERMITES

6.1. ABSTRACT 82



6.3.1. Collection of Experimental Termites 84

6.3.2. Plants Collection and their Extracts Preparation 85

6.3.3. Bioassay 85

6.3.3.1. Toxicity test 85


6.4. RESULTS 86

6.4.1. Microtermes obesi 86

i) Euphorbia helioscopia (leaf extracts) 87

ii) Euphorbia helioscopia (seed extracts) 87

6.4.2. Odontotermes lokanandi 89

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i) Euphorbia helioscopia (leaf extracts) 89

ii) Euphorbia helioscopia (Seed extracts) 90


i) Cannabis sativa (leaf extracts) 91

ii) Cannabis sativa (Seed extracts) 95

6.4.4. Odontotermes lokanandi 96

i) Cannabis sativa (Leaf extracts) 96

ii) Cannabis sativa (Seed extracts) 97


i) Calotropis procera (Leaf extracts) 99

ii) Calotropis procera (Seed extracts) 101

6.4.6. Odontontermes lokanandi 103

i) Calotropis procera (Leaf extracts) 103

ii) Calotropis procera (Seed extracts) 105

6.5. DISCUSSION 106

7 LABORATORY INVESTIGATION OF COPER 111

SULPHATE AND MERCURIC CHLORIDE TO FIND OUT

PROPER CONCENTRATION TO BE USED IN

SLOWACTING TOXICANT BAITS FOR MANAGEMENT

OF TERMITES

7.1. ABSTRACT 111



7.3.1. Studies on the Efficacy of Mercuric Chloride and Copper 113

Sulphate

7.3.2. Bioassay 113

7.3.2.1. Toxicity Test 113


7.4. RESULTS 114

7.4.1. Efficacy of Copper Sulphate and Mercuric Chloride

against Microtermes obesi

114


7.4.2. Efficacy of Copper Sulphate and Mercuric Chloride

against Odontotermes lokanandi

115


7.5. DISCUSSION 116

9

8 SCREENING OF DIFFERENT COMPOUNDS TO FIND OUT

PHAGOSTIMULANT TO MAKE ATTRACTIVE BAIT FOR THE

CONTROL OF SUBTERRANEAN TERMITES

121

8.1. ABSTRACT 121



8.3.1. Studies of different compounds to find out

Phagostimulants

123

8.3.1.1. Determination of Phagostimulant as potential bait

substrates for Microtermes obesi

123

8.3.2. Preparation of Poplar saw Dust Extract 124

8.3.2.1. Determination of poplar saw dust extract as potential

bait substrates for Microtermes obesi

124

8.3.3. Comparative attractancy Test 125

8.4. RESULTS 126

8.4.1. Eualvation of different compounds to find out

phagostimulants

126

i) Effect of different compounds (urea, yeast and

glucose) on bait consumption and survival of Microtermes

obesi

126

ii) Effect of different concentrations of poplar sawdust

extract on bait consumption and survival of Microtermes obesi

127

8.4.2. Comparative attractancy test 1: Distilled water, 0.1% 131

urea, poplar sawdust extract, 3% glucose, and 3% yeast.

8.4.3. Comparative attractancy test 2: Distilled water, 0.1% 131 urea,poplar

sawdust extract, 3% glucose, and 4% yeast

8.4.4. Comparative attractancy test 3: Distilled water, 0.1%

131 urea, poplar sawdust extract, 4% glucose, and 2% yeast

8.4.5. Comparative attractancy test 4: Distilled water, 1% urea, 133

poplar sawdust extract, 2% glucose, and 1% yeast

8.4.6. Comparative attractancy test 5: Distilled water, 1% urea,

poplar sawdust extract, 1% glucose, and 1% yeast

133

8.4.7. Comparative attractancy test 6: Distilled water, 1% urea,

4% yeast, 4% glucose, and poplar sawdust individually and in different

combinations

133

8.5. DISCUSSION 134

9 FORMULATION OF SLOW-ACTING TOXIC BAITS TO

CONTROL SUBTERRANEAN TERMITES

142

9.1. ABSTRACT 142



9.3.1. Formulation of Slow-acting Toxicant Baits 144

9.3.1.1. Experimental Termites 144

9.3.1.2. Choice Feeding Test 145

9.4. RESULTS 146

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9.4.1. Formulation of Palatable toxicant baits for Microtermes

obesi by combining of phagostimulants with different toxicants

146

9.4.2. Comparative percent bait consumption by M. obesi 155

9.5. DISCUSSION 156

10 GENERAL DISCUSSION 162

SUMMARY 178

RECOMMENDATIONS 183

LITERATURE CITED 184

APPENDICES 256

LIST OF TABLES

Table No. Page

3.1 Infestation of NIFA-TERMAPs by O. lokanandi (O) and M. 32 obesi (M) at Islamabad

during 2010 to 2012

3.2 Mean yield (g), mean number of termites in 1.0 gm sample and 34 mean percent

workers in foraging group of M. obesi collected from “NIFA TERMAPs” installed

in Islamabad from

September 2010 to September 2012

3.3 Mean yield (g) and mean number of termites in 1.0 gm sample 35 and mean percent

workers in foraging group of O. lokanandi collected from “NIFA TERMAPs” installed in

Islamabad from

September 2010 to September 2012

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3.4 Correlation between environmental factors and biomass of 39

termite species captured through “NIFA TERMAPs” from

Islamabad

3.5 Correlation between environmental factors (Atmospheric 39 Temperature, Relative

Humidity and Precipitation) and % workers of Termites

6.1 Mean percent mortality in M. obesi at different concentrations 92 of leave and seed

extracts of E. helioscopia

6.2 Mean percent mortality in O. lokanandi at different 93

concentrations of leave and seed extracts of E. helioscopia


extracts of C. sativa


concentrations of leave and seed extracts of C. sativa


extracts of C. procera


concentrations of leave and seed extracts of C. procera

7.1 Mean percent mortality in M. obesi at different concentrations 117 of Copper Sulphate

(CuSo4) and Mercuric Chloride (HgCl2)


concentrations of Copper Sulphate (CuSo4) and Mercuric

Chloride (HgCl2)

9.1 Percent mean mortality in M. obesi offered filter paper baited 153 with different

concentrations of Mercuric Chloride (HgCl2) coated with phagostimulant in choice with

distilled water


concentrations of Copper Sulphate (CuSo4) coated with phagostimulant in choice with

distilled water


concentrations of E. helioscopia coated with phagostimulant in choice with distilled water


concentrations of C. procera (Ak) coated with phagostimulant in choice with distilled water

12


concentrations of C. sativa coated with phagostimulant in choice with distilled water

10.1 Comparison of different studies showing termite’s survey 163

10.2 Comparison of different studies showing foraging behaviour 165

10.3 Comparison of different studies showing termites different dye 168

10.4 Comparison of different plants extract used against termites 172

LIST OF FIGURES

Fig. Page

3.1 Location map of the study areas. The black dots show NIFA- 30 TERMAPS

3.2 Increase in the number of infested traps in relation to temperature 31 and relative

humidity in Islamabad from April to August, 2010

3.3 Effect of atmospheric temperature, relative humidity and 38 precipitation on

Mean±SE number of M. obesi and O. lokanandi collected through “NIFA TERMAPs”

installed in Islamabad during September, 2010 to September, 2012

4.1 Stock baits with different concentration of dyes. A, undyed; B, C 51 and D (Nile

blue-A), E, F and G (Sudan red-7B) at 0.5, 0.25 and

0.125% concentrations, respectively

4.2 Mortality (%) observed in M. obesi after exposure to Nile blue- 54 A at

different concentrations for 15 days

4.3 Mortality (%) observed in O. lokanandi after exposure to Nile 55 blue-A at

different concentrations for 5 days

4.4 Mortality (%) observed in M. obesi after exposure to Sudan red- 58

7B at different concentrations for 9 days

13

4.5 Mortality (%) observed in O. lokanandi after exposure to Sudan 60

red-7B at different concentrations for 5 days

5.1 (A,B,C). Biological stain (Nile blue-A) attained by termite, M. 71 obesi after 4 days at

100 percent relative humidity (H2O); A,

0.125; B, 0.25 and C, 0.5% concentration

5.2 (A,B,C).Biological stain (Nile blue-A) attained by termite, M. 72 obesi after 7

days at 100 percent relative humidity (H2O). A,

0.125; B, 0.25; and C, 0.5% concentration

5.3 (A,B,C). Biological stain (Nile blue-A) attained by termite, M. 73 obesi after 10

days at 100 percent relative humidity (H2O). A,

0.25; B, 0.125 and C, 0.5% concentration

5.4 (A,B,C). Biological stain (Nile blue-A) attained by termite, M. 74 obesi after 7 days at

92 percent relative humidity (Na2Co3). A,


5.5 (A,B,C).Biological stain (Nile blue-A) attained by termite, M. 75 obesi after 7 days

at 76 percent relative humidity (NaCl). A,


5.6 (A,B,C). Biological stain (Nile blue-A) attained by termite, 76 O.lokanandi after 4

day. A, 0.125; B, 0.25; and C, 0.5% concentration

5.7 (A,B,C). Dye, Sudan red visible in termite, M. obesi after 4 days. 77 A, 0.125; B, 0.25

and C, 0.5% concentration

5.8 (A,B,C,D). Sudan red attained by termite, O. lokanandi after 1 78 day at 100 percent

relative humidity (H2O). A, 0; B, 0.125; C,

0.25 and D, 0.5% concentration

5.9 ((A,B,C). Percent number of dyed termites (M. obesi) after 79 specified number of

days, at three concentrations

8.1 Effect of different concentrations of Phagostimulants on the mean 128

percentage survival of Microtermes obesi.

8.2 Effect of different concentrations of Phagostimulants on percent 129 bait consumption

by Microtermes obesi

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8.3 Effect of different concentrations of poplar sawdust extract on the 130 mean percentage

survival and bait consumption by M. obesi

8.4 Response of M. obesi to filter paper soaked in distilled water, 132

0.1% Urea, Poplar saw dust extract, 3% Glucose and 3%Yeast


0.1%Urea, Poplar saw dust, 3% Glucose and 4%Yeast


0.1% Urea, Poplar saw dust, 4% Glucose and 2%Yeast


1% Urea , Poplar saw dust, 2% Glucose and 1%Yeast


0.1%Urea Poplar saw dust, 1% Glucose and 1%Yeast

8.9 Percent mean bait consumption by M. obesi when offered in 139 Choice

chamber

9.1 Choice chamber to formulate slow-acting toxic baits for termites 147

9.2 Percent bait consumption by Microtermes obesi when offered 160 different

baits at high concentrations of toxicants in Choice with distilled water

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ACKNOWLEDGEMENTS

All praises for all Almighty Allah, who is the most merciful and compassionate, the

greatest source of knowledge and wisdom, and who bestowed upon me the skill and

intellect to conduct myself to accomplish the assigned project in the most humble manner.

I would like to express my deepest and heartiest gratitude to my learned and distinguished

supervisors Prof. Dr. Muhammad Naeem, Chairman, Department of Entomology, Pir

Mehr Ali Shah, Arid Agriculture University, Rawalpindi and

Dr. Ehsan-ul-Haq, Program Leader/Principal Scientific Officer, Integrated Pest

Management Program (IPMP), National Agriculture Research Centre, Islamabad, for their

guidance in planning, implementation and completion of this research work. I am thankful

to Dr. Iftikhar Ahmed, Ex-Director General, National Agriculture Research Centre,

Islamabad, for giving me an opportunity and provide research facilities to undertake these

studies.

I feel pleasure to express my gratitude to Mr. Imtiaz Ellahi, Ex-Chairman, Capital

Development Authority, Islamabad, for granting me permission for admission in Pir Mehr

Ali Shah, Arid Agriculture University, Rawalpindi.

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I am thankful to Dr. Zahoor Salihah, Ex-Chief Scientist, Nuclear Institute for Food and

Agriculture (NIFA) and Dr. Abdus Sattar, Dean, Faculty of Agriculture, Abdul Wali

Khan University, Mardan, for identification of termite species and their valuable

suggestions and useful comments during my study.

Thanks are also extended to Prof. Dr. Ata-ul-Mohsin, Department of Entomology and

Prof. Dr. Fayyaz-ul-Hassan, Department of Agronomy, for their encouragement and co-

operation during the course of study. I like to extend my thanks to Mr. Abdul Shakoor,

Ex- Chairman, Department of Statistic, Dr.

Muhammad Asif, Dr. Muhammad Munir and Dr. Muhammad Tariq,

Department of Entomology, Pir Mehr Ali Shah, Arid Agriculture University, Rawalpindi,

for statistical analysis of the data and their valuable suggestions during my study.

Special thanks to Mr. Javeed Khan, Senior Scientific Officer, National

Agriculture Research Centre, Islamabad and Mr. Miskatullah, Research Associate,

Museum of Natural History, Islamabad, for their cooperation during the course of my

study.

I shall be failing in my study if I do not acknowledge the financial support, the inspiration,

well wishing and prays of my affectionate parents and other family members, who provide

moral support during the course of this work.

(ABDUL SATTAR)

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ABSTRACT

The current studies were focused on ecology and management of termites in urban

environment of Islamabad. Effect of temperature, relative humidity and precipitation on

population dynamics, density and foraging activities of

Microtermes obesi and Odontotermes lokanandi were studied from 2010 to 2012. A total

of 1200 poplar wooden stakes was used for monitoring termite activity. Out of 1200 poplar

wooden stakes, only 65 were found infested by M. obesi and O. lokanandi. Positive and

significant correlation was found among atmospheric temperature, precipitation and

population of two species i.e., M. obesi and O. lokanandi; however, a negative correlation

was found between relative humidity and foraging activities of both species.

Dye-markers viz., Nile blue-A and Sudan red-7B at three levels were evaluated against

termite. Results showed that Nile blue-A at high concentration caused 100% mean

mortality in M. obesi, followed by 65.06 and 59.18 % at medium and low concentrations,

respectively. When M. obesi was treated with Sudan red-7B, 100% mean mortality was

found on 9th day at high concentration, followed by 92.10% and 86.89% at medium and

low concentrations, respectively.

Similarly, 100% mean mortality was observed at highest concentration on 5th day of the

trial, when O. lokanandi was treated with Nile blue-A, followed by 71.09% and 61.67% at

medium and low concentrations, respectively. When O.

lokanandi was force-fed on different concentrations of Sudan red-7B; 100% mean mortality

was recorded at high concentration, on 5th day, followed by 57.19% and

42.53% mean mortality at medium and low concentrations, respectively.

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Dye markers (Nile blue and Sudan red) were evaluated to find out proper concentration

that will remains visible for longer period of time. The results revealed that more than 90%

termites retained dye markers in their bodies up to twenty five days for Nile blue-A at 0.25

and 0.125% concentrations and for 15 (fifteen) days at 0.50% concentration; where as 90%

Sudan red was retained for 10 (Ten) days at 0.125 and 0.25% concentrations and for five

days at 0.50% concentration. After 60 days, 59.33% termites were observed blue at 0.125%

concentration, followed by 42% at 0.25% concentration of the same dye; while all the

termites, stained with Sudan red, were found dead on day 60.

Leaf and seed extracts of Euphorbia helioscopia L. (Sun spurge), Cannabis sativa L.

(Bhang) and Calotropis procera (Ait.) (Ak) were tested against M. obesi and O. lokanandi.

Results revealed that all extracts showed moderate toxic effect, however, 100% mortality

in termite (M. obesi) was observed on 11th day; while 100% mortality in O. lokanandi was

noted on 7th day of the trial. In the present studies, Copper Sulphate (CuSo4) and Mercuric

Chloride (HgCl2) at 3 levels were tested against M. obesi and O. lokanandi. The results

revealed that both inorganic insecticides were found palatable and slow-acting toxic.

Glucose, yeast, urea and poplar saw dust extract were tested to find out better

phagostimulants. The results showed that all phagostimulant attracted termites and

maximum survival was observed. The present study was focused to formulate palatable and

slow-acting toxicant baits for an effective control of termites. Baits of Mercuric Chloride,

Copper Sulphate, C. procera, E. helioscopia and C. sativa were tested against termites. The

results showed that baits of Mercuric Chloride, Copper Sulphate were found palatable and

slow-acting; whereas baits of C. procera, E. helioscopia and C. sativa were found repellant.

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Chapter 1

INTRODUCTION

Termites or white ants are eusocial roaches (Inward et al., 2007), belonging

to the order Blattodea. They are moderate sized, thin-skinned, slender insects,

consisting of several castes. They are polymorphic living in colonies that comprise

of reproductive, soldiers and workers. Both winged and wingless individuals occur

in a colony. The queen is very much bigger than the king. Mouthparts are of chewing

type and metamorphosis is simple. Worker termites perform taking care of the brood,

maintaining and repairing the nest, and foraging for food (Krishna,

1969), moreover, they feed other castes i.e., soldiers and functional reproductive

(Grasse, 1939; Noirot and Noirot-Timothee, 1969). Termites are a large group of

organisms of which there are more than 2600 species (Kambhampati and

Eggleton, 2000), these are grouped into seven extant families i.e., Mastotermitidae,

Kalotermitidae, Hodotermitidae, Termopsidae, Serritermitidae, Rhinotermitidae and

Termitidae (Pearce, 1997). In addition, there are 14 sub families and 270 genera in

the order Isoptera (Kambhampati et al., 1996).

Termite colonies live in nests. Nest building is an innate behavior of termites

(Emerson, 1938; Theraulaz et al., 1998, 2003), often resulting in speciesspecific

architectures. Abe (1984) reported that on the basis of nest system and feeding habits

of termites, there are six nesting systems i.e., i) Drywood termites, ii) Dampwood

termites, iii) Intermediate termites, iv) Arboreal termites, v) Subterranean termites,

and vi) Humus feeding termites. These systems were called

1

20

life types, and can be further divided into three main groups of nests: single-piece,

intermediate, and detach types of nests (Abe, 1987).

Studies revealed that subterranean termites infest and damage buildings,

wooden structures, and more than 50 species of living plants (Grace et al., 1996a;

Osbrink et al., 1999; Messenger et al., 2000; Lax and Osbrink, 2003), a variety of

agricultural crops (Dawes-Gromadzki, 2005), moreover, they can also physically

damage non-cellulosic materials such as buried electric and telephone wires and

insulation (Henderson and Dunaway, 1999). Su (2003) reported that termites damage

a total of US $ 22 billion each year world-wide.

The control of subterranean termite is grouped into preventive and curative

measures (Su and Tamashiro, 1987; Su and Scheffrahn, 1990). Several physical

preventive methods have been used to control subterranean termites. Ebeling and

Pence (1957) observed that the use of selected uniform size sand particles as barrier

that could act as physical exclusive device against subterranean termites. The use of

these uniform sized particles is based on the fact that the sand particles were too big

for termites to dislocate with their mouths, however very small enough to stop

termites from piercing through the holes between them (Smith and Rust, 1990; Su

and scheffrahn, 1992). French et al. (2003) reported that “Granitgard” a good

example of the physical barrier. Stainless steel mesh is also termite exclusion device

(Grace et al., 1996b).

Highly effective chemical treatments have been available for many years to

prevent subterranean termite attack and to control infestation. Many termitologists have

studied the effect of organic insecticides to contain subterranean termites

21

(Srivastava, 1978; Kalra, 1979; Thakar et al., 1991; Vidyasagar and Bhat, 1991).

Roonwal and Chatterjee (1961) used BHC (Benzene hexachloride), DDT (Dichloro-

diphenyl-trichloroethane) and Aldrin for the destruction of O. obesus colony. The

frequent use of fast-acting termiticides to contain termites has caused a number of

biological and environmental risks. Increased concern over environmental

contamination and threat to human health by the current termiticides particularly

chlorinated hydrocarbon has led to search for new biological materials and

innovative approaches to termite control. Singh and Saratchandra (2004) reported

that plants and their extracts were used as alternate for synthetic insecticides. These

plant extracts are nerve poisons (Shahid, 1999), and have been used as repellents

since long time (Isman, 1997). Logan et al. (1990) noted that many plants contain

chemicals, but their potential need to be explored. Neem and

Calotropis extract have been used for termites control (Deka and Singh, 2001; Singh

et al., 2002). Plant extracts are less expensive and environmentally safe. Many

farmers had been using plant extracts and wood ash in Asia and Africa for

management of termites (Anonymous, 2000).

Soil insecticide barriers have been the single most important tools for

subterranean termite control of buildings during the last few decades, but limitations

with current soil termiticides have provided the impetus to look for alternatives.

Interest in the use of slow-acting toxicants to suppress the populations of

subterranean termites has been renewed (Su et al., 1982a; Jones, 1984). As suggested

by Beared (1974) the success of slow-acting toxicant bait depends upon its attraction,

palatability, delayed mortality and should be introduced into the colony‟s gallery

system and transferred to unexposed nest-mate by social grooming or trophallaxis.

Su (1982) stressed on the importance of using a slow-acting and non-repellent active

22

ingredient in termite baiting. Jones (1991) was the first to evaluate borate in baits for

population control of the field colonies of Heterotermes aureus. Forschler (1996)

recorded the effect of abamevtin and zinc borate-treated sawdust as a slow-acting

poison against Reticulitermes sp. in the field. As a part of investigation into efficacy

of slow-acting insecticides against termite it is necessary to find a method of marking

termite (Su et al., 1987). There are two alternative methods of detection of

subterranean termites: by using (i) radiotracers and (ii) mark-release-recapture

techniques by using dyes (Su et al., 1982a).

Radiotracers can be used for detecting the nesting system of subterranean

termites. To trace termites in their natural environment, different researchers used

radioisotope that is non toxic, has a short biological half-life, a longer physical half-

life, energetic gamma-rays to allow monitoring through several centimeters of soil,

and would pass through the colony by trophallaxis (Sprag and Fox, 1974; Easey,

1983). The short lived radioisotopes Gold-198, Lanthanum-140 and Iodine131 have

been used widely for determining colony distribution of termites (Kannowski, 1959;

Kloft and Holldbler, 1964; Kloft et al., 1965; Spragg and Fox, 1974; Paton and

Miller, 1980; Huang, 1982; Easey, 1983).

Mark-release-recapture method is another effective way of delineating

foraging territories of termite as well as estimating their populations. Dyes were first

used in Hawaii to measure the distance traveled by Coptotermes formosanus

workers, fed filter paper impregnated with fast green, between interconnected traps

(Fujii, 1975). Lai (1977) and Lai et al. (1983) evaluated nine histological dyes and

reported that Sudan Red-7B was the most persistent and least toxic dietary dye

marker for C. formosanus. Many termitologists used dye such as Nile blue, Sudan

23

red, or flurescent spray paint were used to delineate foraging territory of termite

colonies (Forschler and Ryyder, 1966; Su et al., 1991b; Myles et al., 1994; Su, 1994;

Chambers and Benson, 1995; DeMark et al., 1995).

Keeping in view the economic importance of the termites as common

problematic pests of agricultural crops and buildings, a study was initiated with the

following objectives:

Objectives

1. To study ecology of termites in Islamabad area.

2. To evaluate Dye Markers to know the non-toxic and proper

concentrations of visible dyes.

3. To evaluate insecticidal potential of the plant extracts and in-organic

insecticides against termites.

4. To formulate non repellent and slow-acting toxicant baits for the management

of termites.

Although these objectives focused on the basic concept of the project; a few other

studies were also conducted.

24

Chapter 2

REVIEW OF LITERATURE

Different researchers in the world have studied the various aspects of

subterranean termites and tried to contain these menace creatures by using different

methods. The previous work done by different termitologists can be documented in

the form of review under the following sub-headings:

2.1. ECOLOGICAL STUDIES

Fruit trees, food crops, residentials and other buildings are seriously damaged

by one of the four ecological types of subterranean termites than the other dry wood,

damp wood and harvester termites. As they are more abundant, their cryptic habits

make study of their populations extremely difficult. Their nests or reproductive

centers may be underground or in mounds on the surface, in the stumps or logs,

within or attached to the trunks or branches of trees. A colony may consist of a single

centre or of several interconnected units (Nutting and Johnes,

1990).

Studies revealed that members of Termitidae and Nasutitermitinae are often

numerically dominant in the tropical zones, and these are the most abundant

woodfeeding group (Martius, 1994; Miura et al., 2000; Dawes-Gromadzki, 2005;

Torales et al., 2007). Noirot (1970) reported that some species of termites are

arboreal nesters. Arboreal termitaria use as homes by many other organisms i.e.,

birds (Collias, 1964; Brightsmith, 2000, 2004; Kesler and Haig, 2005), bats

(Dechmann et al., 2004), bees (Barreto and Castro, 2007), and ants (Jaffe et al.,

6

25

1995). Flores-Palacios and Ortiz-Pulido (2005) reported that termites serve as food

for vertebrates i.e., anteaters (Lubin et al., 1977; Lubin and Montgomery, 1981),

lizards (Colli et al., 2006) and invertebrates i.e., ants (Schatz et al.,1999; Souza and

Moura, 2008) and assassin bugs (McMahan, 1982, 1983).

Some termite species benefit nutritionally from wood that has been partially

digested by fungi (Hendee, 1933, 1935), moreover, to digest celloluse all termite

species rely on intestinal or external symbiotic microorganisms (Ohkuma, 2003).

Studies showed that termites accumulate microbes in their nests (Holt, 1998;

Lopez-Hernandez, 2001; Fall et al., 2004; Ndiaye et al., 2004; Dupponois et al.,

2005; Jouquet et al., 2005; Gutierrez and Jones, 2006), termites and soil microbial

communities also compete for similar resources, which may influence termite

nesting or abundance (Holt, 1996). Moreover, entomopathogens, Cordyceps fungi

being some of the most common, heavily loaded tropical soils (Evans, 1982; Schmid-

Hempel, 1998), termites have the sense to detect and avoid pathogens (Zoberi, 1995;

Staples and Milner, 2000; Mburu et al., 2009) and they may potentially select nest

sites on basses of parasites and pathogens (Cruse, 1998).

2.1.1. Population dynamics

Studies on the population dynamics of subterranean termites in field are

complicated by their small size, cryptic nature, and eusocial behaviour. Many studies

have documented their abundance and diversity in tropical regions (Wood and Sands,

1978). Different techniques have been used to estimate subterranean termite i.e.,

habitat sampling (Haverty et al., 1975), radioactive tracers (Spragg and Paton, 1980),

exhaustive trapping (French and Robinson, 1981) and mark-releaserecapture

techniques (Esenther, 1980; Su et al., 1984). Mark-release-recapture methods

26

directly estimate population size and, being non destructive, permit longterm study

of the same termite populations. In addition, by using mark-releaserecapture

techniques for Reticulitermes spp. it has been estimated that foraging populations

ranging from 0.3 to 5 million termites per colony (Esenther, 1980, Grace et al., 1989,

Grace, 1990, Su et al., 1993, Su, 1994; Su et al., 1984; Baroni urbani et al., 1978).

Easey and Holt (1989) used radiotracers Iodine-131 and Gold198 for termite

population study and reported that mark-recapture method is relatively simple and

gives population estimate within the expected range. Populations of termite can be

estimated by using the "Lincoln Index" (Ayre, 1962; Southwood, 1971) or by

"marking-release and recapture methods" (Andrewartha and Birch, 1967). To study

population dynamics different scientists have used excavated nest (Holdaway et al.,

1935; Gay and Greaves, 1940; Rohrmann, 1977; Ohiaqu, 1979; Collins, 1981;

Howard et al., 1982), although this procedure excludes termites in peripheral

foraging galleries.

Termites play an important role in ecosystems in tropical and subtropical regions

(Wood and Sands, 1978). Because of ecosystem engineers, they can affect the

survival of other species (Mills, 1993; Jones et al., 1994; Hansell, 2005; Jouquet et

al., 2006). Many termitologist reported that they are also major decomposers of

organic matter (Holt and Lepage, 2000; Yamada et al., 2005), as a result nutrients

accumulate in their nests that positively influences other soil biota, and increases

primary productivity (Wood and Sands, 1978; Holt and Lepage, 2000; Dupponois et

al., 2005; Jimenez et al., 2006; Barot et al., 2007; Brossard et al., 2007).

2.1.2. Foraging ecology

27

Curtis and Waller (1997) reported that foraging biology of termites is poorly

known. Foraging intensity of subterranean termites is directly related to temperature

and moisture (LaFage et al., 1976). Studies revealed that termites are more likely to

be found in areas with vegetative cover up than open areas (Light,

1934; King and Spink, 1969; Jones et al., 1987). Kofoid (1934) reported that

Reticulitermes species were attracted to dead roots of trees and plants. Subterranean

termite colony can move about several hundred to a few thousand square meters

during their foraging activity (Su et al., 1993; Su, 2001), and contain up to five-to-

seven million foraging termites (Jones, 1988; Su et al., 1993).

Some studies revealed that the foraging activity of subterranean termite

increases as soil temperature gradually increases (Smythe and Williams, 1972;

Delaplane et al., 1991). Kofoid (1934) observed that seasonal fluctuations in the

termites foraging were directly correlated to air temperature. The foraging activity

and feeding rate of Coptotermes formosanus was positively correlated with

temperature (Delaplane et al.,1991). Smith and Rust (1994) mentioned a favorable

temperature range for a desert termite species, Reticulitermes hesperus Banks, he

further told that thermal shadows under vegetation might provide refuge from high

temperatures.

Studies revealed that relative humidity is a vital factor in the survival and

feeding rate of subterranean termites (Kofoid, 1934; Light, 1934; Collins, 1969;

Rudolph et al., 1990; Forschler and Henderson, 1995). Subterranean termites have

high moisture requirements (Kofoid, 1934; Holway, 1941; King and Spink, 1969;

Becker, 1972; Williams, 1977; Puche and Su, 2003). Rain fall that declined soil

temperature and increase soil moisture and relative humidity increasing foraging

28

activity of subterranean termites (Collins et al., 1973; La Fage et al., 1976; Black

and Wood, 1989).

Studies revealed that the foraging activities of termite are intensed during the

summer months, with daily peak in late morning and afternoon, but occurrs around

noon in winter (Nutting et al., 1975; Evans and Gleeson, 2001). Different termite

species response to temperature and moisture is differently, with Reticulitermes

tibialis and R. flavipes preferring forage habitats with cool temperatures and high

moisture. Whereas other termite species i.e., Heterotermes aureus Snyder and R.

hageni, forage during periods of extended heat and relatively low moisture

(Nutting et al., 1975; Jones, 1988; Haagsma and Rust, 1995; Houseman et al.,

2001). Subterranean termites prefer sandy soils than in clay-loams (Jones, 1988).

2.1.3. Species identification

Several identification keys for termit identification are available (Banks and

Snyder, 1920; Banks, 1946; Snyder, 1954; Chaudhry et al., 1972; Gleason and

Koehler, 1980; Nutting, 1990; Scheffrahn and Su, 1994). Identification of species

within the genus Reticulitermes using these published keys has been called into

question (Thorne, 1998; Jones, 2000), this genus has been called for revision

(Weesner, 1970), various authors has come back recently (Haverty and Nelson,

1997; Thorne, 1998; Haverty et al., 1999a; Jones, 2000). Pickens (1934) reported

that identification is complicated by the possibility of hybridization between species

within a genus, specifically between R. tibialis Banks and R. hesperus

Banks, between R. flavipes and R. virginicus (Banks, 1946; Howard and Haverty,

1981). Studies revealed that taxonomic studies of specimens from different colonies

of Reticulitermes currently identified as the same species may have multiple

29

cuticular hydrocarbon phenotypes. Termitologists reported that cuticular

hydrocarbon phenotypes are thought to be species specific among termites (Haverty

et al., 1991; Haverty et al., 1996; Haverty and Nelson, 1997; Haverty et al., 1999b;

Jenkins et al., 2000; Nelson et al., 2001).

2.1.4. Caste composition in termite colony

Various factors i.e., temperature and social conditions can influence caste

differentiation process in social insects (Henderson, 1998; Mao et al., 2005; Scharf

et al., 2007). Subterranean termite colonies exhibit social polymorphism with an

organized caste system. Castes include immatures (larvae), nymphs (intermediates),

soldiers, workers, and reproductives (primary king and queen; neotenics). Colonies

of termite are comprise of multiple social group phenotypes

(Scharf et al., 2007; Miura and Scharf, 2010).

The queen termite is eggs laying machine, depending on her age and size, is

capable of laying eggs at the rate of 36,000 a day for a long as 50 years.The queen

is much larger in size than the king. Worker termites perform taking care of the

brood, maintaining and repairing the nest, and foraging for food (Krishna, 1969),

moreover, they feed other caste i.e., soldiers and functional reproductive (Grasse,

1939; Noirot and Noirot-Timothee, 1969). The primary role of the soldier caste is

defense within the colony. The proportion of soldier in colonies of R. flavipes is

upto 10.8% (Banks and Snyder, 1920; Haverty, 1977).

2.2. EVALUATION OF DYE MARKERS

King and Spink (1969) demonstrated that subterranean termite colonies live

in widespread networks of underground galleries, these galleries lengthen

30

underneath concrete and decorative planting in the urban environment (Grace et al.,

1989). Due to this reason, the demographics of termite colony and foraging activities

are difficult to study under natural conditions. Suitable marker and marking

technique is needed for marking-release and recapture method (Southwood, 1971),

a marker must be: 1) not affect the longevity or behavior of the animals; 2) be

recognizable during the experiment; and 3) not change the behavior of the colony

towards the marked animal. Gosswald and Kloft (1963) recorded that radioactive

items have been used to mark termite colony.

Fujii (1975) reported that dyes were first used in Hawaii to measure the distance

traveled by C. formosanus workers, fed filter paper impregnated with fast green,

between inter-connected traps. Lai (1977) and Lai et al. (1983) screened nine

histological dyes and reported that Sudan red 7B was the most persistent and least

toxic dietary dye marker for C. formosanus.

The technique of mark-release-recapture has been successfully used to study

the population dynamics of Coptotermes formosanus Shiraki (Lai, 1977; Su and

Scheffahn, 1988a) and Reticulitermes flavipes (Kollar) (Esenther, 1980; Grace et al.,

1989; Su and Sheffrahn, 1988b). Studies revealed that termites captured in traps of

wood or corrugated paper, then in the laboratory force fed paper impregnated with

the oil-soluble dye, Sudan red 7B to colour the termites, then released at the same

point in the field site and recaptured at consecutive period (Grace, 1989; Su and

Scheffrahn, 1986). The dye is not passed noticeably by social grooming or

trophallaxis (Grace and Abdallay, 1989; Su et al., 1983). During foraging activities

the distance covered by termites can be measured by the marked individuals in each

sample (Baroni-Urbani et al., 1978; Jackson, 1939). The maximum distance thus

measured of 160 feet was very close to the distances of 165 feet (Ehrhorn, 1934) and

31

200 feet (King and Spink, 1969) mentioned from destructive excavations of

Formosan subterranean termite galleries.

Grace and Abdallay (1989) reported that R. flavipes force fed on Sudan red

7B at 2% concentration or less up to 5 days, is retained for approximately 15-20 days.

Su et al. (1988) reported that an alternative dye marker is required for longer mark-

release- recapture studies. Sudan red 7B was not passed in detectable quantities by

trophallaxis (Su et al., 1983; Lai, 1977), moreover, Sudan red 7B caused delayed

mortality, and with time, the dye faded sufficiently that it could not be seen in an

increasing number of termites (Su et al., 1983; Delaplane et al., 1988). Grace and

Abdally (1989) mentioned that Sudan red 7B could safely be used with shorter 3

week release-recapture cycles with Reticulitermes flavipes.

Neutral Red was also identified by Salih and Logan (1990) as the most promising of

30 dyes listed as markers for Microtermes lepidus sjostedt. The search continues for

additional dye markers, to use either singly or in combination (Grace and Abdally,

1990). Su et al. (1991b) identified Nile Blue as a safe and persistent marker for R.

flavipes.

2.3. PLANTS AND THEIR PRODUCTS

Different type of plants and their extracts are studied for their insecticidal

actions (Isman, 2000; Weaver and Subramanyam, 2000; Koul, 2004; Mordue,

2004; Erturk et al., 2004; Negahban and Moharramipour, 2007). Jbilou et al. (2006)

reported that higher plants contain a rich source of novel natural substances, which

is environmentally safe, and these could be used to control insect. Plant extracts,

essential oils, botanicals and bark, seed, wood, fruit and leaf extracts which are

environmentally safe can be used for termite control (Adams et al., 1998; Singh and

32

Saratchandra, 2004; Verma et al., 2009), these plants contain chemicals that keep

away or kill termites or hamper with their gut flora (Adams et al., 1998;

Boue and Raina, 2003).

Studies revealed that plant and their products could be used to control some

insect pests (Essien, 2004; Erturk et al., 2004; Koona and Dorn, 2005; Chapagain

and Wiesman, 2005). Roy et al. (2005) studied extracts of Shiyalmutra (Blumea

laceera L.) against stored grain pests in the laboratory. Studies revealed that both the

plants and their extracts are toxic to animals and human beings and these can be used

across the world to protect agricultural crops (Zhu et al., 2001; Isman, 2006; Islam

et al., 2011). The crude extract of plants being the mostly used in termite control

(Ogunsina et al., 2009; Upadhyay et al., 2010; Manzoor et al., 2011; Elango et al.,

2012). The Cymbopogon Citratus Stapf (Lemon grass),

Cinnamomum cassia Nees (Cassia leaf), Vetiveria zizaniodes L. (Vetiver)

(Maistrello et al., 2001), Eucalyptus citriodora Hook., Eucalyptus globules Labille.

(Eucalyptus), Cedrus atlantica Glauca (Cedar wood), Syzgium aromaticum L.

(Clove bud) (Zhu et al., 2001), Coleus amboinicus Lour. (Singh et al., 2004),

Isoborneol (Blaske et al., 2003) and Calotropis procera (Ait.) (Singh et al., 2002)

are some of the important plants that can be used for termite control.

Nilanjana and Chattopadhyay (2003) reported that extracts from Tamarindus

indica L., Cynodon dactylon (L.), Rauvolfia serpentine (L.), Adhatoda vasica Nees,

Cleistanthus collinus (Karra), Pongamia pinnata (L.) and Eichhornia crassipes

Antwort controlled the termites, Microcerotermes mycophagus. Research indicated

that thiophenes from four species of Echinops in addition columellarin and

33

sesquiterpene lactone fraction from the heartwood of Australian white cypress

(Callitris glaucophylla J). had shown anti-termitic action against

Coptotermes formosanus Shiraki (Watanabe et al., 2005; Fokialakis et al., 2006).

Drywood termites (Cryptotermes brevis) can be controlled effectively by using the

Piper nigrum L. (Moein and Farrag, 2000).

2.4. PHAGOSTIMULANTS

Feeding behavior of subterranean termites is influence by different compounds

and these compounds are used as phagostimulants. Doi et al. (1999) reported that some

species of woods and their products contain water-soluble compounds that act as

feeding stimulants for subterranean termite. Chen and

Henderson (1996) reported that some proteins can be used as phagostimulants.

Waller and Curtis (2003) observed that Reticulitermes virgin and R. flavipes ate

significantly higher filter paper soaked in 3 percent solutions of sugar than filter

paper soaked in distilled water. different wood-rot fungi, particularly Gloeophyllum

trabeum or brown wood rot fungi attract subterranean termite (Amburgey and

Smythe, 1977; Amburgey, 1979; Esenther and Beal, 1979).

i) Yeast

Waller (1996) conducted experiment on urea, sucrose and yeast solutions to

find out a best phagostimulant. Urea, sucrose and yeast are likely to be encountered

by termites in the soil and decaying wood in much lower concentration in nature

(Anderson, 1962; Martin, 1979). Prillinger et al. (1996) reported that laboratory tests

were carried out for 6 lower termites species to obtain 39 isolates of dimorphic fungi

from the hindgut. Using RAPD-PCR the 39 yeast isolates were assigned to 13

34

different species. Evidence is provided that the yeasts isolated from the hindgut can

be considered symbionts.

ii) Urea

Haifig et al. (2008) described that trehalose and urea solutions are

phagostimulant to Heterotermes tenuis. Heterotermes tenuis fed preferentially on

filter paper treated with 0.03 g/ml trehalose and 0.015 g/ml urea solutions. Martin

(1979) reported that fungi contain urea, on which termites frequently feeding in

decayed wood.

iii) Plant Extracts

Wakako et al. (2005) described that the chemoreception of plant extracts

were evaluated to investigate the water extracts from akamatsu (Pinus densiflora),

neem (Azadirachta indica) and their mixture. The result showed that termites

preference was akamatsu > akamatsu plus neem > neem. Termites showed a strong

preference for the fungus-inoculated sawdust when released in paired choice tests

with fungus-inoculated sawdust against control sawdust (Mary et al., 2004), poplar

wood was more attractive (Salihah et al., 1993).

Termites have the ability to process wood for their nutritional source (La Fage

and Nutting, 1978), some species of wood are preferable (Morales-Ramos and

Guadalupe, 2001, 2003) and their life extend when fed the preferred wood species

(McMahan, 1966; Morales-Ramos and Guadalupe, 2001, 2003). Studies revealed

that ions (Botch et al., 2010), high wood density (Waller et al., 1990), sugar (Waller

and Curtis, 2003; Swoboda et al., 2004; Saran and Rust, 2005) and high levels of

cellulose (Judd and Corbin, 2009) can increase termites baits consumption.

35

Subterranean termites preferred decayed wood by certain species of fungi (Becker,

1976; Amburgey, 1979), and fungus-infected sawdust (Cornelius et al., 2000).

iv) Glucose

Raj and Rust (2005) reported that sugars act as phagistimulants to the termites

and they metabolize carbohydrates such as sucrose, and thus their use in bait may

increase consumption. Anderson (1962) reported that sucrose is found in decomposing

wood and the termites feed on these decomposing wood (Waller et al., 1987).

Abushama and Kambal (1977) recorded that Microtermes traegardhi Sjo¨ stedt

preferred disk soaked in fructose, Heterotermes tenuis Hagen respond to trehalose but

not other sugars (Haifig et al., 2008), Reticulitermes spp. glucose (Swoboda et al.,

2004). Saran and Rust (2005) studied that Reticulitermes spp. showed an increase in

feeding when the food was treated with xylose, ribose, maltose, or fructose.

Reticulitermes virginicus Banks ate significantly high filter paper treated with sugars

glucose, xylose, and sucrose (Waller and Curtis, 2003).

Lenz et al. (2009) recorded that different types of termite species preferred

different type of sugars. Ants and termites show a seasonal response to a variety of

nutrients, including lipids (Ricks and Vinson, 1972), protein (Ricks and Vinson

1972; Stein et al., 1990; Judd, 2005), and carbohydrates (Sudd and Sudd, 1985; Stein

et al., 1990; Judd, 2005). Botch et al. (2010) reported that a seasonal change in

response to phosphates has been noted for Reticulitermes flavipes Kollar. Different

things may provide different nutrients (Shellman-Reeve, 1994). Saran and Rust

(2007) reported that sugars are used as a phagostimulants in baits.

v) Chemicals

36

Yoshimura et al. (1987) concluded that mercuric chloride is a slow-acting

toxicant. The action of heavy metals are slow-acting against termites (Watson and

Lenz, 1990). Seven in-organic compounds i.e., mercuric iodide, zinc sulphate, sodium

arsenate, cadmium chloride, boric acid, lead acetate

and

molybdophosphoric acid can be used against Heterotermes indicola (Sattar, 2000).

Studies revealed that sulfluramid (Su and Scheffrahn, 1988b, 1991; Henderson and

Forshler, 1996), diiodomethyl-p-tolyl sulfone (A-9248) (Su and Scheffrahn,

1988a), dechlorane (mirex) (Esenther and Gray, 1968; Esenther and Beal, 1974;

Ostaff and Gray, 1975; Esenther and Beal, 1978; Paton and Miller, 1980),

hydramethylnon (Su et al., 1982a; Pawson and Gold, 1996) and borates are all

compounds in this class that have been used in termite baits. Foraging activities and

or colony populations were reduced by using A-9248 (Su et al., 1991a),

hydramethylnon (Su et al., 1991a; Pawson and Gold, 1996) and sulfluramid (Su et

al., 1995). Research has revealed that termite mortality occurs by using borate

(Khoo and Sherman, 1979; Ahmed et al., 2004; Kartal and Ayrilmis, 2005). Borates

are acting as metabolic poisons, causing toxicity through biostatic rather than

biocidal mechanisms (Lloyd et al., 1990). The interaction of borates with

nicotinamide adenine dinucleotide (NAD+), riboflavin, coenzyme A and vitamins

B6 and B-12 has been studied (Lloyd et al., 1990; Williams et al., 1990; Woods,

1994). Saghir et al. (2011) reported that A-9248 is also a slow acting compound that

is non-repellent and a biocide.

2.5. BAITS

The concept of a baiting technique for termite pest management dates back

to 1968 (Esenther and Gray, 1968), with subsequent research investigating various

slow-acting active ingredients were used (Beard, 1974; Esenther and Beal, 1974,

37

1978). Placement of stations below or above-ground, each containing cellulose

material, around the perimeter of a structure. In one baiting system, these stations are

initially used to monitor for termite activity. Once activity is detected, chemical bait is

placed in the station. Subterranean termites consumed these baits and passed to other

members of the colony via trophallaxis. The intention of baiting systems, however, is

to suppress colony numbers, disrupt foraging, and potentially eliminate entire colonies.

Bait has necessitated the development of non-toxic and persistent dyes such as Nile

Blue (Su et al., 1991b), Neutral Red (Esenther, 1980), and purple blend (Oi, 2000;

Atkinson et al., 2004).

Su (1982) stressed on the importance of using a slow-acting and nonrepellent

active ingredient in termite baiting. Basically, there are three groups that meet the

requirements of being the appropriate bait toxicant: (i) the metabolic inhibitors, (ii)

biological control agents, and (iii) insect growth regulators (IGRs). Su et al. (1982a)

revealed that the evaluation of bait toxicants could not be based on termite mortality

alone; the behavioural responses of the termites to the insecticides also had to be

considered. This is because termites can seal off, or avoid treated areas and

effectively protect themselves.

i) Metabolic inhibitors

Su et al. (1982a) tested the possibility of using hydramethylnon for the control of

subterranean termite C. formosanus in the laboratory. Jones (1991) was the first to

evaluate borate in baits for population control of field colonies of H. aureus.

Forschler and Townsend (1996) using abamectin and zinc borate-treated sawdust

revealed the potential use of these toxicants against subterranean termites

Reticulitermes sp. in the field. Wooden boards impregnated with sulfluramid at

38

higher concentration were initially accepted by termites (Su and Scheffrahn, 1991;

Su et al.,1995), sulfluramid at the concentration of less than 100 ppm may be

desirable and effective against C. formosanus (Grace et al., 2000). Other potential

toxicants of this group include diiodomethly para-toly sulfone (Su and Scheffrahn,

1988b) and boric acid (Mori, 1987).

ii) Fungi (bioagent)

The possible usage of pathogenic fungi in baits to control Formosanus

subterranean termite C. formosanus was pioneered by Lai (1977) and had been

discussed by Delate et al. (1995) and Jones et al. (1996). However, the conidia of

the fungus were found to have in dormant stage in field colonies which like due to

temperature humidity, inhibition by soil microorganisms and fungistatic secretions

produced by termites. Milner (2001) used M. anisopliae as bait matrix.

iii) Insect Growth Regulators (IGRs)

Insect growth regulators have attracted great attention as promising bait

toxicants. They induced abnormalities in physiological development of insects

(Pallaska, 1997). IGRs are group into juvnoids and juvenile hormones mimics and

chitin synthesis inhibitors (Su and Scheffrahn, 2000). Effect of methoprene were

studied against subterranean termite species C. formosanus and Reticulitermes spp.

(Howard, 1983; Su et al., 1985; Haverty et al., 1989).

Chapter 3

39

IMPACT OF ENVIRONMENTAL FACTORS ON THE

POPULATION DYNAMICS, DENSITY AND FORAGING

ACTIVITIES OF ODONTOTERMES LOKANANDI AND

MICROTERMES OBESI IN ISLAMABAD

3.1. ABSTRACT

Affect of different environmental factors i.e., temperature, relative humidity

and precipitation on population dynamics, density and foraging activities of

Microtermes obesi Holmgren and Odontotermes lokanandi Chatarjee and Thakur

(Isoptera: Termitidae) were studied from March 2010 to July 2012 in Islamabad. A

total of 1200 poplar wooden stakes was used for monitoring the termite activities in

Islamabad. The results showed that 65 out of 1200 poplar wooden stakes were found

infested by two subterranean species of termite i.e., M. obesi and O. lokanandi. Both

were interacting with each other in the experimental field and O. lokanandi was

found significantly dominant. Mean yield per NIFA-TERMAP ranged from 0.83 ±

0.20 to 1.12 ± 0.28 gm and 0.35 ± 0.09 to 0.82 ± 0.19 gm for

M. obesi and O. lokanandi in the field, respectively. M. obesi and O. lokanandi in

1.0 gm sample ranged from 539.83 ± 2.21 to 567.83 ± 9.41 and 407.67 ± 4.75 to

424.5 ± 1.15 individuals, respectively. Population of workers ranged from 93.53 ±

1.73 to 97.68 ± 0.40 and 91.69 ± 1.42 to 98.41 ± 0.50 percent for M. obesi and O.

lokanandi, respectively.

22

Positive and significant correlation was found among atmospheric

temperature, precipitation and both species of subterranean termite i.e., M. obesi and

40

O. lokanandi; however, the correlation was found non significant and negative

between relative humidity and foraging activities of both termite species.

Moreover, correlation was found positive and significant between

atmospheric temperature and percent workers of M. obesi; while negative and

nonsignificant between atmospheric temperature and percent workers of O.

lokanandi. Negative and significant correlation was noted between relative humidity

and percent workers of M. obesi; whereas, positive and significant correlation was

recorded between relative humidity and percent workers of O. lokanandi. Positive

and non-significant correlation was recorded between precipitation and percent

workers of M. obesi; while positive and significant correlation was observed between

precipitation and percent workers of O. lokanand.

3.2. INTRODUCTION

Subterranean termites cause significant building and structural damages

throughout the world, especially in the tropical and sub-tropical regions (Pearce,

1997), they are serious pests of urban structures worldwide (Weesner, 1969;

Edwards and Mill, 1986), and is responsible for over million dollars annually in

treatments and structural repairs (Grace, 1987). Many terminologists reported that

the termites caused considerable damage to buildings in different parts of Pakistan

(Chhotani,1977; Akhtar, 1980; Dawes-Gromadzki, 2005). Moreover, they also

damage in forest and agriculture crops (Dawes-Gromadzki, 2005).

Grace et al. (1989) reported that subterranean termites live in large colonies

and can range from about 0.2- 5 million individuals, the colony grows slowly for

many years (Bignell and Eggleton, 1998). Different techniques have been described

to study the population dynamics of termites. An underground capturing unit

41

comprise of a wooden box inside a short length of polyvinylchloride pipe, with a

plastic cover, that is placed below the soil surface at urban setting to observe

subterranean termites (Su and Scheffrahn, 1986). Esenther (1980) buried corrugated

fiberboard to capture R. flavipes. La Fage et al. (1983) reported a technique of

collecting subterranean termites from infested wood by placing a short length of

polyvinylchloride pipe containing a roll of damped corrugated fiberboard on top of

the wood. Many scientists have used excavated nest to collect data, although this

procedure excludes termites in peripheral foraging galleries

(Holdaway et al., 1935; Gay and Greaves, 1940; Rohrmann, 1977; Ohiaqu, 1979;

Collins, 1981; Howard et al., 1982).

Studies revealed that atmospheric temperature and rainfall have been found

correlated with seasonal foraging activities of termites (Abenserg-Traun, 1991;

Haagsms and Rust, 1995; Rust et al., 1996; Dibog et al., 1998; Evans and Gleason,

2001; Dawes-Gromadski and Spain, 2003; Messenger and Su, 2005; Moura et al.,

2006). Foraging activities of Coptotermes lacteus (Froggat) was found correlated

with both soil and air temperature (Evans and Gleason, 2001). Soil temperature

significantly affected the foraging activity of subterranean termites (Fei and

Henderson, 2004). Feeding at baits was negative correlated with soil moisture for

Coptotermes getroi (Wasmann) and positive correlated with soil moisture for

Heterotermes longiceps (Synder) (Santos et al., 2010). Studies have shown that

seasonal changes in the foraging behavior of subterranean termites may influence the

efficacy of baiting programs due to decline of activities during winter (Ripa et al., 2007;

Haverty et al., 2010).

42

The aim of our study was to determine whether changes in temperature,

relative humidity and precipitation affect the population dynamics, density and

foraging activities of O. lokanadi and M. obesi in Islamabad.

3.3. MATRIALS AND METHODS

3.3.1. Ecological Studies

Ecological study of subterranean termites was conducted in Islamabad, the

Federal Capital of Pakistan. Geographically, it is situated at northern latitudes 33o

42‟ 0‟‟ and eastern longitudes 72o 10‟ 0‟‟ lying at an height of 457 to 610 m higher

than sea level. Its elevation is 507 meters (1,663 feet). Islamabad lies in the

subtropical, sub-humid continental climatic zone. Total area of the Federal Capital

of Pakistan is 906 square Km. The climate of Islamabad is distinguished by cold

winters with some frost events in January and hot summers. The mean maximum

temperature is 40 oC in June; while the mean minimum temperature of January is 3

oC. The mean annual precipitation is about 1000 mm, 70% of which falls during

monsoon season (July, August and September) and remaining 30% falls in winter

(December, January and February). The soil is slightly alkaline, non-saline, loamy

in texture, low in organic matter and major nutrients with exception of available

Potassium (Nizami et al., 2004). The plant community of Islamabad consists of

Justicia adhatoda L., Mangifera indica L., Tamarix aphylla (L.) H. Karst., Acacia

modesta Wall., Dodonaea viscose (L.) Jacq., Zizyphus nummularia (Burm. F.) Wight

and Arn., Pinus roxburghii Sarg., Apluda mutica L., Quercus incana Bartr.,

Woodfordia fruticosa (L.) Kurz., Broussonetia papyrifera (L.) Venten., Fiscus palmata

Forsk. and Dicliptera roxburghiana Nees (Rashid et al., 1987).

3.3.2. Survey

43

Poplar wooden stake survey was carried out in Islamabad from March 2010

to July 2012, followed by the procedure used by (Su and Scheffrahn,1988a). A total

of 1200 monitoring stakes was driven into the soil of infested areas and were checked

fortnightly.

3.3.3. Population Dynamics of Subterranean Termites

Stakes (2.5 x 4 x 28 cm) (thickness width length) of poplar wood were buried

in termite infested areas of Islamabad, and were checked fortnightly. When any stake

was found infested by termite, a “ NIFA-TERMAP” which, consist of a PVC pipe

(8 mm thickness x 15 cm dia x 20 cm length) buried in the soil having a bundle of 5

poplar wooden slices (1.3 x 8 x 15 cm) wrapped in blotting paper covered with

earthen lid (Salihah et al., 1993), was installed on that point. The wooden stakes as

well as “NIFA-TERMAPs” were checked fortnightly and the infested traps were

changed with another new trap. The infested traps were collected and brought to the

laboratory to detach the termites from it. The collected termites were weighed. The

number of soldiers and workers were also determined in one gram termite sample.

The total numbers of termites were obtained by multiplying the number counted in

one gram with the total weight.

3.3.4. Identification of termites

From each trap a sample of 5-10 workers and soldiers were preserved in 80%

alcohol for identification of the species. The samples (soldiers and workers) were

examined under SKT-3BT dissecting steromicroscope. Determination of species

based on keys of Chaudhry et al. (1972).

44

3.3.5. Ecology of foraging termites

Foraging ecology was studied by counting the number of termites captured

by “ NIFA-TERMAP” (Salihah et al., 1993) under the prevailing temperature,

relative humidity and soil moisture of the experimental site. Air temperature and

relative humidity were measured with the help of Hygrotherm and the data of rainfall

was collected from Meteorological Department of Islamabad. The effect of relative

humidity, temperature and rainfall were also studied on the caste composition of

foraging group of termites.

3.3.6. Statistical Analysis

Statistical computation was performed by using MStat-C. Duncan‟s Multiple

Range Test was used to separate the arithmetic means of termites-biomass captured

by NIFA-TERMAPs. The same arithmetic means were compared with each other to

know the population dynamics of termites at different relative humidities and

temperature.

3.4. RESULTS

3.4.1. Ecological studies of termites in urban environment (Islamabad)

3.4.1.1. Survey

A total of 1200 poplar wooden survey stakes were used in Islamabad to

monitor the termite activities followed by the procedure used by Su and Scheffrahn

(1988). Monitoring stakes were driven into the soil of infested areas and were

checked fortnightly. Of the 1200 stakes placed in the ground, typically only 65 were

infested by two termite species i.e., O. lokanandi and M. obesi and the infested traps

were replaced with “NIFA-TERMAPs”.

45

3.4.1.2. Population dynamics

Population dynamics of termites was determined by using poplar wooden

stakes (2.5 x 4 x 28 cm). These stakes were buried in termites infested areas. The

stakes were observed after 15, 30, 45, 60, 75, 90, 105, 120, 135 and 150 days.

Observations showed that after 15 days, termites were detected at 10 stakes having

No. 03, 07, 14, 17, 79, 118, 119, 256, 415, 728; after 30 days, 24 stakes were found

infested at location of stake No. 10, 15, 18, 26, 51, 70, 73, 75, 115, 117, 170, 210,

211, 213, 255, 258, 332, 410, 417, 524, 730, 741, 756, 757; after 45 days 06 more

new infestation was found at stake No. 11, 13, 25, 54, 418, 724; after 60 days 07

more infestation were found at stakes No. 333, 334, 335, 641, 720, 731, 732; after

75 days no new infestation was recorded; after 90 days 03 stakes were observed at

location No. 523, 829, 847; after 105 days 04 more stakes were found at location No.

791, 792, 812, 826; after 120 days no news location was found infested; after

135 days 05 new infested stakes were recorded at location No. 798, 811, 825, 828,

845 and after 150 days 06 more new stakes were found infested at location No.

810, 822, 833, 838, 841 and 844 by termites (Fig-3.1).

A total of 65 stakes out of 1200 was found infested by termites after 150

days. Every infested stake was therefore, replaced by a NIFA-TERMAPs to capture a

huge number of termites from the experimental sites. A total of 10, 34, 40, 47, 47,

50, 54, 54, 59 and 65 NIFA-TERMAPs (Fig-3.2) were set up in Islamabad after 15,

30, 45, 60, 75, 90, 105, 120, 135 and 150 days, respectively. During observations

maximum NIFA-TERMAPs were found infested when the temperature and relative

humidity were recorded maximum, while minimum NIFA-TERMAPs were found

infested in comparatively low temperature and relative humidity.

46

3.4.1.3. Species of termites in the experimental areas

Table-3.1 revealed that two species of termite viz., Microtermes obesi and O.

lokanandi were found in the experimental areas. It was observed that some of the

traps always harbour the same one species and some times a single trap had a mixed

population of both species. When such traps were opened there was a great

antagonistic behaviour that they quarreled up to death of the weaker and fever

members. However, O. lokanandi was found dominant, because the frequency of

capturing O. lokanandi was much higher than that of M. obesi. This showed that O.

lokanandi was a very serious pest in Islamabad.

3.4.1.4. Yield (g) and number of termites in 1.0 gm sample

The termites, collected fortnightly from 65 infested NIFA-TERMAPs, were

weighed in Entomological laboratory of Capital Development Authority,

Islamabad. The number of individuals in 1.0 gm sample were counted.

i) Microtermes obesi

47

Fig-3.1. Location map of the study areas. The black dots show NIFATERMAPS.

Number denotes infested traps

Denotes infested traps detected after 15 days




No new infested traps detected after 75 days



No new infested traps detected after 120 days



48

Fig-3.2. Infestation of “NIFA-TERMAPs” in relation to atmospheric

temperature and relative humidity in Islamabad from April to August,

2010.

Table-3.1. Infestation of NIFA-TERMAPs by Odontotermes lokanandi (O) and

Microtermes obesi (M) at Islamabad during 2010 to 2012.

Trap No. Species of

Termites

Trap No. Species of

Termites

Trap No. Species of

Termites

49

3 O

7 O

10 O+M

11 O

13 O

14 M

15 M

17 O

18 O

25 O

26 O+M

51 O

54 O

70 O+M

73 O

75 O+M

79 M

115 O

117 O

118 O

119 O

210 M

211 M

213 O

255 M

256 O

258 O

332 O

333 O+M

334 M

335 M

410 M

415 O

417 O

418 O+M

523 O

524 O

641 M

720 M

724 O

728 O

730 O

732 O

741 M

756 O+M

757 M

791 O

792 O

798 O

810 O

811 O+M

812 M

822 M

825 M

826 O

828 O

829 O

833 M

838 M

841 O

844 O+M

845 O+M

847 O

50

170 O+M 731 O

Table-3.2 shows that mean ± SE yield of termites varied among each trap,

i.e., it ranged from 0.83 ± 0.20 to 1.12 ± 0.28 gm. Our results indicate that such

variation exists in different foraging sites of a single colony. Similarly, the mean

number of individuals in 1.0 gm sample varied greatly. It ranged from 539.83 ± 2.21

51

to 567.83 ± 9.41 individuals per sample. Variations were also found in mean number

of individuals of Microtermes obesi per gram sample of the different traps.

ii) Odontotermes lokanandi

The result shows that mean ± SE yield (g) of O. lokanandi were recorded

and it ranged from 0.35 ± 0.09 to 0.82 ± 0.19 gm. Our results showed that such

variation exists in foraging sites of different as well as a single colony. Similarly, the

number of individuals in 1.0 gm sample varied greatly. It ranged from 407.67 ± 4.75

to 424.5 ± 1.15 individuals per sample (Table-3.3). This variation is due to the size

and age of the individuals of foraging groups.

3.4.1.5. Foraging Ecology of Subterranean Termites.

The result Table-3.4 and Fig-3.3 shows that correlation was found positive

and significantly different between atmospheric temperature, precipitation and two

species of subterranean termite viz., M. obesi and O. lokanandi, however, the

correlation was recorded negative and non significantly different between relative

humidity and termites species.

Foraging activities of subterranean termite were recorded peaked in summer

months when the temperature and precipitation were high. In summer and fall ground

and atmospheric temperature was favorable for termites foraging. During Table-

3.2. Mean yield (g), mean number of termites in 1.0 gm sample and mean

percent workers in foraging group of Microtermes obesi collected from

“NIFA TERMAPs” installed in Islamabad from September 2010 to

September 2012. Trap Termite Worker

No. Proportion

52

Wt (g) Number (%)

Trap Termite Worker

No. Proportion

Wt (g) Number (%) 10 1.01 ± 0.23 553.67 ± 8.81 97.20 ± 0.57

14 1.05 ± 0.24 548.50 ± 7.57 97.68 ± 0.40

15 0.91 ± 0.23 542.83 ± 8.12 97.51 ± 0.58

26 1.12 ± 0.28 567.83 ± 9.41 97.06 ± 0.69

70 0.83 ± 0.20 560.67 ± 10.15 96.52 ± 1.21

75 0.96 ± 0.23 547.83 ± 8.84 96.02 ± 0.63

79 0.86 ± 0.21 557.00 ± 11.99 96.41 ± 1.11

170 1.05 ± 0.25 555.50 ± 7.42 94.40 ± 1.19

210 0.89 ± 0.21 551.50 ± 7.90 96.53 ± 1.11

211 0.94 ± 0.23 554.00 ± 6.57 96.48 ± 1.00

255 1.02 ± 0.26 549.00 ± 7.19 95.06 ± 1.33

333 1.02 ± 0.26 549.67 ± 7.98 93.53 ± 1.73

334 0.90 ± 0.22 541.83 ± 5.68 93.95 ± 1.36

335 0.90 ± 0.22 550.33 ± 7.87 96.07 ± 1.72

410 0.97 ± 0.23 546.50 ± 5.30 96.07 ± 1.28

418 0.96 ± 0.19 547.00 ± 8.71 95.90 ± 1.16

641 0.85 ± 0.21 563.67 ± 11.76 96.36 ± 1.14

720 1.03 ± 0.24 549.50 ± 5.26 95.25 ± 0.92

741 0.88 ± 0.22 542.83 ± 7.64 95.28 ± 1.42

756 0.93 ± 0.22 554.17 ± 6.64 95.58 ± 1.98

757 0.91 ± 0.22 549.00 ± 8.16 95.99 ± 1.10

811 1.00 ± 0.23 540.17 ± 2.39 94.79 ± 1.15

812 0.93 ± 0.22 560.67 ± 8.49 95.33 ± 1.31

822 0.93 ± 0.21 546.33 ± 5.94 95.39 ± 1.54

825 0.88 ± 0.21 539.83 ± 2.21 96.22 ± 1.61

833 0.93 ± 0.21 557.67 ± 6.45 94.01 ± 1.55

838 0.92 ± 0.24 547.67 ± 4.57 96.67 ± 1.02

844 1.06 ± 0.25 547.17 ± 6.57 96.82 ± 1.14

845 1.04 ± 0.25 542.00 ± 8.05 96.99 ± 0.74

Trap Termite Worker

No.

Proportion

Wt (g) Number (%) Trap Termite Worker

No. Proportion

Wt (g) Number (%) 3 0.62 ± 0.13 418.17 ± 3.09 94.88 ± 1.09

7 0.64 ± 0.14 422.00 ± 2.28 96.76 ± 1.11

10 0.43 ± 0.10 410.33 ± 1.63 95.57 ± 1.37

11 0.70 ± 0.16 413.50 ± 3.56 96.66 ± 1.11

13 0.78 ± 0.17 422.83 ± 3.18 95.90 ± 1.12

17 0.66 ± 0.15 416.67 ± 4.33 95.15 ± 1.41

18 0.77 ± 0.16 419.83 ± 4.95 96.61 ± 1.08

25 0.68 ± 0.15 416.17 ± 4.74 95.56 ± 1.32

26 0.53 ± 0.15 415.83 ± 2.26 95.24 ± 1.09

Table-3.3. Mean yield (g) and mean number of termites in 1.0 gm sample and

mean percent workers in foraging group of Odontotermes lokanandi

collected from “NIFA TERMAPs” installed in Islamabad from

September 2010 to September 2012.

53

51 0.68 ± 0.15 422.67 ± 3.13 96.15 ± 1.12

54 0.75 ± 0.15 418.33 ± 2.47 94.67 ± 1.71

73 0.66 ± 0.14 416.67 ± 3.85 96.01 ± 1.32

75 0.43 ± 0.11 412.50 ± 2.24 91.69 ± 1.42

115 0.71 ± 0.15 414.17 ± 2.50 95.97 ± 1.15

117 0.70 ± 0.16 417.17 ± 4.40 97.61 ± 0.82

118 0.69 ± 0.15 412.17 ± 3.87 97.10 ± 1.32

119 0.63 ± 0.15 418.50 ± 3.10 96.86 ± 1.46

170 0.39 ± 0.09 413.17 ± 2.97 96.56 ± 1.14

213 0.73 ± 0.16 424.50 ± 1.15 98.18 ± 0.55

256 0.82 ± 0.19 417.17 ± 2.58 97.36 ±1.09

258 0.62 ± 0.15 417.00 ± 4.48 98.18 ± 1.24

332 0.59 ± 0.13 423.67 ± 3.17 96.28 ± 0.72

333 0.44 ± 0.10 417.83 ± 3.20 98.41 ± 0.50

415 0.68 ± 0.14 412.00 ± 2.67 96.08

± 1.15

417 0.76 ± 0.16 414.00 ± 2.25 97.15 ±

1.02

418 0.43 ± 0.10 416.17 ± 2.02 96.19 ±

1.23

523 0.71 ± 0.17 419.50 ± 3.36 97.29 ±

0.72

524 0.63 ± 0.15 421.50 ± 3.49 96.76 ±

1.16

724 0.79 ± 0.17 418.17 ± 4.42 96.23 ±

1.16

728 0.65 ± 0.14 416.83 ± 2.63 93.73 ±

1.56

730 0.71 ± 0.14 417.17 ± 2.56 93.56 ±

1.64

731 0.66 ± 0.15 419.50 ± 3.31 94.44 ±

1.13

732 0.73 ± 0.15 416.17 ± 4.94 96.06 ±

1.82

756 0.38 ± 0.09 416.67 ± 2.23 98.34 ± 0.40

791 0.68 ± 0.15 420.17 ± 2.46 98.23 ±

0.59

792 0.64 ± 0.14 418.00 ± 4.67 97.39 ±

1.23

798 0.69 ± 0.15 412.00 ± 2.11 96.77 ±

1.63

810 0.68 ± 0.15 414.33 ± 1.89 97.56 ±

0.71

811 0.45 ± 0.10 417.50 ± 3.02 95.33 ±

1.71

826 0.56 ± 0.13 420.33 ± 2.55 95.25 ±

1.85

828 0.71 ± 0.14 415.67 ± 2.67 94.16 ±

1.62

829 0.66 ± 0.14 418.33 ± 3.40 98.39 ±

0.77

841 0.67 ± 0.15 410.00 ± 3.10 96.76 ±

0.54

844 0.35 ± 0.09 407.67 ± 4.75 96.09 ±

1.62

845 0.41 ± 0.09 417.83 ± 3.32 96.09 ±

0.73

847 0.65 ± 0.15 418.33 ± 3.42 96.40 ± 1.09

54

the evaluation period, precipitation also may have contributed to the termites being

more active. Rainfall makes soil wet, and termites need damp condition to live and

develop.

Moreover, Variations were found in the number of two species per trap on

different dates. No biomass of both species was collected in winter months

(December, January, February and March) when the temperature was low, while the

relative humidity was recorded high. When the temperature increased, maximum

numbers of termite were captured (Fig-3.3).

3.4.1.6. Caste composition of foraging groups of subterranean termites.

Results Table-3.1 and Table-3.2 show the ratio between workers and soldiers

in Microtermes obesi and O. lokanandi. The results reveal that the foragers captured

throughout the observation period were predominantly workers. Mean population of

workers ranged from 93.53 ± 1.73 to 97.68 ± 0.40 and 91.69 ± 1.42 to 98.41 ± 0.50

percent M. obesi and O. lokanandi, respectively. This shows that temperature,

relative humidity and rainfall affect the ratio of workers to soldiers.

Correlation was found positive and significant between atmospheric

temperature and percent workers of M. obesi; while negative and non-significant

between atmospheric temperature and percent workers of O. lokanandi. Negative and

significant correlation was noted between relative humidity and percent workers of

M. obesi; whereas, positive and significant correlation was recorded between relative

55

humidity and percent workers of O. lokanandi. Positive and nonsignificant

correlation was recorded between precipitation and percent workers of

M. obesi; while positive and significant correlation was observed between precipitation

and percent workers of O. lokanand (Table-3.5).

3.5. DISCUSSION

3.5.1. Population dynamics

In our study a total of 1200 poplar wooden survey stakes were used to monitor

the termite activities in Islamabad. Of the 1200 stakes placed in the ground, of which

65 were infested by two termite species i.e., O. lokanandi and M. obesi and the

infested stakes were replaced with “NIFA-TERMAPs”. Many

researchers used ground stakes to monitor termite foraging activities (Esentther and

Beal, 1974, 1978; Su et al., 1982b). Bhanot et al. (1984) used stakes of Kiker (Acacia

arabica) for observing foraging activities and they concluded that M.

unicolor and O. lokanandi were more abundant and frequent foragers.

In the experimental field O. lokanandi and M. obesi were interacting with

each other and it was observed that some traps harbour the same one species, and

some time a single trap may have mixed population of two species. When such traps

were opened there was a great antagonistic behavior that they quarreled up to the

death of the weaker and fever members. Jones (1990) reported that antagonistic

behaviour between H. aureus from different colonies apparently results in the

maintenance of discrete territorial boundaries and demographically closed societies,

except in case of colony subdivision. Many studies have correlated climatic variables

56

such as minimum and maximum temperature and annual rainfall to the range limits

of species (Jeffree and Jeffree, 1996; Bullock et al., 2000).

Fig-3.3.Effect of atmospheric temperature, relative humidity and precipitation

on (Mean ± SE) number of M. obesi and O. lokanandi collected through “NIFA-

TERMAPs” installed in Islamabad during

September, 2010 to September, 2011.

39

Table-3.4. Correlation between environmental factors and biomass of termite species captured through

“NIFA TERMAPs” from Islamabad

Termites species Atmospheric Relative Humidity Precipitation

Temperature (oC) (%) (mm)

M. obesi r =0.717*, P= 0.00 r = -0.030, P=0.889 r = 0.608*, P=0.002

O. lokanandi r = 0.766*, P= 0.00 r = - 0.077, P=0.721 r = 0.557*, P=0.004

* = Significantly different at 5% level of significance

Table-3.5. Correlation between environmental factors (Atmospheric Temperature, Relative Humidity

and Precipitation) and % workers of Termites

Termites Atmospheric Relative Humidity Precipitation

Species Temperature (oC) (%) (mm)

M. obesi r =0.184*, P= 0.005 r = -0.208*, P=0.001 r = 0.069 ns, P=0.292

O. lokanandi r = -0.090 ns, P= 0.084 r = 0.174*, P=0.001 r = 0.159*, P=0.002

* = Significantly different at 5% level of significance. ns = Non-significan

59

In our study a total of 65 stakes out of 1200 was found infested by termites

after 150 days. After detection of termite population by stake method, “NIFA–

TERMAPs” were used to capture a huge number of termites from the experimental

areas. A total of 10, 34, 40, 47, 47, 50, 54, 54, 59 and 65 NIFA-TERMAPs were set

up in Islamabad after 15, 30, 45, 60, 75, 90, 105, 120, 135 and 150 days respectively.

Maximum “NIFA-TERMAPs” were found infested, when the temperature and

relative humidity were recorded maximum, while minimum “NIFA-TERMAPs”

were found infested in comparatively low temperature and relative humidity. Fei and

Henderson (2004) reported that temperature and moisture were the most important

factors in the distribution of subterranean termites. Buxton (1981) also reported that

the seasonal variations of temperature and humidity fluctuated activities of

subterranean termites. Foraging activities of termites are directly correlated with

seasonal variations of environmental factors

(Bouillon, 1970).

Our results showed that M. obesi and O. lokanandi were collected from 29

and 46 “NIFA-TRAPs” respectively in varying ranged Our results indicate that such

variation exists in different foraging sites of a single colony. There seems to be three

factors: i. termites did not like the high moisture content of the soil; ii. the distance

from the colony that worker would travel; iii. the termite soldiers apparently do not

distribute homogeneously within their gallery system. Lower yield of termites was

found in traps, which were installed in wet or irrigated field or away from the colony.

While, higher yield of termites was recorded in traps, which were installed in dry

field or near to the colony. Similarly, the mean number of individuals in 1.0 gm

sample varied greatly. Variations were found in mean number of individuals of M.

60

obesi per gram sample of the different traps. Among termites colonies a considerable

intra-specific variation exist (Su and La Fage,

1984).

Comparison on the number of individuals of the two species per sample

shows a great variation. A significantly greater number of M. obesi was observed as

compared to O. lokanandi. The minimum number of the former species is more than

the mean maximum number of the latter. This variation is due to the different size of

the two species. Individuals of M. obesi are smaller in size than individuals of O.

lokanandi so more individuals were counted in 1.0 gm sample. The two termite

species were also found different greatly in yield per trap and number per 1.0 gm

sample. The maximum yield of M. obesi per trap was 1.12 ± 0.28 gm, while of O.

lokanandi 0.82 ± 0.19 gm. This variation shows that the termite population in the

colony of M. obesi is high as compare to O. lokanandi so more termites come to the

foraging point. The number of individuals in a termite colony varies with species

(Badawi et al., 1984).

3.5.2. Foraging Ecology

In our study foraging behaviour of subterranean termites was studied by

using NIFA-TERMAPS. Correlation between foraging behaviour of M. obesi and O.

lokananadi and a-biotic factors was studied. The results showed that correlation was

found positive and significantly different between atmospheric temperature,

precipitation and both termite species however, the correlation was recorded

negative and non significant between relative humidity and termites species.

Johnson and Whitford (1975) and Ueckert et al. (1976) have reported that foraging

activity is correlated to a considerable extent with soil moisture and temperature.

61

Abushama and Al-Hquty (1988) have also reported positive correlation between

termites activities and soil moisture content. Lenz and Evans (2002) stated that

subterranean habits are widely assumed to reduce adverse effect of weather. Potter,

(2004) reported that termites activities are strong influenced by temperature, in

addition, termites maintain temperature and humidity within their nests.

In our study it was observed that no biomass of both species was collected in

winter months (December, January, February and March) when the temperature was

low, while the relative humidity was recorded high. When the temperature increased,

maximum numbers of termite were captured. Haverty et al. (1999a) also supported

our study and he observed the variation in the population of termites in different

seasons of the year. Studies revealed that high and cold termperature of the soil

surface effect foraging activities of termites (Haverty et al., 1974; La fage et al.,

1976).

In our studies a significatly high number (more than 90%) of workers were

collected as compared to soldiers in every observation. This shows that the worker

termites come to forage in large number as compared to soldiers. In addition, soldiers

in termite colony is comparatively low. During observation 2 to 7% soldiers were

collected from traps. This shows that temperature, relative humidity and rainfall

affect the ratio of workers to soldiers. The caste composition in social insects can be

influenced by environmental conditions such as temperature. (Henderson, 1998;

Mao et al., 2005; Scharf et al., 2007). Caste composition in termite colony or

foraging groups are known to vary with time of day, season, species, and colony size

or age (Bodot, 1970; Sands, 1965; Bouillon, 1964). Nutting (1970) recorded 4%

62

soldiers and 96% non soldiers in a foraging group of H. aureus. A colony of G.

perplexus contain mainly workers and only about 0.4% soldiers (Nutting et al.,

1973).

Chapter 4

63

EFFECT OF DYE-MARKERS I.E., NILE BLUE-A AND SUDAN

RED-7B ON MICROTERMES OBESI AND ODONTOTERMES

LOKANANDI

4.1. ABSTRACT

4-5th instar Soldiers and Workers of Microtermes obesi Holmgren and

Odontotermes lokanandi Chatarjee and Thakur (Blattodea: Termitidae) were force-fed

on different concentrations of dye-markers viz., Nile blue-A and Sudan red-7B in

Entomological laboratory at National Agriculture Research Centre (NARC),

Islamabad. Results showed that Nile blue-A at high concentration caused 100 %

mean mortality in M. obesi after 15 days, followed by mean mortality in M. obesi

observed at medium and low concentrations, respectively; whereas, 100% mean

mortality in M. obesi was found on 9th day at high concentration of Sudan red-7B,

followed by mean mortality in M. obesi recoded at medium and low concentrations,

respectively. However, Nile blue-A and Sudan red-7B caused 100% mean mortality

in O. lokanandi at high concentration after 5th day of the trial, followed by mean

mortality in O. lokanandi at medium and low

concentrations, respectively.

4.2. INTRODUCTION

Study of the population demographics and foraging behavior of

subterranean termites poses difficulties, due to the subterranean gallery system and the

absence of a well-defined nest architecture that is separable from the

64

44

surrounding soil matrix. A marking material is required for studies of population

dynamics of subterranean termites under field conditions. It is therefore, necessary

to find a method of marking termite so that those not exposed to insecticide could be

distinguished from those that had been exposed (Su et al., 1987), marking of

subterranean termites effectively is hard (Evans, 2000). An ideal marking dye should

be in-expensive, durable, easily applied, non-toxic, and clearly identifiable;

moreover, the dye marker should not hamper the insect nor affect its normal biology,

lifespan, growth, or reproduction (Hagler and Jackson, 2001).

The fat-soluble histological dye markers to mark subterranean termites have

been regularly discovered. Many terminologists reported that the use of Nile blue

and Neutral red (Grace and Abdallay, 1989; Evans et al., 1998; Tsunoda et al., 1999;

Stanley et al., 2001). In addition, other dye markers have also been tested, for

example Sudan yellow, Sudan black, Sudan red and Sudan green (Su and Scheffrahn,

1988a; Grace, 1990; Salih and Logan, 1990; Evans,1997). The limited cuticle

sclerotization of termites permits the use of histological markers such as Nile blue,

Neutral red or Sudan red (Su et al., 1991b; Evan, 1997).

Many researchers reported that the use of stains has several disadvantages:

the insects have to ingest the stain diluted in aqueous solution or impregnated in filter

paper, it is time-consuming, some of these substances accelerate termite mortality

(Grace and Abdallay, 1990; Evans, 1997; Nobre et al., 2007) and finally, these

markers do not offer good visual contrast. The main drawbacks of dye markers have

been reported to be heterogeneous colouration, variable fade-out and unintended

transfer to other individuals by trophallaxis, cannibalism and social grooming

(Haagsma and Rust, 1993; Thorne et al., 1996; Curtis and Waller, 1997; Evans et

65

al., 1998; Suarez and Thorne, 2000). However, in several studies Nile blue has

successfully used (Haagsma and Rust, 1993; Evans et al., 1998, 1999; Marini and

Ferrari, 1998; Tsunoda et al., 1999; Evans, 2001; Stanley et al., 2001).

An alternative to the use of stains for marking is the use of external markers,

as used for other insects. There are various types of markers available and examples

used for hymenopterans are enamel paint (Packer, 2005) and numbered plastic discs

(Pereira and Chaud-Netto, 2008), both applied to the thorax of bees, and fluorescent

dye sprays applied to ants (Porter and Jorgensen, 1981). However, according to these

authors, enamel paint alters bee behavior, the plastic discs are adequate only for

insects with large thoraxes and fluorescent dyes require black light to be visible. The

physical fragility of termites and the reduced size of the majority of species are

limitations to the use of external markers. Given such considerations, dye sprays are

an alternative as they do not require handling of the insects. However, the frequency

with which the insects groom themselves and nest mates means that sprayed dyes

persist for only short time periods (Forschler, 1994; Evans, 1997). Brunow et al.

(2005) tested the use of gouache as a topical marker applied individually to

Cornitermes cumulans (Kollar) (Isoptera, termitidae).

The present study was focused to determine the toxicity of Sudan red-7B and Nile

blue-A against Microtermes obesi and O. lokanandi.

4.3. MATERIALS AND METHODS

4.3.1. Biological Stains

Nile blue A (96%) and Sudan red 7B (95%) were evaluated as dye markers.

These compounds were selected from biological stains that are used for dying animal

tissues, lipids or cell granules. Acetone was used as solvent for these dyes.

66

4.3.2. Experimental Termites

Microtermes obesi and O. lokanandi were captured from termite infested building of

Health Directorate, Capital Development Authority, Islamabad by using

“NIFA-TERMAPs” (Salihah et al., 1993). The intested traps were brought to

Entomological laboratory, National Agriculture Research Centre, Islamabad. The

termites were separated from soil and debris by using 5.0, 4.0 and 1.0 mm mesh

sieves in regular sequence. After that the termites along with debris and soil were

placed on the inverted glass Petri dish put on the apparatus set up by NIFA

(Nuclear Institute of Food and Agriculture) termite group consisting of a plastic tub

(dia. 29.5 cm) with inverted glass Petri dish (dia. 15.3 cm). The termites fell down

in the tub without any extra particle. Frequently, the termites and debris on the Petri

dish were disturbed with a camel brush to collect all the termites in the plastic tub

(dia. 29.5 cm). The soil and debris were gently removed and the termites were

introduced in other glass Petri dishes (dia. 15.3 cm) each having two same size filter

papers moistened with distilled water and kept as stock termites in desiccators having

92% Relative Humidity. Identification of termites were done by using the key of

Chaudhry et al. (1972).

4.3.3. Preparation of Dye Attractive Bait

i) Saw dust: Poplar wood saw dust (30 mesh sieved) was kept at 80 0C for two

hours.

ii) Dye solutions: Stock solution of 0.5% was prepared by dissolving 500 mg of

dye in 100 mL of acetone; 0.25% solution was prepared by 1:1 ratio of 0.5% solution

of dye and acetone and 0.125% solution by 1:1 ratio of 0.25% solution of dye and

acetone (Fig-4.1) iii) Staining of saw dust: Hundred grams of saw dust were

67

soaked with 100 mL of each of the dye solutions (0.125, 0.25 and 0.5%) where as

100 grams of saw dust were soaked with acetone only for control series following

the techniques used by (Su et al., 1983). For the solvent evaporation the dyed saw

dust as well as the control series were kept at room temperature for 48 hrs, which

yielded stock of the stained saw dust with dyes concentrations of 0.125, 0.25 and

0.5% and control without dye.

iv) 1 % Agar: A stock solution of 150 mL of 1% agar was prepared by adding

1.5 gms of agar in 148.5 mL of distilled water; boiled in glass flask in a waterbath

with constant stirring for 2 hrs.

v) Mixing of saw dust and 1% agar: Each lot of stained and unstained saw dust

was mixed with 1% of hot agar in the ratio of 2g: 3mL (w/v), respectively. Each Petri

dish (dia. 5.3 cm) half filled with stained bait of different concentration and the other

lot with unstained bait. Two hundred (200) termites (180 workers and 20 soldiers)

were introduced and confined in each Petri dish containing dyed bait (0.125,

0.25 and 0.5%) and was force-fed used the methods described by (Su et al., 1988).

For control series two hundred termites (180 workers and 20 soldiers) were released

on unstained bait. All the experimental units were kept in desiccators having 92%

Relative Humidity. The experiment was designed as a complete randomized with

three replications. Data was taken on daily basis and dead termites were removed

from the experimental units by using forceps. Data was corrected by using Abbot‟s

formula (1925). Statistical analysis was performed by using Co-Stat and Duncan‟s

Multiple Range Test was used to separate the means.

4.4. RESULTS

4.4.1. Toxicity of Biological dyes

68

Studies were under taken on the effect of Nile blue-A and Sudan red-7B against

Micreotermes obesi and O. lokanandi (Blattodea: Termitidae) at

Entomological laboratory, National Agriculture Research Center (NARC),

Islamabad. The results are presented in Figs 4.2 to 4.5.

4.4.2. Nile blue-A


Mean percent mortality at high, medium and low concentrations up to 5 th day

was 14.38 ± 0.41, 13.87 ± 0.57 and 13.37 ± 0.68 respectively. Analysis of variance

revealed that percent mean mortality was found non-significant (P > 0.05) amongst

treatments (Fig-4.2).

Percent mean mortalities on days 6, 7, 8 and 9 at medium and low

concentrations were observed similar (P > 0.05) with each other; while significantly

differed (P < 0.05) from percent mean mortality recorded at high level

(Fig-4.2).

Fig-4.2 shows that percent mean mortality recorded on day 10th was 58.05 ±

0.39 at high concentration followed by 43.15 ± 0.31, 41.61 ± 0.24 at medium and low levels,

respectively. Analysis of variance revealed that percent mean mortality recorded at high, medium and

low concentrations was statistcally differed (P <

0.05) from each other.

Percent mean mortality, i.e., 68.50 ± 0.60, 52.33 ± 0.40 and 46.99 ± 0.31 was

observed at high, medium and low concentrations respectively, on 11 th day.

69

Significant difference (P < 0.05) was found amongst mortalities recorded at all the

concentrations (Fig-4.2).

Percentage mortalities on day 12 recorded at high, medium and low

concentrations were 87.18 ± 0.46, 55.46 ± 0.39 and 50.09 ± 0.23, respectively.

Analysis of variance revealed that percent mean mortality was found significantly

differed (P < 0.05) amongst treatments (Fig-4.2).

Fig-4.2 showed that percent mean mortality recorded on 13 th day at high

concentration was significantly different (P < 0.05) than mortalities recorded at

medium and low concentrations. Moreover, there was also a significantly difference

(P < 0.05) between mortalities recorded at medium and low

concentrations.

On day 14 percent mean mortalities at high, medium and low

concentrations were 97.20 ± 0.46, 62.69 ± 0.33 and 57.62 ± 0.51, respectively. Analysis of

variance revealed that percent mean mortalities recorded at all the

70

Figs-4.1. Stock baits with different concentration of dyes. A, un-dyed bait; B, C

and D ( Nile blue-A), E, F and G (Sudan red-7B) at 0.5, 0.25 and

0.125% concentrations, respectively.

tested concentrations were found statistically differed (P < 0.05) from each other

(Fig-4.2).

Fig-4.2 shows that maximum percent mean mortality (100.00± 0.00) was

recorded at high concentration after 15 day, followed by 65.06 ± 0.27 and 59.18 ±

0.45 mortality at medium and low concentrations, respectively. Analysis of variance

revealed that percent mean mortalities at all doses were found

A

B C D

E F G

71

significantly differed (P < 0.05) from each other.


Fig-4.3 shows that percent mean mortalities recorded at high, medium and

low concentrations were 14.88 ± 0.44, 15.22 ± 0.59 and 14.88 ± 0.31, respectively

on 1st day of the trial. Analysis of variance revealed that the percent mean mortalities

were found non-significant (P > 0.05) from each other.

Percent mean mortalities observed were 31.93 ± 0.20 and 31.60 ± 0.37 at

medium and low concentrations, respectively. Statistically there was no significantly

difference (P > 0.05) between mortalities recorded at these two concentrations.

However, percent mean mortality (33.11 ± 0.22) recorded at high concentration was

found significantly higher (P > 0.05) than mortalities recorded at medium and low

concentrations (Fig-4.3).

On day 3, 58.26 ± 0.47, 46.68 ± 0.25 and 45.49 ± 0.30 percent mean mortalities were

recorded at high, medium and low doses respectively. Analysis of variance revealed that the

percent mean mortalities recorded at all three doses were found significantly differed (P < 0.05)

from each other (Fig-4.3).

On day 4, percent mean mortalities were 81.49±0.80, 55.64±0.54 and

46.85±0.65 at high, medium and low concentrations, respectively. Percent mean

mortalities recorded at all three doses were found statistically different (P < 0.05)

from each other (Fig-4.3).

72

Fig-4.3 shows that maximum percent mean mortality (100 ± 0.00) was

recorded at high concentration after day 5 of the trial, followed by 71.09 ± 0.67 and

61.67 ± 0.72 at medium and low levels, respectively. The results revealed that

percent mean mortalities observed at all three doses were found significantly

different (P < 0.05) from each other.

4.4.3. Sudan red-7B


Fig-4.4 shows that percent mean mortalities up to 3rd day of the trial were

31.15 ± 0.34, 30.64 ± 0.67 and 30.97 ± 0.34 at high, medium and low doses,

respectively. Analysis of variance revealed that percent mortalities were observed

non-significantly different (P > 0.05) at all three doses.

On day 4, the percent mean mortalities recorded were 42.69 ± 0.43, 40.64 ±

0.50 and 40.48 ± 0.50 at high, medium and low levels, respectively. Analysis of

variance revealed that percent mean mortalities recorded at high

73

Fig-4.2. Mortality (%) observed in Microtermes obesi after exposure to Nile

blue-A at different concentrations for 15 days.

74

a

Fig-4.3. Mortality (%) observed in Odontotermes lokanandi after exposure to

Nile blue-A at different concentrations for 5 days.

75

concentration were noted significantly higher (P < 0.05) than other treatments. There

was no significantly difference (P > 0.05) in percent mean mortalities recorded at

medium and low concentrations (Fig-4.4).

On day 5, 58.39 ± 0.24 percent mean mortality was recorded at high

concentration, followed by 55.48 ± 0.38 and 54.62 ± 0.67 percent mean mortalities

at medium and low concentrations, respectively. Mortalities recorded at medium and

low concentrations were found not only significantly lower than that recorded at high

concentration, but there was a statistical difference (P < 0.05) in the mortality

recorded at these two concentrations (Fig-4.4).

On day 6, significantly different (P < 0.05) mortalities were recorded

amongst the tree treatments. The highest mortality of 68.28 ± 0.45 was recorded at

high concentration, followed by 62.58 ± 0.42 and 60.86 ± 0.04 percent mortalities at

medium and low concentrations, respectively (Fig-4.4).

Fig-4.4 shows that percent mean mortalities recorded on 7 th day of the trial

were 84.44 ± 0.50, 75.34 ± 0.69 and 74.30 ± 0.81 at high, medium and low doses,

respectively. Percent mortality observed at high concentration was significantly

higher (P < 0.05) than other treatments. Statistically there was no difference (P >

0.05) in the mortalities recorded at medium and low concentrations.

The results shows that percent mean mortalities recorded on 8 th day were

76

94.86 ± 0.47, 85.82 ± 0.46 and 82.62 ± 0.40 at high, medium and low doses, respectively. Analysis of

variance revealed that percent mean mortalities were observed significantly differed (P < 0.05) at all

doses (Fig-4.4).

Maximum (100.00 ± 0.00) percent mortality was recorded at high

concentration after 9 day, followed by 92.10 ± 0.49 and 86.89 ± 0.83 percent mean

mortalities at medium and low concentrations, respectively. Analysis of variance

revealed that percent mean mortalities were observed significantly different (P <

0.05) amongst treatments (Fig-4.4).


Fig-4.5 shows that percent mean mortalities of O. lokanandi up to 2nd day

were 16.61 ± 0.17, 16.61 ± 0.12 and 16.60 ± 0.54 at high, medium and low doses,

respectively. The results revealed that percent mean mortalities were observed

nonsignificant (P > 0.05) amongst treatments.

On day 3, percent mean mortality was 31.77 ± 0.23 at high concentration,

which was significantly differed (P < 0.05) from percent mortalities 27.87 ± 0.24 and

27.67 ± 0.62 recorded at medium and low concentrations, respectively. Percent mean

mortalities recorded at medium and low concentrations were found nonsignificant (P

> 0.05) (Fig-4.5).

On day 4, maximum percent mean mortality (43.69 ± 0.23) observed at high

concentration followed by 36.48 ± 1.11 and 25.22 ± 0.11 percent mortalities at

medium and low concentrations, respectively. Analysis of variance revealed that

77

percent mean mortalities were observed significantly different (P < 0.05) at all doses

(Fig-4.5).

b

a

a

Fig-4.4. Mortality (%) observed in Microtermes obesi after exposure to

78

Sudan red-7B at different concentrations for 9 days.

On day 5, 100.00 ± 0.00 percent mortality was observed at high concentration

followed by 57.19 ± 0.65 and 42.53 ± 1.67 percent mortalities recorded at medium

and low concentration, respectively. Analysis of variance revealed that percent mean

mortalities recorded at all doses were found significantly differed (P < 0.05) amongst

each other (Fig-4.5).

4.5 DISCUSSION

Our study was a trial to screen out the best concentrations of stains for

marking Microtermes obesi and O. lokanandi. Hundred percent mortality was

recorded at higher concentration after 15 days by using Nile blue-A against M. obesi;

followed by mortality recorded at medium and lower concentrations, respectively;

while 100 percent mortality was noted at high dose after 5 th day of the experiment

when O. lokanandi was tested, followed by mortality noted at medium and low

concentrations, respectively. Our results showed that O. lokanandi was more

sensitive than M. obesi against Nile blue-A. Our results tally with those of Su et al.

(1991b), who reported that Nile blue-A was safe and persistent marker for R. flavipes.

79

Salih and Logon (1990) uesd 30 dyes against M. lepidus, and reported that neutral

red was safe and give the termites a persistent, clear colour which was not transferred

between them.

Our results revealed that Sudan red-7B caused 100% mortality in M. obesi

after 9 days at high concentration, followed by mortality recorded at medium and

lower concentrations, respectively; while Sudan red-7B caused 100% mortality in O.

lokanandi at high concentration after 5 days, followed by mortality mortalities

a

80

Fig-4.5. Mortality (%) observed in Odontotermes lokanandi after exposure to

Sudan red-7B at different concentrations for 5 days.

recorded at medium and low concentrations, respectively. Our study showed that M.

obesi was also more resistance to Sudan red-7B as compare to O. lokanandi.

Many researchers observed that Sudan red had been considered as a suitable

biological stain for C. formosanus (Lai, 1977; Su et al., 1983, 1988; Delaplane et

al., 1988; Delaplane and La Fage, 1989), but not appropriate for making R. flavipes.

Grace and Abdally (1989) reported that Sudan red-7B could safely be used with

shorter release and recapture cycle with R. flavipes. Grace and Abdallay (1989)

demonstrated that low concentrations of Sudan red-7B are rapidly excreted by R.

flavipes, and that extended feeding periods result in high mortality.

In our study, Microtermes obesi was found more resistance to both dye

markers i.e., Nile blue-A and Sudan red 7B, because Nile blue-A caused lower

mortality in termites and was retained well for maximum period of time; while O.

lokanandi was found sensitive to both dye markers.

81

Chapter 5

INVESTIGATION A PROPER CONCENTRATION OF DYE

MARKERS WHICH REMAIN VISIBLE IN THE BODY OF

TERMITES AT DIFFERENT RELATIVE HUMIDITIES.

5.1. ABSTRACT

Experiments were focused to screen out the best relative humidity for

staining of termites. The relative humidities used were 100% (H2O), 92% (Na2Co3)

and 76% (NaCl). Two termite species i.e. Microtermes obesi and Odontotermes

lokanandi were force-fed on bait containing Nile blue-A and Sudan red-7B. Three

concentrations i.e. 0.5, 0.25 and 0.125% of each dye were tested against both species.

The results showed that M. obesi gained Nile blue colour in 100% relative humidity

after 10 days at all concentrations; while at 92 and 76% relative humidities; termites

gained slight colour only at 0.5% concentration after 7 and 10 days. The results

82

indicated that maximum blue O. lokanadi were observed at 0.25% concentration

after 4 dys, but this concentration was found toxic to O.

lokanadi.

The results revealed that M. obesi did not get any colour under all the relative

humidity (100, 92 and 76%) after 4 days when they treated against Sudan red-7B at

0.125, 0.25 and 0.5% concentrations. Almost the same situation was observed in O.

lokanandi.

Retention time of Nile blue and Sudan red was recorded against M. obesi

62

for eight weeks. Nile blue-A at 0.125% caused lower mortality and it was retained well

for eight weeks in more than 59% termites. Sudan red-7B caused

comparatively more mortality.

5.2. INTRODUCTION

To develop an effective control strategy for subterranean termites, a

systematic information of the basic ecology and biology is essential. A critical factor

in containing subterranean termites is having a regular knowledge of their spreading

patterns. A key component of any termite dispersal study is a viable marker. Fujii

(1975) mentioned that dyes were first used in Hawaii to measure the distance

traveled by Coptotermes formosanus workers. The ideal marker for insects is

durable, cheap, non-toxic, easy to apply and easy to identify; it should also not

hamper movement, irritate the individual or affect its behavior (Hagler and Jackson,

83

2001). Evans (1997) reported that marking should not alter interindividual behaviors

involving the marked individuals, particularly with social

insects.

Studies revealed that oil-based dye markers viz., Sudan red, neutral red,

coloured glues and Nile blue have been used for tracing of subterranean termites

(Grace and Abdallay, 1989; Jones, 1990; Haagsma and Rust, 1993; Su et al., 1991b;

Loreto et al., 2009), biological studies of Termite require more than one distinctive

dye markers (Evans et al., 1998, 1999; Evans, 2001, 2002), these dye markers are

available in different colours such as green, red, yellow, black and blue

(Evans et al., 1998).

Some studies have documented that the retention of dye markers in

subterranean termites fluctuate greatly between both the type of dye which is applied

and the termite species viz., Nile blue and Neutral red have proven valuable for long

biological studies such as three months for certain species of termite (Haagsma and

Rust, 1993; Oi, 2000; Su et al., 1991b, 1993). Lai (1977) and Lai et al. (1983)

screened nine histological dyes and identified that Sudan red as the most persistent

and least toxic dietary dye marker for C. formosanus. Sudan red-7B caused delayed

mortality, and with time, the dye faded sufficiently that it could not be seen in an

increasing number of termites (Su et al., 1983; Delaplane et al.,1988). Sudan red-7B

could safely be used with shorter 3 week release-recapture cycles with Reticulitermes

flavipes (Grace, 1989,1990; Grace and Abdally, 1989). Neutral red was also

identified by Salih and Logan (1990) as the most promising of 30 dyes listed as

markers for Microtermes lepidus sjostedt. Su et al. (1991b) reported that

Nile blue was safe and persistent marker for R. flavipes.

84

Keeping in view the importance of dye marker, the present study was

conducted to search visibility of dye Nile blue A and Sudan red 7B in termite bodies

at different relative humidities.


5.3.1. Experimental Termites

Microtermes obesi and O. lokanandi were captured from termite infested

building of Health Directorate, Capital Development Authority, Islamabad by using

“NIFA-TERMAPs” (Salihah et al., 1993). The intested traps were brought to

Entomological laboratory, National Agriculture Research Centre, Islamabad. The

termites were separated from soil and debris by using 5.0, 4.0 and 1.0 mm mesh

sieves in regular sequence. After that the termites along with debris and soil were

placed on the inverted glass Petri dish put on the apparatus set up by NIFA

(Nuclear Institute of Food and Agriculture) termite group consisting of a plastic tub

(dia. 29.5 cm) with inverted glass Petri dish (dia. 15.3 cm). The termites fell down

in the tub without any extra particle. Frequently, the termites and debris on the Petri

dish were disturbed with a camel brush to collect all the termites in the plastic tub

(dia. 29.5 cm). The soil and debris were gently removed and the termites were

introduced in other glass Petri dishes (dia. 15.3 cm) each having two same size filter

papers moistened with distilled water and kept as stock termites in desiccators having

92% Relative Humidity. Identification of termites were done by using the key of

Chaudhry et al. (1972).

5.3.2. Visibility of dye markers in the body of termites under at different relative

humidity

85

Experiment was conducted to screen out the best relative humidity for

staining termites in Entomological laboratory, National Agriculture Reasearch

Centre, Islamabad. The relative humidities used were 100% (H2O), 92% (Na2Co3)

and 76% (NaCl). Saturated solutions of all those salts were prepared in desiccators

and covered with their lids placed in laboratory at 28 ± 2 0C (temperature) and

60±5% (relative humidity) for 48 hours to maintain the required humidities. After

that the already prepared glass Petri dishes having baits with dyes (Nile blue and

Sudan red) at 0.5, 0.25 and 0.125% concentrations each with 100 termites were kept in desiccators

having 92% relative humidity. Colour of the termites bodies was observed daily and when ever there

was clear change in the dye, gained by termites under any humidity, photographs of termites were

taken. The number of survivors was observed on daily basis up to 15 days.

5.3.3. Retention test

A culture of termite i.e., Microtermes obesi was collected from the field and

acclimatized in the laboratory for 48 hours. Three concentrations (0.5, 0.25 and

0.125%) each of two dyes i.e., Nile blue-A and Sudan red 7B were prepared. In each

concentration a filter was soaked. For the solvent evaporation, the filter papers were

kept at room temperature for 48 hrs. Then a cluster of M. obesi was released to each

concentration and force-fed for 24 hours. Then the same stained culture was used for

retention studies. Fifty (50) stained worker termites from each concentration were

selected at random and transferred to Petri dish (5.3 cm dia) containing un-dyed bait

(prepared as mentioned earlier). In addition, five numbers each un-dyed worker and

soldier termites were also added to the same Petri dish. There were three replicates

for each treatment. Number of stained termites and survivors were recorded after 5,

10, 15, 20, 25, 30, 35, 40, 45, 50, 55 and 60 days.

Statistical computing was performed using Co-Stat.

86

5.4. RESULTS

5.4.1. Visibility of dye markers i.e., Nile blue-A and Sudan red-7B in the body of

termites at different relative humidities

5.4.1.1. Nile blue-A


100% relative humidity

After 4 days

The termites feeding on bait containing dyes at 0.25 and 0.125%

concentrations did not gain any colour; whereas at 0.5% concentration the termites gained

negligible colour (Fig-5.1).

After 7 days

The termites feeding on bait having dye at 0.5% concentration got maximum colour

followed by 0.25 and 0.125% concentrations (Fig-5.2).

After 10 days

The termites feeding on the bait having 0.5% concentration got sufficient stain

followed by those feeding at 0.25 and 0.125% concentrations (Fig-5.3).

92% Relative Humidity

After 4 days

The termites feeding continuously on the baits containing dyes at 0.5, 0.25 and

0.125% concentrations did not get any colour up to 4 days of experiment.

After 7 days

87

The termites feeding continuously on the baits having dye at 0.125 and

0.25% concentrations did not get any colour in their bodies; however, at 0.5% a very

slight indication of colour was observed (Fig-5.4).

After 10 days

Almost same situation was observed at all the three concentrations (0.125,

0.25 and 0.5%) as after 10 days; however, a slight increase of dye (colour) was

recorded in termites that fed on the bait containing dye at 0.5% concentration.

76% Relative Humidity

After 4 days

The termites feeding on the baits containing dyes at 0.5, 0.25 and 0.125% concentrations did

not gain any colour.

After 7 days

The termites feeding on the baits containing dyes at 0.125 and 0.25%

concentrations did not get any colour; however, at 0.5% concentration, the termites

got some amount of dye in their bodies (Fig-5.5).

After 10 days

Same situation was observed after 10 days.

The results indicated that termites feeding on baits containing Nile blue at

0.5% concentration was found the best when kept under at 100% relative humidity,

but still it was not suitable, because it had taken much time to get some dye in their

body (Fig-5.3).

88


This group of termites when kept at 100, 92 and 76 % humidities, the result

showed that this species was more sensitive than M. obesi. A greater percent of

termites was found dead just after 4 days, however, the maximum longevity (4 days)

was recorded only under 100% relative humidity.

Photography of the termites after 4 days showed that at 0.25%

concentration they got prominent blue color, but they were found dead. It showed that

0.25% concentration was toxic to the termites (Fig-5.6).

5.4.1.2. Sudan red-7B


Microtermes obesi, when kept on baits containing Sudan red-7B at 0.125, 0.25 and

0.5% concentrations. They did not get any colour under all the relative

humidity (100, 92 and 76%) after 4 days (Fig-5.7).


This group of termite when kept on different relative humidities (100, 92 and

76%) and force-fed on baits having different concentrations of sudan red. They were

found dead after 24 hours (Fig-5.8). Moreover, they did not show indication of red

colouration in their bodies.

5.4.2. Retention of dye

When the dyed termites (M. obesi) transferred to un-dyed attractive bait. The

results (Fig-5.9) revealed that more than 90% termites, having 0.25 and 0.125%

89

concentrations of Nile blue, retained dye markers in their bodies up to twenty five

days and for fifteen (15) days having 0.50% concentration; whereas more than 90%

termites, having 0.125 and 0.25% concentrations of Sudan red, retained dye markers

in their bodies for Ten (10) days and for five days having 0.50% concentration. After

60 days, maximum (59.33%) termites were observed blue having 0.125%

concentration of Nile blue, followed by 42% having 0.25% concentration of the same

dye; while all the termites, stained with Sudan red, were found dead on day 60.

5.5. DISCUSSION

In our study, we focused to investigate different concentrations of Nile blue-A and

Sudan red under at different relative humidity for staining of M. obesi. The results

showed that at 100% relative humidity the termites did not gain any colour after 4

days; however, they gained maximum stain on 7th as well as on 10th day at all tested

concentrations of Nile blue-A; while at 92 and 76% relative humidities, termites did

not gain any noticeable stain after 4, 7 and 10 days at 0.25

and 0.125% concentrations, however at 0.5% concentration slight colour was

observed in termites bodies after 7 and 10 days. These results tally with those of Su

et al. (1991b), who reported that Nile blue-A was safe and persistent marker for

Reticulitermes flavipes Kall. Nile blue-A at 0.25% concentration against

Heterotermes indicola (Wasmann) was found non toxic, long persistent and best

marker in laboratory as well as in the field (Salihah et al., 1994, 1995, 1996 and

1997).

Our study showed that maximum O. lokanadi population was found dead

after 4 days by using different concentrations of Nile blue-A. Maximum blue termite

was observed at 0.25% concentration, but this concentration was also found toxic to

O. lokanadi, because all the prominently stained termites were found dead. Nile blue-

A and Neutral red, can persist for different times in different species and these

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different species in turn have different tolerances to these substances (Su et al., 1993;

Evan, 1997). Our results revealed that M. obesi did not get any colour under all the

relative humidity (100, 92 and 76%) after 4 days when they treated

against Sudan red-7B at 0.125, 0.25 and 0.5% concentrations.

0.125% (A)

0.25% (B)

0.5% (C )

91

Figs-5.1. (A, B, C). Biological stain (Nile blue-A) attained by termite, Microtermes

obesi after 4 days at 100 percent relative humidity

(H2O); (A), 0.125; (B), 0.25 and (C), 0.5% concentrations.

0.125%(A)

0.25%(B)

0.5%(C)

Figs-5.2. (A, B, C). Biological stain (Nile blue-A) attained by termite,

92

Microtermes obesi after 7 days at 100 percent relative humidity

(H2O). (A), 0.125; (B), 0.25; and (C), 0.5% concentrations.

0.25%(A)

0.125%(B)

0.5%(C)



(H2O). (A), 0.25; (B), 0.125 and (C), 0.5% concentrations.

93

0.125%(A)

0.25%(B)

0.5%(C)



(Na2Co3). (A), 0.125; (B), 0.25; and (C), 0.5% concentrations.

94

0.125% (A)

0.25% (B)

0.5% (C)

Figs-5.5. (A, B, C). Biological stain (Nile blue-A) attained by termite, Microtermes

obesi after 7 days at 76 percent relative humidity

(NaCl). (A), 0.125; (B), 0.25; and (C), 0.5% concentrations.

95

0.125% (A)

0.25% (B)

0.5% (C)


Odontotermes lokanandi after 4 day. (A), 0.125; (B), 0.25; and (C),

0.5% concentrations.

96

0.125% (A)

0.25% (B)

0.5% (C)

Figs-5.7. (A, B, C). Dye, Sudan red visible in termite, Microtermes obesi after 4

days. (A), 0.125; (B), 0.25 and (C), 0.5% concentrations.

97

Control (A)

0.125% (B)

0.25% (C)

0.5% (D)

Figs-5.8. (A, B, C, D). Sudan red attained by termite, Odontotermes lokanandi

after 1 day at 100 percent relative humidity (H2O). (A), Control; (B), 0.125;

(C), 0.25 and (D), 0.5% concentrations.

98

Figs-5.9. (A, B, C). Percent number of dyed termites (Microtermes obesi) after

specified number of days, at three concentrations.

Almost the same situation was observed in O. lokanandi. Sudan red-7B was

found to reside the longest in and cause the least mortality in Coptotermes

formosanus Shiraki (Lai et al., 1983), it has been successfully used for estimating

99

the population size of C. formosanus field colonies (Lai, 1977). Grace and Abdallay

(1989) demonstrated that low concentrations of Sudan red-7B are rapidly excreted

by R. flavipes, and that extended feeding periods result in high mortality. The amount

of Sudan red-7B in termites decreases immediately after the termites stopped feeding

on the stained paper (Su et al., 1983).

Retention time of Nile blue and Sudan red was recorded by using M. obesi

for eight weeks. Nile blue-A (0.125%) caused lower mortality and was retained well

for eight weeks in more than 59% termites. and it is recommended to use against M.

obesi for long studies. Sudan red-7B caused comparatively more mortality and can

be used in short term of studies. The decrease in number of dyed termite in both cases

with the passage of time was due to the mortality of the termites, but not due the

trophallactic transfer of dye. Salihah et al. (1994, 1995, 1996 and 1997) reported that

Sudan red-7B at 0.25% concentration was non toxic to H. indicola and gave

prominent pink colour to termite, but its retention period in field was lesser (42 day)

than that of Nile blue-A (1 year and 3 months). Nile blueA retained in R. flavipes

and H. formosanus species throughout the 15 days period and did not cause

significant mortality (Su et al., 1991b).

In our study, we found that 100% (H2O) relative humidity was best for

staining of termites. Two termite species i.e. Microtermes obesi and Odontotermes

lokanandi were force-fed on bait containing Nile blue-A and Sudan red-7B. Three

concentrations i.e., high, medium and low of each dye were tested against both

species. Our results showed that both species of termites gained colour in 100%

relative humidity at high and medium concentrations. Retention time of Nile blue

and Sudan red was recorded against M. obesi for eight weeks. Nile blue-A caused

100

lower mortality, and it was retained well for eight weeks in termites. Sudan red-7B

caused comparatively more mortality.

Chapter 6

SCREENING OF PLANT EXTRACTS TO FIND OUT PROPER

101

CONCENTRATION FOR DEVELOPMENT OF SLOW-ACTING

TOXICANT BAITS TO MANAGE TERMITES

6.1. ABSTRACT

Workers and soldiers (4-5th instar) of Microtermes obesi and Odontotermes lokanandi

were tested against Leaf and seed extracts of Euphorbia helioscopia L.,

Cannabis sativa L., and Calotropis procera (Aiton) in Entomological laboratory of

National Agriculture Research Centre, Islamabad. Three concentrations (high,

medium and low) of leaves and seeds extrats of Euphorbia helioscopia, Cannabis

sativa and Calotropis procera were prepared and tested against Microtermes obesi

and O. lokanandi. Results revealed that all extracts showed moderate toxic effect.

100% mortalities were found in M. obesi and O. lokanandi on day 11 and 7

respectively.

6.2. INTRODUCTION

Subterranean termites are more abundant and their populations extremely

vicious polyphagous insect pests (Hickin, 1971), they damaged goods, plants and

agricultural crops (Manzoor and Mir, 2010). Tsunoda (2003) reported that a huge

amount of economy are used up annually to control termites and repair their damages

throughout the world. After world war second, synthetic insecticides such as

persistent organo-chlorine (OC) and organophosphate (OP), carbomate and

pyrethriod were discovered and the management of termites has been totally based

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on these fast-acting insecticides (Anonymous, 2000; Venkateswara et al., 2005), but

soon it was realized that these fast-acting poisons caused residual effects, resistance,

102

adverse effects on human health and environmental hazards that hinder extensive

use of pesticides (Coats, 1994).

Logan et al., (1990) reported fast-acting synthetic insecticides were replaced

with bio-pesticides, which were universal acceptable and practical approach across

the world. Some plants and their extracts contain a variety of chemical compounds

with many potential uses. Studies revealed that plant extracts play a vital role in

human and animal health safety, forest, agriculture, store grain and household pest

management (Pascual and Robledo, 1999; Scott et al., 2004), plant extracts have

been tested for their possible insecticidal, anti-feedant and repellent properties

(Saxena, 1998; Zhu et al., 2001; Blaske and Hertel, 2001; Isman, 2006).

Phytochemcials act on termites and other insects in several ways, including growth

retardation (Breuer and Schmidt, 1995), suppression of behaviour (Khan and Saxena,

1986), feeding inhibition (Wheeler and Isman, 2001), toxicity (Hiremath et al.,

1997), oviposition avoidance (Zhao et al., 1998) and reduction of fecundity and

fertility (Muthukrishnan and Pushpalatha, 2001).

Some plants contain a rich source of chemicals that keep away or kill termites

or hamper with their gut flora (Adams et al., 1998; Boue and Raina, 2003; Cheng et

al., 2004; Park and Shin, 2005; Verma et al., 2009), termite can be controlled with

few plant species such as Diospyros sylvatica Roxb, Lysitoma seemnii L.,

Pseudotsuga menziesii (Mirb.), Tabebuia guayacan (Seem.) (Ganapaty et al., 2004),

Euphorbia kansuii GanSui and Curcuma aromatica Salisb. (Shi et al., 2008),

Eucalyptus citrodora (Hook.), Eucalyptus globules L. (Zhu et al.,

2001), Taiwania cryptomerioides Hay. (Chang et al., 2001), Dodonaea viscose (L.)

Jacq. Purple hop bush (Anonymous, 2001), Cinnamomum camphora (L.) Nees and

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Eberm., Rosmarinus officinalis L., Ocimum basilicum L., Cymbopogon winterianus

Jowitt. (Sbeghen et al., 2002) and Coleus ambionicus (Lour.) (Singh et al., 2004)

have been discovered for their anti-feedant and insecticidal activities. Studies

revealed that many plants contain termiticides (Sakasegawa et al., 2003; Park and

Shin, 2005; Jembere et al., 2005; Cheng et al., 2007; Ding and Hu, 2010; Supriadi

and Ismanto, 2010).

The aim of present study was to investigate proper concentrations of

Euphorbia helioscopia L., Cannabis sativa L., and Calotropis procera (Aiton) for

development of slow-acting toxicant baits to manage termites.


6.3.1. Collection of Experimental Termites

Microtermes obesi and O. lokanandi were captured from termite infested

building situated in Rawal Town, Islamabad by using “NIFA-TERMAPs” (Salihah

et al., 1993). The intested traps were brought to Entomological laboratory, National

Agriculture Research Centre, Islamabad. The termites were separated from soil and

debris by using 5.0, 4.0 and 1.0 mm mesh sieves in regular sequence. After that the

termites along with debris and soil were placed on the inverted glass Petri dish put

on the apparatus set up by NIFA (Nuclear Institute of Food and Agriculture) termite

group consisting of a plastic tub (dia. 29.5 cm) with inverted glass Petri dish (dia.

15.3 cm). The termites fell down in the tub without any extra particle. Frequently,

the termites and debris on the Petri dish were disturbed with a camel brush to collect

all the termites in the plastic tub (dia. 29.5 cm). The soil and debris were gently

removed and the termites were introduced in other glass Petri dishes (dia. 15.3 cm)

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each having two same size filter papers moistened with distilled water and kept as

stock termites in desiccators having 92% Relative Humidity.

Identification of termites were done by using the key of Chaudhry et al. (1972).

6.3.2. Plants Collection and their Extracts Preparation

i) Euphorbia heliocsopia L. ( Sun spurge)

Healthy and fresh plants of Euphorbia helioscopia L. (Sun spurge),

Cannabis sativa L. (Bhang) and Calotropis procera (Ait.) (Ak), were picked from

Islamabad and brought in Entomological Laboratory of National Agriculture

Research Centre, Islamabad where seeds and leaves were removed from plants and

were chopped in pestle and motar finely. Following the technique of Aboaba et al.

(2006) with some modifications; aqueous extracts of seeds and leaves of Euphorbia

helioscopia, Cannabis sativa and Calotropis procera were prepared in three levels

i.e., 50, 33 and 25% (high, medium and low). These extracts were stored in

refrigerator.

6.3.3. Bioassay

6.3.3.1 Toxicity test

Following the technique used by Smith (1979), forced feeding test was carried out.

Sterlized Petri dishes (dia. 5.5 cm) were taken, and used as experimental units. Circular

blotting papers were cut according to the bottom of Petri dish. In each Petri dish, two filter

papers were placed in the bottom and one in the cap. In each Petri dish, Filter papers placed

in the bottom were damped with 0.4 mL of the respective extract dose to the extent that it

was fully absorbed. Medical syringe was used for soaking the filter papers. New syringe was

used for each dose.

105

Distilled water was used for control. Each treatment was replicated three times. Then

a population of 50 termites (45 workers and 05 soldiers) was released to each

experimental unit (Petri dish). These experimental units (Petri dishes) were kept in

the desiccators having 92% relative humidity and these desiccators were placed in

the laboratory at temperature (27 ± 3 0C) and relative humidity (60 ± 5% ). Daily

observations were taken and the dead termites were removed from each Petri dish

through forceps.


Completely randomized designed was used and the data obtained was converted to

percentage as:

Total number of dead termites after treatment x 100 Percent

Mortality =

Total number of termites before treatment

Then Abbot,s formula (1925) was used to correct the data (percent mortality).

Co-stat was used for data analysis. Least Significant Difference ( LSD) at P<0.05

was used for mean separation.

6.4. RESULTS

6.4.1. Microtermes obesi

The effects of different doses (high, medium and low) of Euphorbia

helioscopia aqueous leaves and seeds extracts on percent mean mortality in M. obesi

are presented in table-6.1.

i) Euphorbia helioscopia (leaf extracts) on 1st day, percent mean mortality in M.

obesi at high, medium and low doses was 3.40 ± 0.68, 3.40 ± 0.68 and 2.72 ± 0.68

106

respectively. The results indicate that there were no significantly (P > 0.05)

difference among aqueous extracts of Euphorbia helioscopia on day 1st (Table-6.1).

On second day of the experiment, the percent mean mortality in M. obesi was

6.25 ± 1.20 and 5.56 ± 0.69 recorded at high and medium doses, respectively, which

were found similar to each other (P > 0.05), while differed (P < 0.05) significantly

from percent mean mortality of 1.39 ± 0.69 noted at low

concentration.

Percent mean mortality in M. obesi on days 3rd , 4th and 8th were recorded at

high, medium and low doses. The analysis revealed that the percent mean mortalities

were found significantly (P < 0.05) higher amongst all treatments

(Table-6.1).

On days 5th to 7th and 9th the percent mean mortalities in M. obesi were

recorded at high, medium and low doses. The results (Table-6.1) revealed that

percent mean mortality in M. obesi recorded at medium doses was observed non

significant (P > 0.05) from percent mean mortality found at high and low aqueous

concentration, while the percent mean mortality found at high was found

significantly higher (P < 0.05) from mortality found at low dose.

Maximum percent mean mortalities in M. obesi were recorded on days 10th

and 11th (Table-6.1). The percent meant mortalities recorded at high, medium and

low doses on 10th and 11th day were 87.83 ± 3.50, 76.14 ± 1.04, 72.75 ± 2.09; 100.00

± 0.00, 91.06 ± 3.16 and 85.82 ± 2.17, respectively. The results indicate that the

107

percent mean mortality at medium and low doses was observed nonsignificant (P >

0.05), but significantly lower from high dose.

ii) Euphorbia helioscopia (seed extracts)

Results (Table-6.1) revealed that percent mean mortality in M. obesi on days 1st

and 2nd at high, medium and low doses was observed non-significant (P >

0.05).

The mean percent mortalities in M. obesi were 11.03 ± 1.29, 9.55 ± 1.44, and

5.86 ± 1.91 at high, medium and low doses on day 3rd , respectively. The analysis of

variance revealed that the percent mean mortality noted at high and medium doses

were remain non-significant (P > 0.05), but significantly (P < 0.05) higher from

mortality found at low dose (Table-6.1).

On days 4th , 5th , 7th and 10th the percent mean mortalities in M. obesi were

recorded at high, medium and low aqueous doses. The analysis revealed that percent

mean mortalities were found significantly different (P < 0.05) amongst all treatments

(Table-6.1).

On days 6th, 8th and 9th; the percent mean mortalities in M. obesi were

recorded at high, medium and low aqueous doses.The results (Table-6.1) indicated

that the mean percent mortalities recorded at medium and low aqueous

concentrations were observed non-significant (P > 0.05), but significantly lower (P <

0.05) from high dose.

108

Table-6.1 showed that on day 11, percent mean mortalities in M. obesi

recorded were 100.00 ± 0.00, 100.00 ± 0.00 and 94.17 ± 3.06 at high, medium and

low aqueous concentrations, respectively, which were found non-significant (P >

0.05).

6.4.2. Odontotermes lokanandi

The effects of different doses (high, medium and low) of Euphorbia

helioscopia aqueous leaves and seeds extracts on percent mortality of O. lokanandi

are presented in table-6.2.

i) Euphorbia helioscopia (leaf extracts)

Table-6.2 shows that percent mean mortality in O. lokanandi at high, medium

and low aqueous doses was observed non-significant (P > 0.05) on 1st day; while

percent mean mortality at high and medium aqueous doses was observed non-

significantly different (P > 0.05), but significantly higher (P < 0.05) from low dose

on 2nd and 4th days.

On days 3rd and 5th the percent mean mortality in O. lokanandi was recorded

19.99 ± 1.31, 16.67 ± 0.87, 12.48 ± 1.38; 54.52 ± 1.55, 48.54 ± 0.85 and 40.48 ±

2.62 at high, medium and low aqueous doses, respectively. The analysis of variance

indicated that percent mean mortality at high and medium doses was observed non-

significant (P > 0.05), but significantly higher (P < 0.05) from low dose (Table-6.2).

Table-6.2 shows that the percent mean mortalities in O. lokanandi were 80.07

± 3.04, 68.80 ± 2.92 and 61.22 ± 0.89 at high, medium and low aqueous doses on

6th day. The analysis of variance indicates that percent mean mortalities at meium

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and low doses was recorded non-significant (P > 0.05), but significantly lower (P <

0.05) from high dose.

On 7th day, maximum mean mortality in O. lokanandi was found. The

analysis revealed that the percent mean mortalities were 100 ± 0.00, 93.32 ± 3.35

and 78.83 ± 5.29 at high, medium and low aqueous concentrations, respectively. The

mean percent mortality was noted non-significant (P > 0.05) at high and medium

doses, but significantly higher (P < 0.05) from mean percent mortality recorded at

low dose (Table-6.2).

ii) Euphorbia helioscopia (Seed extracts)

Percent mean mortalities in O. lokanandi exposed to different doses i.e., high,

medium and low of seed were recorded. The table-6.2 shows that maximum mean

mortalities were found at high concentration as compared to medium and low

concentrations on 1st day. Statistically percent mean mortalities recorded at high

concentrations was found non significant (P > 0.05) from percent noted at medium

concentration, but differed from percent mortality observed at low dose.

On days 2nd and 3rd the mean percent mortalities in O. lokanandi were 24.96

± 2.45, 13.89 ± 2.43, 8.81 ± 1.25; 37.46 ± 2.07, 31.21 ± 2.60 and 20.30 ± 1.48 at high, medium and

low aqueous concentrations, respectively. The analysis of variance revealed that percent mean

mortality was significantly different (P < 0.05) from each other on days 2nd and 3rd at high, medium

and low concentrations (Table-6.2).

Table-6.2 shows that the percent mean mortalities in O. lokanandi were

110

60.40 ± 2.43, 41.96 ± 2.87, 37.66 ± 2.62; 68.88 ± 4.50, 52.42 ± 1.64 and 51.13 ±

4.34 on 4th and 5th days at high, medium and low concentrations, respectively. The

results show that the percent mean mortalities at medium and low concentrations

were found non-significant (P > 0.05) on days 4th and 5th, but significantly different

from percent mean mortality recorded at high dose.

Maximum percent mean mortality in O. lokanandi was observed on day 6th. The

results show that percent mean mortalities were 100.00 ± 0.00, 76.58 ± 4.12,

75.15 ± 7.08 were at high, medium and low concentrations, respectively. Statistically

the percent mean mortalities at medium and low concentrations were observed non-

significant (P > 0.05), while significantly lower (P < 0.05) from percent mean

mortality recorded at high concentration (Table-6.2).


The effects of different doses (high, medium and low) of Cannabis sativa

aqueous leaves and seeds extracts on percent mean mortality in M. obesi are shown

in tabe-6.3.

i) Cannabis sativa (leaf extracts)

Table-6.3 shows that the percent mean mortalities in M. obesi at high,

Table-6.1. Mean percent mortality in Microtermes obesi at different

concentrations of leaf and seed extracts of Euphorbia helioscopia.

Leaf Seed

After days High Medium Low High Medium Low

1 3.40 ± 0.68a 3.40 ± 0.68a 2.72 ± 0.68a 2.05± 1.18a 2.07 ± 0.01a 1.37 ± 0.69a

111

2 6.25 ± 1.20a 5.56 ± 0.69a 1.39 ± 0.69b 5.59 ± 1.37a 4.89 ± 0.68a 2.79 ± 0.69a

3 18.70 ± 0.67a 15.09 ± 1.14b 11.49 ± 1.82c 11.03 ± 1.29a 9.55 ± 1.44a 5.86 ± 1.91b

4 27.99 ± 1.64a 24.22 ± 1.23b 21.94 ± 1.26c 19.82 ± 1.89a 16.01 ± 1.22b 12.19 ± 1.44c

5 38.98 ± 1.96a 34.93 ± 1.24ab 32.47 ± 1.69b 36.00 ± 0.29a 31.20 ± 1.40b 25.61 ± 0.92c

6 53.42 ± 1.86a 48.29 ± 1.13ab 45.77 ± 0.38b 52.87 ± 1.62a 36.36 ± 1.42b 33.88 ± 0.27b

7 57.01 ± 1.65a 53.26 ± 0.92ab 50.45 ± 0.94b 64.99 ± 1.27a 53.04 ± 1.57b 49.59 ± 1.11c

8 66.61 ± 2.05a 62.62 ± 0.12b 58.54 ± 1.16c 79.46 ± 1.74a 72.90 ± 0.82b 69.08 ± 2.11b

9 74.68 ± 1.32a 69.21 ± 2.01ab 64.61 ± 2.54b 88.91 ± 2.44a 83.69 ± 0.82b 79.61 ± 0.55b

10 87.83 ± 3.50a 76.14 ± 1.04b 72.75 ± 2.09b 95.70 ± 2.15a 92.40 ± 2.08b 86.87 ± 1.67c

11 100.00 ± 0.00a 91.06 ± 3.16b 85.82 ± 2.17b 100.0 ± 0.00a 100.0 ± 0.00a 94.17 ± 3.06a

Significant difference (P < 0.05) is shown by different letters within a row

112

Table-6.2. Mean percent mortality in Odontotermes lokanandi at different

concentrations of leaf and seed extracts of Euphorbia helioscopia.


Leaf After days High Medium Low

Seed High Medium Low

1 8.97 ± 0.72a 8.97 ± 0 .72 a 6.55 ± 0.30a

2 a 15.67 ± 0.11 a 16.41 ± 0.69 11.94 ± 0.70 b

3 a 19.99 ± 1.31 16.67 ± 0.87 ab b 12.48 ± 1.38

4 a 34.55 ± 0.82 a 31.82 ± 0.81 b 25.63 ± 1.34

5 a 54.52 ± 1.55 48.54 ± 0.85 ab 40.48 ± 2.62 b

6 80.07 ± 3.04 a 68.80 ± 2.92 b b 61.22 ± 0.89

7 100 .00 ± 0.00a 93.32 ± 3.35 a 8 8 . 3 ± 5.29b

8.99 ± 2.46a 2.33ab 6.19 ± 1.37b 2.75 ±

24.96 ± 2. a 45 b 13.89 ± 2.43 8.81 ± 1.25c

37.46 ± 2.07 a b 31.21 ± 2.60 20.30 ± 1.48 c

a 60.40 ± 2.43 b 41.96 ± 2.87 b 37.66 ± 2.62

68.88 ± 4.50 a 52.42 ± 1.64 b 51.13 ± 4.34 b

a 100.0 ± 0.00 76.58 ± 4.12 b 75.15 ± 7.08 b

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medium and low concentrations were observed non-significant (P > 0.05) on day 1st.

Percent mean mortalities were recorded from 2nd to 6th days at high, medium and low

doses. The analysis of variance shows that the percent mean mortalities at high

concentration was observed non-significant (P > 0.05) from percent mean mortality

recorded at medium level, but significantly differed (P < 0.05) from percent mean

mortality noted at low concentration from days 2nd to 6th.

On days 7th and 8th the percent mean mortalities in M. obesi were recorded at

high, medium and low doses (Table-6.3). The analysis of variance indicates that

percent mean mortality recorded at high dose was observed non-significant (P >

0.05) from percent mean mortality noted at medium dose, but significantly differed

(P < 0.05) from percent mean mortality observed at low concentration on 7 th and 8th

days. Moreover, the percent mean mortalities recorded at high, medium and low

concentrations were found significantly different (P < 0.05) amongst all treatments

on 9th day.

Table-6.3 shows that the percent mean mortalities in M. obesi recorded at

high, medium and low concentrations were 97.17 ± 17, 89.63 ± 0.90 and 87.75 ±

0.82, respectively on 10th day. The results indicated that percent mean mortality

recorded at medium dose was noted non-significant (P > 0.05) from percent mean

mortality observed at low dose, but significantly (P < 0.05) lower from percent mean

mortality noted at high aqueous concentration.

Maximum percent mean mortality in M. obesi was recorded on 11th day of the

trial. Table-6.3 shows that the percent mean mortalities in M. obesi were 100.00 ± 0.00,

114

98.01 ± 0.10 and 95.00 ± 0.98 at high, medium and low concentrations, respectively.

Analysis revealed that percent mean mortality recorded at high dose was observed non-

significant (P < 0.05) from percent mean mortality recorded at medium concentration,

but significantly different (P < 0.05) from observation recorded at low level.

ii) Cannabis sativa (Seed extracts)

Table-6.3 shows that the percent mean mortalities in M. obesi at high,

medium and low concentrations were found non-significant (P > 0.05) amongst

treatments on 1st, 2nd, 4th and 5th days, however the percent mean mortalities observed

on 3rd day were 21.50 ± 1.19, 19.41 ± 1.60 and 18.03 ± 1.19 at high, medium and

low doses, respectively. The results revealed that the percent mean mortality noted

at high level was observed non-significant (P > 0.05) from percent mean mortality

recorded at medium, but the percent mean mortality recorded at high concentration

was found significantly higher (P < 0.05) from percent mean mortality recorded at

low level on 3rd day.

On day 6th the percent mean mortalities in M. obesi were 47.66 ± 2.31,

43.04 ± 2.35 and 38.38 ± 2.53 at high, medium and low doses, respectively. Analysis

revealed that percent mean mortality observed at high concentration was found non-

significant (P > 0.05) from percent mean mortality recorded at medium

concentration, but the percent mean mortality recorded at high dose was found

significantly higher (P < 0.05) from percent mean mortality observed at low

concentration (Table-6.3).

On days 7th , 8th and 9th, percent mean mortalities in M. obesi were recorded

at high, medium and low. Results (Table-6.3) revealed that the percent mean

115

mortality recorded at medium dose was observed non-significant (P > 0.05) from

percent mean mortality found at low dose, but was noted significantly different (P

< 0.05) from percent mortality observed at high dose.

The percent mortality in M. obesi was 94.76 ± 1.00, 89.48 ± 1.00 and 85.28

± 2.02 at high, medium and low doses, respectively on 10 th day. The results indicate

that the percent mean mortality recorded at high, medium and low doses was

observed significantly different (P < 0.05) amongst treatments (Table-6.3).

Table-6.3 shows that maximum percent mean mortality in M. obesi was noted

on 11th day. 100 (hundred) percent mean mortality was observed at high level, which

was statistically non-significant (P > 0.05) from percent mean mortality observed at

medium dose and significantly different (P < 0.05) from percent mean mortality

observed at low level.

6.4.4. Odontotermes lokanandi

The effects of different doses (high, medium and low) of Cannabis sativa

aqueous leaves and seeds extracts on percent mortality in O. lokanandi are shown in

table-6.4.

i) Cannabis sativa (Leaf extracts)

Table-6.4 shows that the percent mean mortalities in O. lokanandi were 9.15

± 1.38, 7.03 ± 1.39 and 2.80 ± 0.68 at high, medium and low doses, respectively on

1st day. The results indicated that the percent mean mortality observed at high dose

was found no-significant (P < 0.05) from percent mean mortality noted at medium

116

dose, but significantly higher (P < 0.05) from percent mean mortality noted at low

dose.

O n days 2nd and 3rd, the percent mean mortality in O. lokanandi was 17.04 ±

0.58, 12.40 ± 0.79, 10.04 ± 1.45; 28.70 ± 2.73, 21.15 ± 1.40 and 16.87 ± 1.91,

respectively at high, medium and low concentrations. The results showed that

percent mean mortality noted at medium dose was observed non-significant (P >

0.05) from percent mortality found at low dose, but significantly lower (P < 0.05)

from percent mortality recorded at higher dose on 2nd and 3rd days (Table-6.4).

Table-6.4 indicates that the percent mean mortality observed at high, medium

and low doses was found significantly different (P < 0.05) amongst each other on 4th,

5th and 6th days. 100 (hundred) percent mortality was noted at high dose on 7 th day,

which is non significant (P > 0.05) from percent mean mortality observed at medium

dose, but significantly higher (P < 0.05) from percent mortality recorded at low dose.

ii) Cannabis sativa (Seed extracts)

Table-6.4 shows that percent mortalities in O. lokanandi were 7.85 ± 1.40,

6.43 ± 0.05, 2.13 ± 1.23; 21.18 ± 1.30, 19.69 ± 0.56, 15.17 ± 0.93; 65.00 ± 3.83,

57.64 ± 0.72 and 45.54 ± 2.13 on 1st , 2nd and 5th days, respectively at high, medium

and low concentrations. Analysis indicate that percent mean mortality recorded at

high dose was non-significant (P > 0.05) from percent mean mortality observed at

117


concentrations of leaf and seed extracts of Cannabis sativa.


118

medium dose, but significantly differed (P < 0.05) from percent mean mortality noted at

low on 1st and 2nd days.

The percent mean mortalities in O. lokanadi noted on 3rd , 4th and 6th days

were 29.47 ± 2.59, 25.12 ± 3.13, 16.41 ± 2.84; 43.61 ± 2.68, 38.73 ± 2.82, 30.94 ±

3.72; 83.41 ± 3.00, 76.17 ± 2.41 and 66.72 ± 1.86, respectively at high, medium and

low doses. Analysis of variance shows that the percent mean mortality observed at

high, medium and low doses were observed significantly different (P <

0.05) amongst each other on 3rd, 4th and 6th days (Table-6.4).

On day 7th the percent mean mortalities in O. lokanandi were 100.00 ± 0.00,

93.31 ± 3.35 and 80.13 ± 2.32 at high, medium and low doses, respectively. The

result indicates that percent mean mortality noted at high dose was observed

nonsignificant (P > 0.05) from mortality recorded at medium concentration, but

significantly different (P < 0.05) from percent mean mortality noted at low dose

(Table-6.4).


The effects of different doses (high, medium and low) of Calotropis procera

aqueous leaves and seeds extracts on percent mortality in Microtermes obesi are

shown in table-6.5.

i) Calotropis procera (Leaf extracts)

The results (Table-6.5) indicated that the percent mean mortality in

Microtermes obesi observed non-significant (P > 0.05) at high, medium and low

doses on 1st, 2nd and 4th days. The percent mean mortalities noted on days 3rd and 5th

119

were 13.85 ± 1.86, 11.66 ± 1.38, 8.76 ± 0.06; 28.71 ± 2.18, 23.92 ± 1.97 and 19.97

± 1.95, respectively at high, medium and low doses. Results showed that the percent

mean mortality at high dose was observed non-significant (P > 0.05) from percent

mean mortality noted at medium dose, but significantly different (P < 0.05) from

percent mortality recorded at low level on 3rd and 5th days.

On days 3rd, 6th and 9th the percent mean mortalities in M. obesi were noted

at high, medium and low doses. The analysis revealed that the percent mean mortality

observed at medium dose was found non-significant (P > 0.05) from percent mean

mortality noted at low dose, but significantly lower (P < 0.05) from percent mean

mortality observed at high dose on days 3rd, 6th and 9th (Table-6.5).

Table-6.5 shows that the percent mean mortalities were 62.89 ± 1.30, 55.13

± 1.41, 50.79 ± 2.24; 80.37 ± 0.71, 75.91 ± 1.36 and 71.43 ± 0.80 on days 7 th and 8th,

respectively at high, medium and low doses. Results revealed percent mean mortality

was significantly different (P < 0.05) amongst treatments on days 7 th and

8th.

Maximum percent mean mortalities were recorded on 10th and 11th days. The

analysis indicated that the percent mean mortalities were 97.03 ± 1.70, 95.12 ±

0.92, 89.24 ± 0.84; 100.00 ± 0.00, 100.00 ± 0.00 and 95.80 ± 1.03 on days 10 th and

11th , respectively at high, medium and low doses. The results showed that percent

mean mortality at high dose was observed non-significant (P > 0.05) from percent

mortality recorded at medium dose, but significantly differed (P < 0.05) from percent

mean mortality found at low dose on days 10th and 11th (Table-6.5).

120

ii) Calotropis procera (Seed extracts)

The table-6.5 shows that the percent mean mortality found on days 1st, 2nd and 3rd

at high, medium and low doses was observed non-significant (P > 0.05).

The percent mean mortalities were 32.03 ± 2.44, 29.76 ± 1.13, 25.18 ± 1.16; 71.91

± 0.98, 67.55 ± 0.51 and 64.09 ± 1.80 on days 4 th and 7th , respectively at high,

medium and low doses. The results showed that the percent mean mortality recorded

at high dose was found non-significant (P > 0.05) from mortality noted at medium

dose, but significantly different (P < 0.05) from percent mean mortality observed at

low dose, moreover the percent mean mortality observed at medium dose was

observed non-significantly (P > 0.05) from percent mean mortality found at low dose

on days 4th and 7th (Table-6.5).

The results (Table-6.5) shows that the percent mean mortalities were 46.86 ±

1.05, 42.97 ± 0.67 and 35.94 ± 0.68 at high, medium and low doses, respectively on

day 5th. Results showed that the percent mean mortality was observed

significantly different (P < 0.05) amongst treatments on day 5 th.

On days 6th, 8th and 9th; the percent mean mortality in M. obesi was observed

at high, medium and low doses of seed extract of Calotropis procera. Results showed

that the percent mean mortality recorded at medium dose was noted non-significant

(P > 0.05) from percent mean mortality found at low dose, but significantly lower (P

< 0.05) from percent mean mortality recorded at high dose on 6 th, 8th and 9th days

(Table-6.5).

Table-6.5 indicates that the percent mean mortalities were 100.00 ± 0.00,

121

96.04 ± 1.05 and 94.14 ± 1.63 at high, medium and low doses on 10th day, Table-6.4.

Mean percent mortality in Odontotermes lokanandi at different

concentrations of leaf and seed extracts of Cannabis sativa.

Leaf

After days High Medium Low

Seed

High Medium Low

1

2

3

4

5

6

7

9.15 ± 1.38a

17.04 ± 0.58a

28.70 ± 2.73a

41.55 ± 0.67a

57.87 ± 2.10a

80.73 ± 2.69a

100.0 ± 0.00a

7.03 ± 1.39a

12.40 ± 0.79b

21.15 ± 1.40b

32.54 ± 2.60b

47.86 ± 3.93b

65.16 ± 3.31b

94.21 ± 3.22a

2.80 ± 0.68b

10.04 ± 1.45b

16.87 ± 1.91b

26.59 ± 2.76c

39.29 ± 2.79c

46.48 ± 3.59c

81.58 ± 2.30b

7.85 ± 1.40a

21.18 ± 1.30a

29.47 ± 2.59a

43.61 ± 2.68a

65.00 ± 3.83a

83.41 ± 3.00a

100.0 ± 0.00a

6.43 ± 0.05a

19.69 ± 0.56a

25.12 ± 3.13b

38.73 ± 2.82b

57.64 ± 0.72a

76.17 ± 2.41b

93.31 ± 3.35a

2.13 ± 1.23b

15.17 ± 0.93b

16.41 ± 2.84c

30.94 ± 3.72c

45.54 ± 2.13b

66.72 ± 1.86c

80.13 ± 2.32b


respectively. 100 (hundred) percent mean mortality was observed at high dose, which

was non-significant (P > 0.05) from percent mean mortality noted at medium dose,

122

but significantly different (P < 0.05) from percent mortality noted at low

concentration. Moreover, the percent mean mortality observed at medium and low

doses was also noted non-significant.

6.4.6. Odontontermes lokanandi

The effects of different doses (high, medium and low) of Calotropis procera

aqueous leaves and seeds extracts on percent mortality in O. lokanand are shown in

table-6.6.

i) Calotropis procera (Leaf extracts)

Table-6.6 shows that percent mean mortalities in Odontotermes lokanandi

were 8.26 ± 1.14, 4.12 ± 1.17, 3.43 ± 1.35 at high, medium and low doses,

respectively on 1st day. The results showed that the percent mean mortality at

medium dose was non-significantly different (P<0.05) from percent mean mortality

noted at low dose, but significantly lower (P < 0.05) from percent mean mortality

observed at high dose.

On day 2nd and 3rd the percent mean mortality in O. lokanandi was observed

at high, medium and low doses. The results showed that percent mean mortality was

observed non-significant (P > 0.05) amongst treatments on days 2nd and 3rd of the

trial (Table-6.6).

Table-6.6 shows that the percent mean mortalities in O. lokanandi were

66.37 ± 2.35, 61.95 ± 1.57 and 55.79 ± 0.97 at high, medium and low doses, Table-

6.5. Mean percent mortality in Microtermes obesi at different

concentrations of leaf and seed extracts of Calotropis procera.

123


respectively on 5th day. The analysis of variance revealed that percent mean mortality

at high dose was noted non-significant (P > 0.05) from percent mean mortality found

at medium dose, but significantly differed (P < 0.05) from percent mean mortality

noted at low dose.

124

Results (Table-6.6) indicates that the percent mean mortalities in O.

lokanandi were 50.00 ± 1.19, 45.20 ± 1.84, 40.47 ± 1.19; 87.70 ± 1.98, 82.06 ± 1.04,

74.52 ± 1.67; 100.00 ± 0.00, 93.71 ± 1.83 and 87.43 ± 2.03 on days 4 th, 6th and 7th,

respectively at high, medium and low doses. Analysis of variance showed that

percent mean mortality observed at high dose was observed significantly different (P

< 0.05) from percent mean mortality noted at medium and low dose.

ii) Calotropis procera (Seed extracts)

On 1st, 2nd 3rd and 4th days of the trial, the percent mean mortality in O.

lokanandi found at high, medium and low doses. The results indicated that the

percent mean mortality recorded at high dose was found non-significant (P > 0.05)

from percent mean mortality noted at medium dose, but significantly different (P <

0.05) from percent mean mortality recorded at low dose on 1st, 2nd , 3rd and 4th days

(Table-6.6).

The results of the percent mean mortalities in O. lokanandi on seed extracts

were 68.89 ± 2.71, 62.11 ± 1.81, 52.35 ± 2.54; 91.90 ± 4.23, 80.97 ± 3.37 and 68.65

± 3.25 on 5th and 6th days, respectively. The analysis revealed that the percent mean

mortality recorded at high dose was observed significantly different (P <

0.05) from percent mean mortality recorded at medium and low doses. The percent mean mortality

noted at medium and low doses was also observed significantly different (P < 0.05) (Table-6.6).

On day 7th , the percent mean mortalities in O. lokanandi were 100.00 ±

0.00, 100.00 ± 0.00 and 91.16 ± 1.15 at high, medium and low doses, respectively.

Hundred percent mortality was observed at high and medium doses, which was

125

found significantly different (P < 0.05) from percent mean mortality found at low

dose (Table-6.6).

6.5. DISCUSSION

Microtermes obesi and O. lokanadi were force-fed on different levels of leaf

and seed aqueous extracts of E. helioscopia. Our results indicated that E. helioscopia

contained chemical components that can be used for insect control. Percent mean

mortality in both species of termites were found concentrations depended. When leaf

and seed extracts of E. helioscopia were presented to M. obesi, percent mortality

range in M. obesi was from 2.72 ± 0.68 to 100 ± 0.00 and 1.37 ± 0.69 to 100.00 ±

0.00, respectively; however mortality ranged from 6.55 ± 0.30 to 100 ± 0.00 and

2.75 ± 1.37 to 100 ± 0.00 when leaf and seed extracts of E. helioscopia were offered

to O. lokanandi. In the present trial, it was found that O. lokanandi was more

susceptible to E. helioscopia that M. obesi. 100 % mortalities in two species of

termites were noted at higher dose. Studies reveal that essential oils and plant extracts

are an important natural reservoir of larvicides (Jacobson, 1983; Adebayo et al.,

1999; Murty and Jamil, 1987) or insecticides (Raguraman and Singh, 1997; Gbolade,

2001) or insect repellents (Sadik, 1973; Thorsell et al.,

1998; Oyedele et al, 2000). Verena and Hertel (2001) reported that some plants and Table-6.6. Mean

percent mortality in Odontotermes lokanandi at different concentrations of leaf

and seed extracts of Calotropis procera.

Leaf Seed


1 8.26 ± 1.14a 4.12 ± 1.17b 3.43 ± 1.35b 10.88 ± 0.68a 9.52 ± 0.68a 5.44 ± 0.68b

2 20.71 ± 1.35a 17.85 ± 0.66a 16.42 ± 1.37a 22.31 ± 0.81a 19.41 ± 1.12a 10.05 ± 1.85b

126

3 29.85 ± 1.93a 28.35 ± 1.39a 24.63 ± 0.19a 32.04 ± 0.93a 28.11 ± 1.17a 17.16 ± 1.44b

4 50.0 ± 1.19a 45.20 ± 1.84b 40.47 ± 1.19c 46.11 ± 2.03a 43.57 ± 0.83a 49.7 ± 2.57b

5 66.37 ± 2.35a 61.95 ± 1.57a 55.79 ± 0.97b 68.89 ± 2.71a 62.11 ± 1.8b 52.35 ± 2.54c

6 87.70 ± 1.98a 82.06 ± 1.04b 74.52 ± 1.67c 91.90 ± 4.23a 80.97 ± 3.37b 68.65 ± 3.25c

7 100.0 ± 0.00a 93.71 ± 1.83b 87.43 ± 2.03c 100.0 ± 0.00a 100.0 ± 0.00a 91.16 ± 1.15b


their extracts contain insecticides and these can be used for the management of

termites. Stoll (2001) reported that many higher plants were evaluated to be effective

against harmfull insect and diseases of various agriculture crops. In our study, it was

found that extracts of E. helioscopia, tropical herbal plants, contain insecticidal

properties, which can be used against M. obesi and O. lokanandi.

In our trail, percent mortality in M. obesi ranged from 3.39 ± 0.65 to 100.00

± 0.00 and 7.36 ± 1.74 to 100.00 ± 0.00 when leaf and seed extracts of C. sativa,

respectively were used; while lethal ranged from 2.80 ± 0.68 to 100.00 ± 0.00 and

127

2.13 ± 1.23 to 100.00 ± 0.00 when O. lokanandi were force fed on leaf and seed

extracts of C. sativa, respectively. Our results indicated that extract of C. sativa

contains insecticidal properties and percent mean mortality in two species of termites

were found dose depended. In our study it was found that extract of seeds were more

lethal that extracts of leaves of C. sativa. Badshah et al., (2004) reported that seed

extracts of Cannabis sativa L. and Polygonum hydropiper L. against Heterotermes

indicola and Coptotermes heimi were observed more lethal than leaf extracts in two

species of termites. Jalees et al. (1993) evaluted Cannabis sativa against the larvae

of Culex quinquefasciatus, Aedes aegypti and Anophles stephensi and they reported

that Cannabis sativa contain chemicals, which can be used

against insect.

The present study showed that percent mortalities in M. obesi by using

aqueous extracts of leaf and seed of Calotropis procera ranged from 2.73 ± 0.67 to

100.00 ± 0.00 and 3.42 ± 0.67 to 100.00 ± 0.00, respectively; while percent mortality

in O. lokanandi by forced feeding on leaf and seed extracts of Calotropis procera

ranged from 3.43 ± 1.35 to 100.00 ± 0.00 and 5.44 ± 0.68 to 100.00 ± 0.00,

respectively. In our study, it was found that the insecticidal activities of leave extracts

of Calotropis procera were significantly lower as compared with insecticidal

potency of seed extracts. Our results also showed that O. lokanandi was more

susceptible than M. obesi. Our results tally with the results of Badshah et al. (2004),

who indicated that extracts of seed and leaf of Calotropis procera (Ait.) had lethal

effects on H. indicola. Many termitologists reported that termites were successfully

controlled by using leaves extracts of Polygonum hydropiper (L) and

128

Pogostemon paviflorus (Benth) (Rehman et al., 2005), Aleurits fordii Hemsl, (Tung

tree) extracts (Hutchins, 2006), Garlic (Allium sativum L.), Calotropis procera

(Giridhar et al., 1988; Parihar, 1994), Diospyros sylvatica Roxb.

(Ganapaty et al., 2004) and Euphorbia kansuii GanSui. (Shi et al., 2008), Calotropis

procera (Ait.), D. stramonium L. and Datura alba Nees were also found the most

effective against the termites (Bajwa and Rajpar, 2001; Ayodele and Oke, 2003).

Our study revealed that all tested plant extracts had moderate lethal effect on

M. obesi and O. lokanandi. Mortalities in termites were found concentration

dependent. Euphorbia helioscopia, Cannabis sativa and Calotropis procera are

common weed almost every where in Islamabad. Being very chief source, farmers

can easily handle and apply these materials for the mamagement of termites and other

insect pest.

Traditionally soil treatment with insecticides of high repellency and long residual

effect have been used for flooding the target area is costly, inefficient and environmentally

unsafe. In the following experiment we attempted to test inorganic insecticides (Mercuric

Chloride and Copper Sulphate) against termites to find out slow-acting toxicant to formulate

baits that could be palatable, attractive and slow-active.

129

Chapter 7

LABORATORY INVESTIGATION OF COPPER SULPHATE AND

MERCURIC CHLORIDE TO FIND OUT PROPER CONCENTRATION TO

BE USED IN SLOW-ACTING

TOXICANT BAITS FOR MANAGEMENT OF TERMITES.

7.1. ABSTRACT

Two compounds viz. Copper Sulphate (CuSo4) and Mercuric Chloride

(HgCl2) at three different concentrations (high, medium and low) were evaluated

against Microtermes obesi and Odontotermes lokanandi to screen potential

slowacting toxicants.

After12 days, Mercuric chloride at high dose caused 100.00 ± 0.00 mortality

in M. obesi followed by 74.70 ± 1.43 and 64.62 ± 1.20 at medium and low

130

concentrations, respectively; whereas Copper Sulphate at high, medium and low

doses caused 86.90 ± 1.00, 69.18 ± 1.38 and 61.69 ± 0.58 mortality

respectively.

After 5th days, Mercuric chloride at high dose caused 100.00 ± 0.00 mortality

in O. lokanandi followed by 86.14 ± 1.60 and 54.53 ± 2.74 at medium and low

concentrations, respectively; while Copper Sulphate at high, medium and low

concentrations caused 100.00 ± 0.00, 79.79 ± 0.87 and 65.45 ± 2.46 mortality,

respectively.

7.2. INTRODUCTION


111

prevent subterranean termite attack and to control infestation. The regular application

of fast-acting termiticides for the management of subterranean termites has caused

many environmental and biological hazards. Interest on the use of slowacting

toxicants to suppress the populations of subterranean termites has been renewed (Su

et al., 1982a; Jones, 1984). Beared (1974) reported that the success of a slow acting

toxicant bait depends upon its attraction, palatability, delayed mortality and should

be introduced into the colony‟s gallery system and transferred to unexposed nest-

mate by social grooming or trophallaxis. Suppression of subterranean termite

populations reduces their damaging potential to near-by structures and may provide

long-term control. Moreover, a successful bait toxicant technique will drastically

reduce insecticides applications. It was first observed in the early 1900's that the

slow-acting arsenic dusts could be used to control subterranean termites (Randall

131

and Doody, 1934). Su et al. (1982) reported that the slow-acting quality of a toxicant

is very important, because increasing dead termites at the acquisition site will keep

away other colony-members from approaching the toxicant. Studies showed that

avermectin B1, hydramethylnon

R

(Amdro ) (Su et al., 1987), sulfluramid (Su and Scheffrahn, 1988b), A-9248 (Su and

Scheffrahn, 1988b), and insect growth regulators (IGRs) such as fenoxycarb, S-

31183, methoprene have indicated slow-acting characteristic against C. formosanus

and R. flavipes (Kollar) (Jones, 1984; Su et al., 1985; Haverty et al., 1989),

imidacloprid was found slow-acting poison against subterranean termites, (Thorn

and Breisch, 2001; Kard, 1998). Scheffrahn et al. (2001) studied the efficacy of

imidacloprid for the prevention of colony formation of subterranean termite.

The aim of present study was to investigate the efficacy of Cupper Sulphate

(CuSo4) and Mercuric Chloride (HgCl2) against subterranean termites (Microtermes obesi and

Odontotermes lokanandi).


7.3.1. Studies on the Efficacy of Mercuric Chloride and Copper Sulphate

Three concentrations i.e., high, medium and low of Mercuric Chloride

(HgCl2) were prepared by dissolving 100mg, 50mg and 25 mg of Mercuric Chloride

in 100 mL distilled water. Concentrations of Copper Sulphate (CuSo4) were also

prepared in the same way as Mercuric Chloride.

7.3.2. Bioassay

7.3.2.1. Toxicity Test

132

Following the technique of Smith (1979), forced feeding tests were

conducted. Sterlized Petri dishes (dia. 5.5 cm) were taken, and used as experimental

units. Circular blotting papers were cut according to the bottom of Petri dish. In each

Petri dish, two filter papers were placed in the bottom and one in the cap. In each

Petri dish, Filter papers placed in the bottom were damped with 0.4 mL of the

respective toxicant doses to the extent that it was fully absorbed. Medical syringe

was used for soaking the filter papers. New syringe was used for each dose. Distilled

water was used for control. Each treatment was replicated three times. Then a

population of 50 termites (45 workers and 05 soldiers) was released to each

experimental unit (Petri dish). These experimental units (Petri dishes) were kept in

the desiccators having 92% relative humidity and these desiccators were placed in

the laboratory at temperature (27 ± 3 0C) and relative humidity (60 ± 5% ). Daily

observations were taken and the dead termites were removed from each Petri dish

through forceps.


Completely randomized designed was used and the data obtained was converted to

percentage as:

Total number of dead termites after treatment x 100 Percent

Mortality =

Total number of termites before treatment

Then Abbot,s formula (1925) was used to correct the data (percent mortality).

Co-stat was used for data analysis. Least Significant Difference (LSD) at P<0.05 was

used for mean separation.

7.4. RESULTS

133

7.4.1. Efficacy of Copper Sulphate and Mercuric Chloride against M. obesi


i) Copper Sulphate (CuSo4)

Percent mean mortalities] in M. obesi at high, medium and low up to 4th day

were 17.14 ± 1.86 16.41 ± 0.69 and 14.92 ± 1.43, respectively. Analysis of variance

indicates that the percent mean mortality was found non-significant (P>0.05)

amongst treatments (Table-7.1).

On days 5th to 12th percent mortalities in M. obesi recorded at high, medium and

low were observed significantly differed (P<0.05) from each other. Maximum mean

mortality (86.90 ± 1.00%) was recoreded on 12th day at high concentration

(Table-7.1).

ii) Murcuric Chloride (HgCl2)

Results (Table-7.1) shows that percent mean mortalities in M. obesi were

32.13 ± 1.04, 29.99 ± 1.05 and 29.29 ± 0.84 at high, medium and low concentrations,

respectively up to 4th day. Analysis of variance revealed that percent mean mortality

was found non-significant (P>0.05) amongst each other.

On day 5th to 8th, percent mean mortalities in M. obesi at medium and low

concentrations were 38.05 ± 1.06, 48.10 ± 1.36, 54.03 ± 1.46, 59.47 ± 1.08; 34.34 ±

134

1.01, 42.72 ± 1.72, 53.19 ± 1.97, and 55.15 ± 1.26, respectively. Analysis of variance

showed that the percent mortality in M. obesi was found similar (P>0.05) to each

other, but significantly lower (P<0.05) from percent mortality found at high dose

(Table-7.1).

On day 9th to 12th percent mortality in M. obesi recorded at high, medium and low

was noted significantly differed (P<0.05) amongst each other. 100.00 ±

0.00% mortality was recorded on 12th day at high concentration (Table-7.1).

7.4.2. Efficacy of Copper Sulphate and Mercuric Chloride against O.

lokanandi


i) Copper Sulphate (CuSo4)

Table-7.2 shows that percent mean mortalities in O. lokanandi up to 2nd day

at high, medium and low were 47.13 ± 1.77, 44.65 ± 2.36 and 43.11 ± 1.72,

respectively. Analysis of variance revealed that percent mean mortality was found

non-significant (P>0.05) amongst each other.

On days 3rd to 5th , percent mortality in O. lokanandi recorded at high, medium and

low was found significantly different (P<0.05) from each other.

100.00 ± 0.00% mean mortality was recorded in 5th day at high concentration (Table-7.2).

ii) Mercuric Chloride (HgCl2)

135

Table-7.2 shows that percent mean mortalities in O. lokanandi up to 2nd day

at high, medium and low were 29.85 ± 0.66, 28.37 ± 0.87 and 29.09 ± 1.11,

respectively. Analysis of variance revealed that percent mean mortality was found

non-significant (P>0.05) amongst each other.

On days 3rd to 5th, percent mean mortalities in O. lokanandi recorded at high,

medium and low were observed significantly differed (P<0.05) amongst each other.

100.00 ± 0.00% mean mortality was recorded on 5th day at high

concentration (Table-7.2).

7.5. DISCUSSION

Our results showed that Mercuric chloride and Copper Sulphate at high

concentrations caused 100.00 ± 0.00 and 86.90 ± 1.00 mortalities, resprectively in

M. obesi after 12 days; whereas 100.00 ± 0.00 mortalities were found in O.

lokanandi after 5th day by using Mercuric chloride and Copper Sulphate at high


concentrations of Copper Sulphate (CuSo4) and Mercuric Chloride

(HgCl2)

Copper Sulphate (CuSo4)

After days High Medium Low

Mercuric Chloride (HgCl2)

High Medium Low

136

1

2

3

4

5

6

7

8

9

10

11

12

5.47 ± 1.35a

9.72 ± 0.69a

14.37 ± 1.33a

17.14 ± 1.86a

27.41 ± 0.94a

41.27 ± 0.79a

57.24 ± 1.14a

61.03 ± 0.51a

64.66 ± 0.56a

71.3 ± 1.54a

81.76 ± 1.37a

86.90 ± 1.00a

4.79 ± 0.67a

9.03 ± 0.69a

12.95 ± 0.09a

16.41 ± 0.69a

18.14 ± 1.0b

28.57 ± 1.37b

33.88 ± 0.27b

44.89 ± 1.97b

49.98 ± 1.30b

58.23 ± 1.82b

66.06 ± 1.76b

69.18 ± 1.38b

4.10 ± 1.17a

9.02 ± 0.69a

12.94 ± 1.16a

14.92 ± 1.43a

14.28 ± 0.33c

23.02 ± 0.79c

28.20 ± 1.37 c

31.37 ± 1.00c

38.80 ± 0.34c

49.55 ± 1.15c

55.64 ± 1.57c

61.69 ± 0.58c

4.78 ± 1.35a

9.58 ± 0.62a

19.73 ± 0.78a

32.13 ± 1.04a

47.76 ± 0.64a

64.11 ± 0.93a

71.76 ± 1.03a

80.18 ± 0.70a

82.29 ± 0.97a

87.26 ± 0.97a

93.38 ± 0.98a

100.00 ± 0.00a

4.10 ± 1.17a

7.54 ± 0.71a

19.00 ± 1.10a

29.99 ± 1.05a

38.05 ± 1.06b

48.10 ± 1.36b

54.03 ± 1.46b

59.47 ± 1.08b

61.95 ± 0.77b

68.17 ± 1.08b

72.65 ± 0.85b

74.70 ± 1.43b

4.11 ± 0.03a

6.85 ± 0.66a

19.02 ± 1.23a

29.29 ± 0.84a

34.34 ± 1.01b

42.72 ± 1.72b

53.19 ± 1.97b

55.15 ± 1.26b

56.63 ± 0.76c

60.01 ± 0.55c

63.23 ± 1.34c

64.62 ± 1.20c

Significant Difference (P < 0.05) is shown by different letters within a row.

Table-7.2. Mean percent mortality in Odontotermes lokanandi at different

concentrations of Copper Sulphate (CuSo4) and Mercuric Chloride

(HgCl2).

Copper Sulphate (CuSo4) Mercuric Chloride (HgCl2 )


1 11.50 ± 0.62a 10.07 ± 0.69a 10.07 ± 0.69a 6.41 ± 1.20a 5.70 ± 0.67a 6.41 ± 1.20a

137

2 47.13 ± 1.77a 44.65 ± 2.36a 43.11 ± 1.72a 29.85 ± 0.66a 28.37 ± 0.87a 29.09 ± 1.11a

3 57.92 ± 1.82a 50.45 ± 0.94b 44.84 ± 0.99c 50.79 ± 1.48a 40.79 ± 1.61b 36.62 ± 1.70c

4 76.29 ± 0.94a 63.89 ± 1.39b 54.64 ± 0.90c 77.63 ± 1.25a 59.25 ± 1.07b 38.85 ± 0.87c

5 100.00 ± 0.00a 79.79 ± 0.87b 65.45 ± 2.46c 100.0 ± 0.00a 86.14 ± 1.60b 54.53 ± 2.74c

Significant Difference (P < 0.05) is shown by different letters within a row.

concentration. Heavy metals are acting slow-acting poison against subterranean

termites (Watson and Lenz, 1990). Yoshimura et al. (1987) concluded that Mercuric

Chloride is a slow-acting poison. Saghir et al. (2011) reported that A9248 is also a

slow acting compound that is non-repellent and a biocide. Borates are acting as

metabolic poisons, causing toxicity through biostatic rather than biocidal

mechanisms (Lloyd et al., 1990). Coated and Sheasby (1971) reported that termites

can be controlled by using 5% solutions of Copper Sulphate. Chen et al. (1986)

treated blocks pine with 0.2 or 2% N-methyldioctylamine-boric acid complex (a) or

138

N-dioctylamine-boric complex (b) and Copper Sulphate in 1:2 molar ratio and used

against termites. Roomi et al. (1992) found that wooden pickets for fir (Abies

pindrow), coated with 2% Mercuric chloride to be resistance to mound-building and

subterranean termites in field test in Pakistan. In our experiment, we used this

chemical in much smaller concentration so as it may work as slow acting toxicant

rather than as repellent. Mortality and insecticidal activity are dose dependent even

with toxicants with delayed toxicity (Su et al., 1987;

Haagsma, 2003; Remmen and Su, 2005, Rust and Saran, 2006; Saran, 2006).

In our study, it was observed that at various concentrations of chemicals the

termites soldiers died earlier than the workers. This early death of soldiers may be

explained on the basis that they are depended upon workers for their food and as the

workers were stressed due to the effects of chemicals, they may not have fed the

soldiers adequately and the soldiers were thus first to die.

Our study revealed that Mercuric Chloride and Copper Sulphate were

palatable, attractive and slow-acting. These compounds could be used to formulate

slow-acting toxicant bait for management of subterranean termites.

139

Chapter 8

SCREENING OF DIFFERENT COMPOUNDS TO FIND OUT

PHAGOSTIMULANT TO MAKE ATTRACTIVE BAIT FOR

THE CONTROL OF SUBTERRANEAN TERMITES

8.1. ABSTRACT

Urea, yeast, glucose and saw dust extract were tested for their phagostimulant

properties on the subterranean termite, Microtermes obesi Holmgren (Blattodea:

Termitidae). The termites were attracted to all the compounds tested and they

survived for a long period of time. Maximum percent survival was 4% for glucose,

followed by 1% for yeast and 4% for urea. The highest consumption was for a bait

140

having 4% glucose, followed by 3% yeast, 3% urea and distilled water. Maximum

termite survival was for filter paper soaked in an extract of saw dust extract that had

been boiled for 25 minutes, followed by filter paper boiled for 20, 15 and 10 minutes,

respectively. Lower survival of termites was recorded on filter paper that was soaked

in sawdust extract that had not been boiled. Maximum bait consumption also was

found for filter paper soaked in poplar sawdust extract that had been boiled for 25

minutes; whereas lower consumption was found for sawdust extract that had not been

boiled.

8. 2. INTRODUCTION

Studies have shown that termites choose foods which contain higher levels of

nutrients and nutrients could be added to a termite bait matrix to increase its

121

palatability for termites (Smythe and Carter, 1970; Waller, 1988; Delaplane, 1989;

Oi et al., 1996; Doi et al., 1999).

Several studies have investigated that ions (Botch et al., 2010), high wood

density (Waller et al., 1990), sugar (Waller and Curtis, 2003; Swoboda et al., 2004;

Saran and Rust, 2005), and high levels of cellulose (Judd and Corbin, 2009) can

increase termite‟s food consumption. Abushama and Kambal (1977) reported that

Microtermes traegardhi Sjo¨ stedt preferred fructose, Heterotermes tenuis Hagen

respond to trehalose (Haifig et al., 2008); Reticulitermes spp. showed preference on

xylose, ribose, maltose, or fructose (Saran and Rust, 2005). Concentrations of agar

and sawdust have also been varied to increase the palatability of termite bait

(Spragg and Fox, 1974; Paton and Miller, 1980; Spragg and Paton, 1980; Su et al.,

141

1982; French and Robinson, 1984; Holt and Easey, 1985; Easey and Holt, 1989;

Miller, 1990; Su, 1994). Body extracts of termites in ether and acetone have been

tested for attractancy against termites by Lund (1966); in hexane (Matesumura et at.,

1969; Howard, 1980; Tokoro et al., 1990). In termite diet nitrogen compounds are

deficient and several proteins were tested to determine their potential as termite

phagostimulants or deterrents (Hingate, 1941; Potrikus and Breznak, 1981; Waller,

1988; Reinhard and Kaib, 2001). Henderson et al. (1994) described that urea were

found to increase feeding by Coptotermes formosanus in the laboratory. Akhtar and

Raja (1985) studied the effect of wood and wood extracts of Albizia procera

(Roxb.) and Bauhinia variegate L. on the survival and feeding response of

Bifiditermes beesoni. Waller et al. (1999) reported that yeast was found best

phagostimulant for termites, 39 yeast isolates from the hind gut of termites by using

RAPD-PCR (Prillinger et al., 1996). Different baits were screened in laboratory and

indicated that saw dust from Eucalyptus tetrodonta F. Muell. gave most satisfactory

attraction to Matotermes darwiniesis (Spragg and Fox, 1974), damp sawdust is

preferred food than pine sawdust (Vigil, 1979).

The main objective of the present study was to test different compounds (urea,

yeast, glucose, and poplar sawdust extract) to find out phagostimulants for

management of Microtermes obesi Holmgren.


8.3.1. Studies of different compounds to find out Phagostimulants

8.3.1.1. Determination of different compounds (urea, yeast and glucose) as

potential bait substrates for Microtermes obesi

142

To determine the impact of different compounds (urea, yeast and glucose) on

the survival of Microtermes obesi, eight concentrations (0.1, 1, 2, 3, 4, 5, 6, and 7%)

of urea, yeast and glucose were prepared in distilled water. Then, 0.8 mL of each

treatment was pipetted onto two previously weighed filter papers (Whatman no. 42,

4.2 cm diam), which were placed in a glass Petri dish (5.5 cm dia). Distilled water

was used as the control. Termites (Microtermes obesi) were captured from building

of National Agriculture Research Centre, Islamabad. These termites were

acclimatized in Entomological laboratory of National Agriculture Research Centre,

Islamabad for forty eight (48) hours. Then a cluster of fifty (50) termites (4 th -5th

instar; 45 workers and 5 soldiers) were introduced into each Petri-dish. Following

the procedure of Smith (1979), termites were fed for 20 days, and survival was

recorded each day. The experimental units were kept in controlled laboratory

conditions at 28 ± 20C and 60 ± 5% R.H. Survival of the termites was recorded daily

for twenty days. After twenty days, the experiment was terminated and filter papers

were dried in an oven for two hours at 80 0C and weighed. Percent bait consumption

was calculated by using the following formula:

Weight of the control sample − Weight of the tested sample ×100

% bait consumption =

Weight of the control sample

The experiment was arranged as completely randomized block design

(RCB) with four treatments (compounds) at eight levels (concentrations) plus a water

control. The data were analyzed by using Co-Stat (CoHort Software,

Monterey, CA) at the 5% level of significance. Means were separated by using

Turkey‟s HSD (honest significant difference) test at the 5% level.

8.3.2. Preparation of poplar saw dust extract

143

Poplar sawdust, the most attractive food for termite species (Salihah et al.,

1993), was taken from a saw mill and sterilized at 80 0C for 2 hrs in an oven. Then,

it was passed through a 30-mesh sieve to obtain very fine particles, which were

mixed with distilled water in the ratio of 1:10 (w/v) in a conical glass flasks (i.e., 10

g poplar sawdust was mixed in 100 mL distilled water). The sawdust was boiled for

0, 5, 10, 15, 20, or 25 minutes, and filtered through filter paper (Whatman no. 42) in

separate flasks covered with airtight lids. The filtrates were kept in a refrigerator (10

0C) until used for experiments.

8.3.2.1. Determination of poplar saw dust extract as potential bait substrates for

Microtermes obesi

For this study, we followed the methodology of Grace and Yates (1992) with

some modifications. Twenty grams of sterilized sand and 3 mL of distilled water

were placed into each of 18 clean and sterilized graduated beakers (4 cm diam). Two

filter papers (Whatman no. 42, 4.2 cm diam), one soaked in extract and the other in

distilled water, were weighed and placed vertically at opposite sides of the beaker in

such a way that half of each filter paper was covered in sand. Fifty termites (4 th -5th

instar; 45 workers and 5 soldiers) were added to each beaker. Daily observations

were taken and dead termites were removed with forceps. Survival of the termites

was recorded daily for 20 days. After 20 days, the filter papers were separated from

sand, washed thoroughly in water, dried in an oven for 2 hrs at 80 0C, and weighed.

The percent bait consumption was calculated using the formula presented above.

The experiment was arranged as completely randomized block design (RCB)

with six treatments (concentrations) and a control. Each treatment was replicated

144

three times. The data were analyzed by using Co-Stat at the 5% level of significance.

Means were separated by using Turkey‟s HSD test at the 5% level of significance.

8.3.3. Comparative attractancy test

For these experiments, we followed the procedures of Waller et al. (1999), with

some modifications. We used clean, sterilized choice chambers (dia. 18.4 x 3.3 cm

high) that had been internally divided in to five equal compartments by three plastic

walls (7 mm high). Twenty grams of sterilized sand (80 0C for 24 hrs) with 3 mL

distilled water were added to each compartment. Filter papers (Whatman no. 42)

were soaked in distilled water, yeast, urea, glucose, or sawdust extract (boiled for 25

minutes) and were placed in the choice chambers in such a way that half of the filter

paper was covered in sand. Then, 250 termites (225 workers and 25 soldiers) were

added to each choice chamber, and the experiment was replicated three times. Daily

observations of the termites were recorded, and after16 days, the experiment was

terminated and the filter papers were re-weighed.

The percent bait consumption was determined using the formula given above.

8.4. RESULTS

8.4.1. Eualvation of different compounds to find out phagostimulants

i) Effect of different compounds (urea, yeast and glucose) on bait consumption

and survival of Microtermes obesi

The urea and yeast treatments significantly reduced M. obesi survival at all

concentrations, and no insects survived above 5% urea or 6% yeast (Fig-8.1).

Maximum survival was 67.33 ± 1.15% for the 4% urea treatment and 70.0 ± 1.33%

145

for the 1% yeast treatment compared with 74.67±0.67% survival in the water control.

At all concentrations, glucose either increased (5 concentrations) or had no effect (3

concentrations) on survival of M. obesi. The lowest survival was for the 5% glucose

treatment (72.67±0.67%), while maximum survival was recorded at

4% glucose (84.67±0.67%).

Except for the two highest concentration of urea (6 and 7%), all treatment baits

(urea, yeast, and glucose) had increased consumption over the water control (Fig-8.

2). The highest overall bait consumption was for 4% glucose (27.19±0.24%). The highest consumption

for yeast was 21.05±0.21% (3%), and for urea it was 15.32±0.54 (3%), compared with the water

control 4.47±0.16%. Bait consumption was only 7.58±0.75% for 0.1% glucose.

ii) Effect of different concentrations of poplar sawdust extract on bait

consumption and survival of Microtermes obesi

Termite survival was significantly higher (P < 0.05) for all concentrations of

poplar sawdust extract than it was for the water control (Fig-8.3). Maximum survival

(83.33 ± 0.67) was recorded for filter paper that had been boiled for 25 minutes,

followed by 82.00 ±1.15, 80.67 ± 0.67, 77.33 ± 0.67, and 63.33 ± 0.67 for termites

fed on filter paper soaked in sawdust extract boiled for 20, 15, 10, and 5 minutes,

respectively. Termite survival was 60.0 ± 0.00 for filter paper soaked in sawdust

extract but not boiled and 70.67 ± 0.67% for filter paper and distilled water

(control).

Termites ate significantly more filter paper soaked in sawdust extract than they

did filter papers soaked only in water (control) (Fig-8.3). Average weight loss (i.e.,

amount consumed by termites) of filter papers soaked in poplar sawdust extract but

146

not boiled was 5.98 ± 0.41g, which was not significantly different (P > 0.05) from

the control (5.22±0.29 g). However, weight losses of filter paper soaked in poplar

sawdust extracts boiled for 5, 10, 15, 20, and 25 minutes were significantly greater

(P < 0.05) than the control. The maximum percent bait consumption (20.69 ± 0.92)

was for the longest boiling time (Fig-8.3).

Fig-8.1. Effect of different concentrations of Phagostimulants on the mean

percentage survival of Microtermes obesi.

b b

a a

d

c

a b

c

b

a a

c c

a

b

c

d

a

b

b

c

a a

c

d

a

b

c c

a b

147

Fig-8.2. Effect of different concentrations of Phagostimulants on percent bait

consumption by Microtermes obesi.

a a

b

c

a ab

b

c

a b

a

c

c

b

a

d

c

b

a

d

c

b

a

d c

b

a

c c

b

a

c

148

Fig-8.3. Effect of different concentrations of poplar sawdust extract on the

mean percentage survival and bait consumption by Microtermes

obesi.

f e

e

d

c

c

b

b

d

e

a

a ab

ab

149

8.4.2. Comparative attractancy test 1: Distilled water, 0.1% urea, poplar sawdust

extract, 3% glucose, and 3% yeast

When termites were allowed to feed in choice chamber on filter papers

soaked in different phagostimulants i.e., 0.1% urea, poplar saw dust extract, 3%

glucose, 3% yeast and control for sixteen days; the results showed that 3% yeast was

found more attractive, followed by 3% glucose, saw dust extract and 0.1% urea.

Minimum attraction was noticed in control.

Termites consumed the maximum amount of filter paper that had been

soaked in 3% yeast, followed by filter papers with 3% glucose, poplar sawdust

extract, and 0.1% urea, respectively (Fig-8.4). These treatments all were

significantly different (P < 0.05) from the control.



When termites were offered choices to feed in choice chamber on filter papers

soaked in distilled water (control), 0.1% urea, poplar saw dust extract, 3% glucose

and 4% yeast for sixteen days; termites consumed the maximum amount of filter

paper that had been soaked in 3% glucose, followed by filter papers with 4% yeast,

sawdust extract, and 0.1% urea, respectively (Fig-8.5). These treatments all were




150

When termites were given choice to feed in choice chamber on filter papers

soaked in distilled water, 0.1% urea, saw dust extract, 4% glucose and 2% yeast for

sixteen days; termites consumed the maximum amount of filter paper that had been

Fig-8.4. Response of Microtermes obesi to filter paper soaked in distilled water,

0.1% Urea, Poplar saw dust extract, 3% Glucose and 3% Yeast

151

soaked in 4% glucose, followed by filter papers soaked in 2% yeast, 0.1% urea, and

sawdust extract, respectively (Fig-8.6). These treatments all were significantly

different (P < 0.05) from the control.

8.4.5. Comparative attractancy test 4: Distilled water, 1% urea, poplar sawdust


When the termites were offered choice to feed in choice chamber on filter

paper soaked in distilled water (control), 1% urea, poplar saw dust extract, 2%

glucose and 1% yeast for sixteen days; termites consumed the maximum amount of

filter paper that had been soaked in 1% urea, followed by filter papers soaked in

sawdust extract, 1% yeast, and 2% glucose, respectively (Fig-8.7). These treatments

all were significantly different (P < 0.05) from the control.

8.4.6. Comparative attractancy test 5: Distilled water, 1% urea, poplar sawdust


When termites were offered choice to feed in choice chamber on filter paper

soaked in distilled water (control), 1% urea, poplar saw dust extract, 1% glucose and

1% yeast for sixteen days; termites consumed the maximum amount of filter paper

that had been soaked in 1% urea, followed by filter paper soaked in sawdust extract,

1% yeast, and 1% glucose, respectively (Fig-8.8). These treatments were all


8.4.7. Comparative attractancy test 6: Distilled water, 1% urea, 4% yeast, 4%

glucose, and poplar sawdust individually and in different combinations

There were significant (P < 0.05) treatment effects among the combinations of

treated filter papers bioassayed (Fig-8.9). The maximum consumption of treated

filter papers was for a bait having 4% glucose + 4% yeast + 1% urea + sawdust,

152

followed by filter papers having 4% glucose + 4% yeast +1% urea, 4% glucose + 1%

urea + sawdust, 1% urea + 4% yeast, sawdust extract + 4% yeast, sawdust extract,

4% yeast + 4% glucose, 4% glucose, 4% yeast, 4% urea, and distilled water,

respectively (Fig.8.9). Consumption of the 4% glucose + 4% yeast + 1% urea +

sawdust extract was significantly different from the other treatment combinations

and control.

8.5. DISCUSSION

Our results showed reduced survival of Microtermes obesi at all concentrations

of urea and yeast, with zero survival of termites at the highest concentrations of urea

(6% and 7%) and yeast (7%). However, glucose did not reduce survival of M. obesi,

even at the highest concentration (7%) concentration, and maximum survival for any

treatment was recorded for 4% glucose (84%). At most concentrations, glucose had

a stimulant effect on termite feeding. Higher concentrations of carbohydrates have

been reported to kill the gut protozoan of termites, which can lead to reduced survival

(Kanai et al., 1982).

Termites consumed significantly more filter-paper baits that had been treated

with urea, yeast, or glucose than they did filter papers treated with distilled water.

The highest bait consumption for each component was 27.19, 21.05, and 15.32 for

4% glucose, 3% yeast, and 3% urea, respectively. This increased consumption might

have been due to the phagostimulant effects of these components on termite feeding.

Our results agree with those of Reinhard and Kaib (2001), who determined that

glucose acted as feeding stimulants for R. santonensis. Waller and Curtis

(2003) found that baits treated with the highest concentration of glucose were

153


0.1% Urea, Poplar saw dust, 3% Glucose and 4% Yeast

154



155


1% Urea, Poplar saw dust, 2% Glucose and 1% Yeast

156



157

Fig-8.9. Percent mean bait consumption by Microtermes obesi when offered in

Choice chamber

significantly preferred by subterranean termites in choice evaluations. Waller et al.

(1999) observed that significantly greater numbers of termites were recruited to yeast

and sucrose chambers than they were to the control.

Our results showed that survival of termites feeding on baits with sawdust

extract was significantly higher than on untreated baits. Maximum survival of

Percent bait consumption by termites

3.36 7.2

7.65

8.95

9.27

9.97

10.19

12.61

15.43

17.82

22.38

Control

Urea (1%)

Yeast (4%)

Glucose (4%)

Yeast (4%)+ Glucose (4%)

Sawdust extract

Poplar saw dust extract + Yeast (4%)

Urea (1%) + Yeast (4%

Glucose (4%) + Urea (1%) + Poplar saw dust extract

Glucose (4%) + Yeast (4%) + Urea (1 % )

Glucose (4%) + Yeast (4%) + Urea (1 %) + Poplar saw dust extract

158

termites was recorded at filter paper boiled for 25 minutes, followed by filter paper

soaked in poplar sawdust extract boiled for 20, 15, and 10 minutes, respectively.

Termites show a preference for certain species of wood (McMahan 1966;

MoralesRamos and Guadalupe, 2001) and even show higher survivorship on the

preferred wood (McMahan 1966; Morales-Ramos and Guadalupe, 2003).

When M. obesi were offered choices of filter paper soaked in different

components in a five-compartment choice chamber, maximum attraction and bait

consumption was found for filter paper soaked in glucose, followed by yeast, poplar

sawdust extract, and urea. Minimal attraction and bait consumption was recorded for

the control (filter paper soaked in distilled water). It was concluded that glucose,

yeast, poplar sawdust extract, and urea are phagostimulants. These results confirm

the results obtained for 0.1% urea by (Waller, 1996), poplar sawdust extract

(Badshah, 2003), 3% yeast (Waller, 1999), and 3% glucose (Sattar, 2000).

Subterranean termites are known to regularly consume nitrogen in the form of uric

acid when they consume the bodies of nest mates (Hingate, 1941; Potroikus and

Breznak, 1981). Lysine (protein) was found as a feeding stimulant for both

Coptotermes formosanus (Chen and Henderson, 1996) and Reticulitermes

santonensis (Reinhard and Kaib, 2001). Galactose has been reported to significantly

increase consumption of baits by Reticulitermes spp. (Swoboda, 2004). Populations

of R. flavipes differ in their response to potential phagostimulants (Su and La Fage,

1984; Creffield et al., 1985; Deheer and Kamble, 2008; Lenz et al., 2009).

Different compounds i.e., urea, yeast, glucose and saw dust extract were

tested to find out phagostimulant that was acceptable and palatable for subterranean

159

termite, Microtermes obesi. The results revealed that all four tested compounds

attracted termites and it was proved that these compounds were phagostimulants.

In the following experiment we attempted to combine different

concentrations of Mercuric Chloride, Copper Sulphate, Calotropis procera,

Euphorbia helioscopia and Cannabis sativa with phagostimulants (urea, yeast,

glucose and saw dust extract) and these baits were tested against termites to

formulate slow-acting toxicant baits that could be palatable, attractive and slowactive

for termites.

Chapter 9

FORMULATION OF SLOW-ACTING TOXIC BAITS TO

CONTROL SUBTERRANEAN TERMITES

160

9.1. ABSTRACT

Five slow-acting toxicant baits i.e., Mercuric Chloride, Copper Sulphate,

Calotropis procera, Euphorbia helioscopia and Cannabis sativa were formulated

and tested against Microtermes obesi. The results showed that maximum percent

mortalities were observed at bait of Mercuric Chloride, followed by bait of Copper

Sulphate. However, baits of Euphorbia helioscopia, Calotropis procera and

Cannabis sativa showed moderate toxicity for Microtermes obesi. The result

revealed that highest percent bait consumption was observed containing Mercuric

Chloride, followed by baits of Copper Sulphate, Euphorbia helioscopia, Calotropis

procera and Cannabis sativa, respectively.

9.2. INTRODUCTION

Subterranean termites are the most destructive pest of agriculture, forest and

buildings, causing economic damage annually throughout the world, especially in

the tropical and sub-tropical regions (Pearce, 1997). Management of termites require

a total of US $22 billion each year world-wide (Su, 2003). Termite control often

generally emphasized on the control of the subterranean termites, because of its more

destructive nature than other termite groups (Lee and Chung, 2003). Both preventive

as well as curative measures are adopted to control subterranean termites (Su-and

Tamashiro, 1987; Su and Scheffrahn, 1990). Subteranean termites make their

conlonies in the ground, a regular information on organization of termite colonies is

required to plan successful methods of control (Spragg and Fox,

142

1974). Highly effective chemical treatments have been available for many years to

prevent subterranean termite attack and to control their infestation. The regular use

of fast-acting termiticides for the management of termites has caused a number of

161

environmental and biological hazards. Increased concern over environmental

contamination and threat to human health by the current termiticides particularly

chlorinated hydrocarbon has led to search for new biological materials and

innovative approaches to termite control.

Interest to suppress the populations of subterranean termites, terminologists

have used a new methodology of slow-acting toxicants (Su et al., 1982b; Jones,

1984). The novel concept of the pest management of subterranean termites utilizing

termite baiting systems is a model in the pest control field. Su and Scheffrahn (1996,

1998) reported that bait stations are designed to facilitate the consumption of a bait-

toxicant and its transfer to the rest of the colony; the goal is termite population

reduction or elimination.

The success of a slow-acting toxicant bait depends upon its attraction,

palatability, delayed mortality and these should be introduced into the colony‟s

gallery system and transferred to unexposed nest-mate by social grooming or

trophallaxis (Beared, 1974). Baits containing slow-acting toxicants to control

termites are promising both in terms of efficacy and minimizing environmental

impact. Because of its ability to eliminate termite colonies, baiting technology can

be a stand-alone measure for long-term protection of structures (Thorne and

Forschler, 2000; Grace and Su, 2001). A successful bait toxicant must be both non

repellent to promote feeding, and slow-acting to permit distribution through the

colony by trophallaxis and other social contacts (Su,1982; Su et al., 1987).

Esenther and Gray (1968) first reported successful control using baits

impregnated with the slow-acting and non-repellent insecticide dechlorane (mirex),

and development of the baiting techniques using this compound has continued in

162

China (Gao, 1987) and Australia (French, 1988). The effectiveness of baits depends

on termites locating the bait and the rate at which they consume and distribute it to

other colony members. Many researchers observed that a considerable amount of

research over years has focused on devising more attractive baiting systems and bait

matrices (Mauldin and Rich, 1975; Becker, 1976; Esenther and Beal, 1974, 1978;

Lenz et al., 1991; Chen and Henderson, 1996; Suoja et al.,

1999: Rojas and Morales-Ramos, 2001; Reinhard et al., 2002; Cornelius, 2003;

Morales-Ramos and Rojas, 2003). The success of baiting systems termite control

depend on the regular feeding of termites on baits (Su, 1991, 1994: Haagsma and

Bean, 1998). Many terminologists have tested heavy metals that is murcury, lead,

arsenical and boron as slow-acting toxicants against termites (Randall et al., 1934;

Reierson, 1966; Brill et al., 1987; Williams and Amburgey, 1987; Grace and

Abdallay, 1990; Khatoon et al., 1993).

The present project mentions a series of laboratory studies conducted in order to

formulate slow-acting toxicant baits for the management of M. obesi.


9.3.1. Formulation of Slow-acting Toxicant Baits

9.3.1.1. Experimental Termites

Termites (Microtermes obesi) were captured from an old structure situated

in Rawal Town, Islamabad with the help of “NIFA-TERMAPs” (Salihah et al.,

1993). The termite infested traps were brought to Entomological laboratory of

National Agriculture Research Centre, Islamabad. The traps were opened and the

termites along with the soil particle and rubbish were passed through 5.0, 4.0 and 1.0

mm mesh sieves in regular series. Then the termites along with rubbish and soil were

163

put on the inverted glass Petri-dish placed on the apparatus designed by NIFA

(Nuclear Institute for Food and Agriculture) termite group, comprising of a plastic

tub (dia. 29.5 cm) with inverted Petri-dish (dia.15.3 cm). Frequently the termites and

rubbish on the Petri dish were disturbed gently with a camel brush to fell down all

the termites in the tub. The Petri-dish along with the left over rubbish was gently

removed and the termites were released in other glass Petri-dishes (dia.15.3 cm) each

having two same size filter papers moistened with distilled water. Petri dishes were

placed in desiccators having 92% relative humidity.

9.3.1.2. Choice Feeding Test

Following the procedure used by (Waller et al., 1999), studies were

conducted to formulate slow-acting toxic bait to control subterranean termites by

combining different toxicants i.e., Mercuric Chloride, Copper Sulphate, Euphorbia

helioscopia (Sun spurg), Calotropis procera (Ak), Cannabis sativa (Bhang) and

phagostimulants i.e., 4% glucose + 4% yeast + 1% urea + poplar sawdust extract (1:1

ratio). Choice chamber consisting of a central Petri-dish (dia. 9 x 1.5 cm high)

attached by clear plastic tubing, having dia 0.68 cm and 14 cm length, to other eight

Petri-dishes (dia. 9 x 1.5 cm high) (Fig-9.1). The central Petri-dish was left empty

while the other seven attached Petri-dishes contained filter papers (already weighted

with electrical balance) soaked in distilled water and the 8th one contained weighed

filter paper soaked in solution of HgCl2 coated with a mixture of phagostimulants

i.e., 4% glucose + 4% yeast + 1% urea + poplar saw dust extract (1:1 ratio). Each

Petri-dish was covered with a led. Then a population of 250 termites (4 th -5th instar)

including 225 workers and 25 soldiers was placed to the central Petri-dish and was

allowed to forage through the tubing to the attached Petri-dishes. Each concentration

was replicated three times. Daily observations were taken and 01- 02 mL of distilled

164

water (equal amounts in all the treatments) was sprayed on the filter paper of all the

Petri-dishes on every 2nd or 3rd day to maintain the moisture content of the filter

paper. After sixteen days, the experiment was terminated and the final weight of filter

paper was taken from which the consumed portion of filter paper was determined.

Weight loss in filter paper was considered as palatability for the said chemical, while

mortality showed its toxicity.

Similarly for Copper Sulphate (CuSo4), Euphorbia helioscopia, Calotropis

procera and Cannabis sativa the same procedure was applied as was explained for

Mercuric Chloride (HgCl2) and these experiments were also terminated after 16 days

and data were tabulated. In all these tests, termites were allowed to feed according

to their choice either on chemical treated or control filter papers inside the chambers.

The data were analyzed by using Co-State at 5% level of significance. Duncan,s

Multiple Range Test was used to separate the means.

9.4. RESULTS

9.4.1. Formulation of Palatable toxicant baits for Microtermes obesi by

combining of phagostimulants with different toxicants

165

Fig-9.1. Choice Chamber to formulate slow-acting toxic baits for termites.

Chambers 1 to 7th: Petri dishes contained filter papers soaked in distilled water.

Chamber 8th : Chamber contained filter paper soaked in toxicant coated with

a mixture phagostimulants.

Chamber 9th: Termite releasing chamber.

i) Mercuric Chloride

Results (Table-9.1) showed that percent mortality of termites in baits having

different concentrations of Mercuric chloride were found significant (P<0.05) on 4 th

day. Maximum (28.4 ± 0.46) mortality was recorded in bait having 0.1% Mercuric

1

8

7

6

5

4

3

2

9

166

chloride, followed by 6.8 ± 0.23 and 5.6 ± 0.23 at 0.05 and 0.025% respectively.

Minimum (0.93 ± 0.13) mortality was recorded in control. Percent mortality was

directly proportional to concentration.

On 8th day of the trial, percent mortalities were 54.13 ± 0.71, 17.73 ± 0.35,

14.67 ± 0.35 and 2.13 ± 0.13 in baits having 0.1, 0.05, 0.025% concentrations of

Mercuric chloride and control, respectively. Analysis of variance revealed that the

percent mortality of termites differed significantly (P<0.05) from each other

(Table-9.1).

Percent mean mortalities were 79.33 ± 0.35, 29.87 ± 0.35, 26.93 ± 0.13 and 3.2 ±

0.23 in baits having 0.1, 0.05, 0.025% concentrations of Mercuric chloride and

control, respectively on 12th day of the trial. Analysis of variance revealed that

percent mean mortality was significantly differed (P<0.05) amongst each other

(Table-9.1).

On day 16th, the maximum (92.53 ± 0.35) mean mortality was observed in bait

containing 0.1% concentration of Mercuric chloride, followed by 44.27 ± 0.35 and

35.6 ± 0.23 at 0.05 and 0.025%, respectively. The minimum mortality (7.2 ±

0.23) was found in control. Analysis of variance revealed that percent mean mortality was

found significantly different from each other (Table-9.1).

ii) Copper Sulphate

Results (Table-9.2) shows that percent mortality of termites was

significantly different (P<0.05) in baits having different concentrations of Copper

167

Sulphate on day 4. Percent mortalities of termites were 19.73 ± 0.35, 13.87 ± 0.35

and 10.80 ± 0.23 in baits having 0.1, 0.05 and 0.025% concentrations of Copper

Sulphate, respectively. Minimum percent mean (3.20 ± 0.23) was noted in control.

On day 8, the percent mean mortalities in baits having 0.1, 0.05 and

0.025% concentrations of Copper Sulphate and control were 40.67 ± 0.35, 30.00 ±

0.23, 26.67 ± 0.48 and 10.00 ± 0.23, respectively. Analysis of variance shows that


(Table-9.2).

Table-9.2 showed that percent mean mortality was found statistically

different (P<0.05) amongst each other on 12th day of the trial. Maximum

(74.00±0.40) percent mean mortality was noted at 0.1% concentration of Copper

Sulphate, followed by 39.73 ± 0.35 and 34.53 ± 0.35 in baits having 0.05 and 0.025%

concentration, respectively; while minimum (15.07 ± 0.13) percent mean mortality

was observed in control.

Maximum (90.93 ± 0.13) mean mortality was recorded in bait having 0.1%

concentration of Copper sulphate, followed by 57.73 ± 0.35 and 42.13 ± 0.35 in baits

containing 0.05 and 0.025% concentrations, respectively; while minimum (18.13 ±

0.35) percent mean mortality was observed in control on 16th day of the experiment.

The (Table-9.2) shows that percent mean mortality in all treatments were observed

significantly differed (P<0.05) amongst each other.

168

iii) Euphorbia helioscopia

Results (Table-9.3) showed that maximum percent mean mortality was 7.20 ± 0.23

in bait containing 50% concentration of Euphorbia helioscopia, followed by 5.87 ±

0.35, 5.33 ± 0.13 and 4.12 ± 0.13 in baits having 33 and 25% concentrations of

Euphorbia helioscopia and control, respectively on 4th day of the experiment.

Analysis of variance revealed that percent mean mortality in baits having 33 and

25% doses was observed non-significant (P>0.05), but significantly differed

(P<0.05) from bait having 50% concentration and control.

On day 8, the percent mean mortalities in baits containing 50, 33 and 25%

concentrations of Euphorbia helioscopia and control were 15.73 ± 0.35, 14.93 ±

0.35, 14.00 ± 0.23 and 8.80 ± 0.23, respectively. Analysis of variance shows that


(Table-9.3).

Results (Table-9.3) showed that percent mean mortalities in baits containing 50 and 33%

concentrations of Euphorbia helioscopia were 31.20 ±

0.23 and 30.67 ± 0.35, respectively on 12th day of the trial, which were observed non-significant

(P>0.05) between each other, but significantly higher (P<0.05) from percent mortality recorded in bait

having 25% concentration and control.

Results (Table-9.3) showed that maximum (45.87 ± 0.35) mean mortality was

recorded in bait containing 50% concentration of Euphorbia helioscopia, followed

by 42.80 ± 0.23 and 39.47 ± 0.48 percent mean mortalities noted in baits having 33

and 25% concentrations respectively, while minimum (18.00 ± 0.23) percent mean

mortality was observed in control on 16th day of the experiment. Table-9.3 showed

169

that percent mean mortality in all doses were found significantly differed (P<0.05)

amongst each other.

iv) Calotropis procera (Ak)

Results (Table-9.4) shows that percent mean mortalities were 8.00 ± 0.23,

7.87 ± 0.48 and 7.47 ± 0.35 in baits containing 50, 33 and 25% concentrations of

Calotropis procera on 4th day, which were non-significantly differed (P>0.05), but

significantly higher (P<0.05) from percent mean mortality (4.67 ± 0.35) recorded in

control.

On day 8, the percent mean mortalities in baits containing 50 and 33%

concentrations of Calotropis procera were 21.60 ± 0.40 and 21.20 ± 0.46, which

were found non-significantly differed (P>0.05) amongst each other, while

significantly higher (P<0.05) from percent mean mortalities recorded in bait having

25% concentration and control (Table-9.4).

On day 12, percent mean mortalities were 38.80 ± 0.46, 37.20 ± 0.46 and

34.53 ± 0.35 in baits containing 50, 33 and 25% concentrations of Calotropis

procera, respectively. Analysis of variance revealed that percent mean mortalities

recorded in baits having 50, 33 and 25% concentrations of Calotropis procera were

found significantly differed (P<0.05) amongst each other. Minimum (17.47 ± 0.35)

percent mean mortality were observed in control, which were found significantly

lower from percent mortalities recorded in baits having 50, 33 and 25%

concentrations (Table-9.4).

170

Results (Table-9.4) showed that percent mean mortality in all baits were

found significantly higher (P<0.05) amongst each other on 16th day of the

experiment. Maximum (54.40 ± 0.23) mean mortality was recorded in bait

containing 50% concentration of Calotropis procera, followed by 50.00 ± 0.46 and

42.67 ± 0.35 percent mean mortalities noted in baits having 33 and 25%

concentrations, respectively, while minimum (20.67 ± 0.35) percent mean mortality

was observed in control.

v) Cannabis sativa

On 4th day, the highest percent mean mortality was 10.00 ± 0.23 in bait containing

50% concentration of Cannabis sativa, followed by 8.67 ± 0.35 and 8.00 ± 0.40 in

baits having 33 and 25% concentrations, respectively; while the lowest percent mean

mortality (6.53 ± 0.35) was observed in control. Analysis of variance revealed that

percent mean mortalities in baits having 33 and 25% concentrations of Cannabis

sativa were observed non-significant (P>0.05), but statistically differed (P<0.05)

from bait having 50% concentration and control

(Table-9.5).

153

Table-9.1. Percent mean mortality in Microtermes obesi offered filter paper baited with different concentrations of

Mercuric Chloride (HgCl2) coated with phagostimulant in choice with distilled water

Phagostimulant Concentration

Termites mortality (%) over time (mean ± SE)

4th day 8th day 12th day 16th day

Glucose (4%) +

Yeast (4%) +

Urea (1%) +

Poplar sawdust

extracts in 1:1

ratio

Control 0.93 ± 0.13 d 2.13 ± 0.13 d 3.2 ± 0.23 d 7.2 ± 0.23 d

0.025% 5.6 ± 0.23 c 14.67 ± 0.35 c 26.93 ± 0.13 c 35.6 ± 0.23 c

0.05% 6.8 ± 0.23 b 17.7 3± 0.35 b 29.87 ± 0.35 b 44.27 ± 0.35b

0.1% 28.4 ± 0.46 a 54.13 ± 0.71 a 79.33 ± 0.35 a 92.53 ± 0.35 a

Value in the same column with different letter shows significantly difference (P<0.05)

154

Table-9.2. Percent mean mortality in Microtermes obesi offered filter paper baited with different concentrations of Copper

Sulphate (CuSo4) coated with phagostimulant in choice with distilled water




Glucose (4%) +

Yeast (4%) +

Urea (1%) +

Poplar sawdust

extracts in 1:1

ratio

Control 3.20 ± 0.23d 10.00 ± 0.23 d 15.07 ± 0.13 d 18.13 ± 0.35 d

0.025% 10.80 ± 0.23 c 26.67 ± 0.48 c 34.53 ± 0.35 c 42.13 ± 0.35 c

0.05% 13.87 ± 0.35 b 30.00 ± 0.23 b 39.73 ± 0.35 b 57.73 ± 0.35 b

0.1% 19.73 ± 0.35 a 40.67 ± 0.35 a 74.00 ± 0.40 a 90.93 ± 0.13 a

Value in the same column with different letter shows significant difference (P<0.05)

155

Table-9.5 shows that percent mean mortalities recorded in baits containing

50, 33, 25% concentrations of Cannabis sativa and control were 21.33 ± 0.35,

19.47 ± 0.35, 17.87 ± 0.27 and 12.53 ± 0.35, respectively on 8 th day of the trial.

Analysis of variance revealed that percent mean mortalities in all treatments were

found significantly different (P<0.05) amongst each other.

The highest percent mortality was 34.13 ± 0.35 in bait having 50%

concentration of Cannabis sativa, followed by 30.93 ± 0.48 and 28.40 ± 0.23 found

in baits having 33 and 25% concentrations of Cannabis sativa on 12th day. The lowest

(18.27 ± 0.35) percent mortality was noted in control. Results (Table-9.5) showed

that percent mean mortalities in all treatment were found statistically differed

(P<0.05) amongst each other.

Results (Table-9.5) showed that percent mean mortality recorded in all

treatments are significantly higher (P<0.05) from each other on 16 th day of the trial.

Maximum (52.27 ± 0.48) mean mortality was recorded in bait having 50%

concentration of Cannabis sativa, followed by 48.80 ± 0.69 and 38.20 ± 0.69 percent

mean mortalities noted in baits having 33 and 25% concentrations of Cannabis

sativa, respectively, while minimum (24.13 ± 0.48) percent mean mortality was

observed in control.

9.4.2. Comparative percent bait consumption by Microtermes obesi when

offered different baits at high concentrations of toxicants i.e., 0.1% Mercuric

Chloride, 0.1% Copper Sulphate, 50% Euphorbia helioscopia, 50% Calotropis

procera (Ak), 50% Cannabis sativa coated with a mixture of phagostimulants

156

i.e., 4% Glucose + 4% Yeast + 1% Urea + Poplar saw dust extract in Choice

with distilled water

Different baits having highest concentrations of Mercuric chloride, Copper

sulphate, E. helioscopia, C. procera, C. sativa coated with a mixture of

phagostimulants i.e., 4% glucose + 4% yeast + 1% urea + poplar saw dust extract in

Choice chamber. The results (Fig-9.2) showed that maximum bait consumption was

observed in chamber having Mercuric chloride followed by bait consumption noted

in chambers of Copper sulphate, Euphorbia helioscopia, Calotropis procera (Ak),

Cannabis sativa and water (control), respectively. Analysis of variance revealed that

percent mean bait consumption was found non-significant (P>0.05) between bait

having Mercuric chloride and Copper sulphate, while significantly higher (P<0.05)

from percent bait consumption having E. helioscopia, C. procera, C. sativa and water

(control).

9.5. DISCUSSION

Our results indicated that maximum mortalities were observed in baits having high

(0.1%) concentration of Mercuric Chloride and Copper Sulphate after

16th day followed by mortalities at 0.05 and 0.025% concentrations, respectively.

Minimum mortality was found in control. This shows that the level of mortality of

termites were depended on concentration. Highest (92.53 ± 0.35) percent mortalities

were observed at Mercuric Chloride followed by mortality (90.93 ± 0.13) at Copper

Sulphate after 16 days indicate a good slow-acting toxicant characteristics. This

confirms the results of (Brill et al., 1987; Yoshimura et al.,

1987; Sattar, 2000) who concluded that heavy metals are slow-acting toxicants.

177

Table-9.3. Percent mean mortality in Microtermes obesi offered filter paper baited with different concentrations of Euphorbia

helioscopia coated with phagostimulant in choice with distilled water




Glucose (4%) +

Yeast (4%) +

Urea (1%) +

Poplar sawdust

extracts in 1:1

ratio

Control 4.12 ± 0.13 c

8.80 ± 0.23 d

12.80 ± 0.23 c

18.00 ± 0.23 d

25% 5.33 ± 0.13 b

14.00 ± 0.23 c

28.13 ± 0.35 b

39.47 ± 0.48 c

33% 5.87 ± 0.35 b

14.93 ± 0.35 b

30.67 ± 0.35 a

42.80 ± 0.23 b

50% 7.20±0.23a

15.73±0.35a

31.20 ± 0.23 a

45.87 ± 0.35 a


178

Table-9.4. Percent mean mortality in Microtermes obesi offered filter paper baited with different concentrations of Calotropis

procera (Ak) coated with phagostimulant in choice with distilled water




Glucose (4%)

+ Yeast (4%) +

Urea (1%) +

Poplar sawdust

extracts in 1:1

ratio

Control 4.67 ± 0.35 b 10.93 ± 0.35 c 17.47 ± 0.35 d 20.67 ± 0.35 d

25% 7.47 ± 0.35 a 18.80 ± 0.23 b 34.53 ± 0.35 c 42.67 ± 0.35 c

33% 7.87 ± 0.48 a 21.20 ± 0.46 a 37.20 ± 0.46 b 50.00 ± 0.46 b

50% 8.00 ± 0.23 a 21.60 ± 0.40 a 38.80 ± 0.46 a 54.40 ± 0.23 a


Table-9.5. Percent mean mortality in Microtermes obesi offered filter paper baited with different concentrations

of Cannabis sativa coated with phagostimulant in choice with distilled water




179

Glucose (4%) +

Yeast (4%) +

Urea (1%) +

Poplar sawdust

extracts in 1:1

ratio

Control 6.53 ± 0.35 c 12.53 ± 0.35 d 18.27 ± 0.35 d 24.13 ± 0.48 d

25% 8.00 ± 0.40 b 17.87 ± 0.27 c 28.40 ± 0.23 c 38.20 ± 0.69 c

33% 8.67 ± 0.35 b 19.47 ± 0.35 b 30.93 ± 0.48 b 48.80 ± 0.69 b

50% 10.00 ± 0.23 a 21.33 ± 0.35 a 34.13 ± 0.35 a 52.27 ± 0.48 a


180

Fig-9.2. Percent bait consumption by Microtermes obesi when offered

different baits at high concentrations of toxicants in Choice with

distilled water

Our results showed that baits developed from plant extracts showed moderate

toxic effect. Maximum (54.40 ± 0.23) mortalities were recorded in baits having

181

Calotropis procera (Ak), followed by Euphorbia helioscopia (Sun spurg) and

Cannabis sativa (Bhang), respectively. Our results showed that the food

consumption and attraction of termites were always more in choice chambers having

filter paper soaked in distilled water (control). It revealed that the plant extracts had

strong repellent activities. Termites did not attract to the chamber where toxic baits

were placed. Plants extracts were found repellent when used against subterranean

termites during choice test, while in No-choice test, extracts proved to be toxic by

direct contact and indirect exposure (Farkhanda et al., 2011).

In Our results Mercuric Chloride and Copper Sulphate were found palatable,

attractive and non-repellent. Termites consumed Maximum bait of Mercuric Chlorid,

followed by bait of Copper Sulphate, while consumption of baits having plants

extract found low as compared to control. Heavy metals are slowacting, palatable

and non-repellent toxicant for termites control (Watson and Lenz, 1990), while plant

extracts were found repellent for subterranean termites (Blaske and Hertel, 2001).

Chapter 10

182

GENERAL DISCUSSION

In our study, 1200 poplar wooden survey stakes were installed in Islamabad

to monitor the termite activities. Of the 1200 stakes placed in the ground, typically

only 65 were infested by two termite species i.e., Odontotermes lokanandi and

Microtermes obesi. Researchers used different survey techniques (Table-10.1).

During observations, it was found that some traps harbour the same one species, and

some time a single trap may have mixed population of two species of different

genera. When such traps were opened there was a great antagonistic behavior that

they quarreled up to the death of the weaker and fever numbers. Antagonistic

behaviour between different colonies actually results in the maintenance of discrete

territorial boundaries and demographically closed societies (Jones, 1990). Studies

have correlated climatic variables such as minimum and maximum temperature and

annual rainfall to the range limits of species (Jeffree and Jeffree, 1996; Bullock et

al., 2000).

In our study a total of 65 stakes out of 1200 was found infested by termites.

The infested stakes were replaced by “NIFA–TERMAPs” to capture a huge number of

termites from the experimental areas. A total of 10, 34, 40, 47, 47, 50,

54, 54, 59 and 65 NIFA-TERMAPs were set up after 15, 30, 45, 60, 75, 90, 105, 120,

135 and 150 days, respectively. Throughout the experimental period, fluctuations

were found in environmental factors. Maximum traps were found infested, when the

temperature and relative humidity were recorded maximum. Fei and Henderson

(2004) reported that temperature and moisture were the most important factors in the

distribution of subterranean termites.

162

Table-10.1. Comparison of different studies showing termite’s survey

183

Reference Survey techniques

Lee and Wood (1971) From soil by hand sorting

Baroni-Urbani et al. (1978) From soil by hand sorting

Southwood (1978) Tullgren-Berlese funnels

Gentry and Whitford (1982) Wooden blocks

Johston et al. (1971) Plagues or “ground board

Howard and Haverty (1981) Bouts

Wood (1974) Litter bages

La fage et al. (1973) Rolls of toilet paper

Lafage et al. (1983) Fiber board

French et al. (1986) Beer mats and even cork

Esentther and Beal (1974, 1978) Ground stakes

Su et al. (1982b) Ground stakes

Bhanot et al. (1984) Stakes of Kiker (Acacia Arabica)

Sattar et al. (2013) Poplar wooden stakes

In our study, M. obesi were collected from 29 sites, while O. lokanandi were

trapped from 46 points in varying ranged. There seems to be three factors: i. termites

did not like the high moisture content of the soil; ii. the distance from the colony that

184

workers would travel; iii. the termite soldiers apparently do not distribute

homogeneously within their gallery system. Lower yield of termites was found in

traps, which were installed in wet or irrigated field or away from the colony. While,

higher yield of termites was recorded in traps, which were installed in dry field or

near to the colony. Similarly, the mean number of individuals in 1.0 gm sample

varied greatly. Su and La Fage (1984) reported that a considerable intra-specific

variation exist among termites colonies.

Number of individuals of the two species per sample shows a great variation.

A significantly greater number of M. obesi was observed as compared to O.

lokanandi. Minimum number of termites per sample of the former species was found

more than the maximum number of the latter. This variation was due to the different

size of the two species. Individuals of M. obesi are smaller in size than individuals

of O. lokanandi so more individuals were counted in 1.0 gm sample. The two termite

species were also found different greatly in yield per trap. The maximum yield of M.

obesi and O. lokanandi per trap were 1.12 ± 0.28, 0.82 ±

0.19 gm, respectively. This variation shows that termite population in the colony of

M. obesi is high than O. lokanandi so more termites (M. obesi) population come to

the foraging point. The number of individuals in a termite colony varies with species

(Badawi et al., 1984).

Table-10.2. Comparison of different studies showing foraging behaviour

Insect Reference Techniques for behaviour study

Subterranean termites Wood et al. (1977) Soil core method

185

//

Lafage et al. (1973) Baiting

//

Haverty et al. (1975) Baiting

//

Hosney and Said, (1980) Baiting

//

Badawi et al. (1984) Paper rolls

// Sattar et al. (2013) NIFA-TERMAPS

The cryptic nature of the subterranean termites make behavioural studies

more difficult. In our study foraging behaviour of subterranean termites was

observed by using NIFA-TRAPS. Many researchers have used different techniques

for behaviour study of subterranean termites (Table-10.2).

Our results showed that correlation was found positive and significant

between atmospheric temperature, precipitation and both termite species i.e., M.

186

obesi and O. lokanandi; however, the correlation was recorded negative and non

significantly different between relative humidity and both termites species. Evans

and Gleason (2001) concluded that foraging activities of termites have been

correlated with both air temperature and rainfall. Foraging activities of subterranean

termite were recorded peaked in summer months when the temperature and

precipitation were recorded high. In summer and fall, ground and atmospheric

temperature is favorable for termites foraging. No biomass of both species was

collected in winter months (December, January, February and March) when the

temperature was low, while the relative humidity was recorded high. When the

temperature increased, maximum numbers of termite were captured. Haverty et al.

(1999a) supported our study and they observed variation in the population of termites

in different seasons of the year. Foraging activities of subterranean termites are

affected by too hot or too cold temperature of the soil surface (Haverty et al., 1974;

La fage et al., 1976).

In our study, more workers were collected than soldiers in every observation.

This shows that the worker termites come to forage in large number as compared to

soldiers. In addition, soldiers in termite colonies are comapartively low. Moreover,

the temperature, relative humidity and rainfall affect the ratio of the workers and

soldiers. Studies revealed that the caste composition in social insects can be

influenced by environmental conditions such as temperature. (Henderson, 1998; Mao

et al., 2005; Scharf et al., 2007). Nutting (1970) recorded 4% soldiers and 96% non

soldiers in a foraging group of H. aureus. A colony of G. perplexus contain mainly

workers and only about 0.4% soldiers (Nutting et al., 1973).

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Due to concealed nesting structures of subterranean termites, demonstration

of their population suppression or colony elimination is difficult. Mark-

releaserecapture technique can be used for detection of the foraging territories of

subterranean termites. A wide variety of markers have been used to assess insect

population dynamics, dispersal, territoriality, feeding behavior, trophic-level

interactions, and other ecological interactions. The ideal marker should persist

without inhibiting the insect biology. Furthermore, the marker should be

environmentally safe, easy to apply, clearly identifiable, inexpensive, durable and

nontoxic (Hagler and Jackson, 2001). Dyes were first used in Hawaii to measure the

distance traveled by Coptotermes formosanus workers (Fujii, 1975).

Our study was focused to screen out dye markers for M. obesi and O.

lokanandi. The results showed that maximum mortality of M. obesi was recorded at

higher concentration after 15 days by using Nile blue-A, followed by medium and

lower concentrations, respectively, while maximum percent mortality of O.

lokanandi was recorded at high concentration after day 5 of the trial, followed by

mortality at medium and low concentrations, respectively. Our results showed that

Table-10.3. Comparison of different studies showing termites different dye

Termites species Reference Dyes used

R. flavipes Su et al. (1991b) Nile blue-A

H. indicola Salihah et al. (1994, 1995, Nile blue-A

1996 and 1997)

M. lepidus Salih and Logon (1990) 30 dyes

M. obesi Sattar this study Nile blue-A

O. lokanandi Sattar this study Nile blue-A

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C. formosanus Lai (1977) Sudan red-7B

C. formosanus Su et al. (1983, 1988) Sudan red-7B

C. formosanus Delaplane et al. (1988) Sudan red-7B

C. formosanus Delaplane and La Fage (1989) Sudan red-7B

R. flavipes Grace and Abdally (1989) Sudan red-7B

M. obesi Sattar this study Sudan red-7B

O. lokanandi Sattar this study Sudan red-7B

O. lokanandi was found more susceptible than M. obesi against Nile blue-A. Many

researchers used Nile blue-A for staining different species of termites (Table-10.3).

Similarly, Sudan red-7B caused maximum mortality in M. obesi after 9 days

at high concentration, followed by mortality at medium and lower concentrations,

respectively, however, Sudan red-7B caused maximum mortality in O. lokanandi at

high concentration after 5 days, followed by mortalities at medium and low

concentrations, respectively. The results show that M. obesi is also more resistance

to Sudan red-7B as compare to O. lokanandi. Many researchers observed that Sudan

red had been considered as a suitable biological dye (Table-10.3).

Our study was focused to screen out the the best relative humidity for staining

M. obesi. The results showed that the termites did not gain any colour at 100%

relative humidity after 4 days, however, they gained maximum stain in 7 th day as

189

well as in 10th day at the different concentrations of Nile blue-A. At 92 and 76%

relative humidities, termites did not gain any noticeable stain after 4, 7 and 10 days

at 0.25 and 0.125% concentrations, however at 0.5% concentration slight colour was

observed in termites bodies after 7 and 10 days. Our results tally with those of Su et

al. (1991), who reported that Nile blue-A was safe and persistent marker for R.

flavipes. Our results showed that maximum mortality was recorded after 4 days when

Nile blue-A was used against O. lokanadi. Maximum stain was observed at 0.25%

concentration, but this concentration was found toxic to O. lokanandi, because all the

prominently stained termites were found dead. Nile blueA and Neutral red, can

persist for different times in different species and these different species in turn have

different tolerances to these substances (Su et al., 1993; Evan, 1997). Our results

revealed that M. obesi did not get any colour under all the relative humidity (100, 92

and 76%) even after 4 days, when they treated against Sudan red-7B at 0.125, 0.25

and 0.5% concentrations. Almost the same situation was observed in O. lokanandi.

Sudan red-7B had been used for marking C. formosanus for over a decade (Begon,

1979). Sudan red-7B was found to reside the longest in and cause the least mortality

of the Formosan subterranean termite, Coptotermes formosanus Shiraki (Lai et al.,

1983), it has been successfully used for estimating the population size of C.

formosanus field colonies (Lai, 1977).

Retention time of Nile blue and Sudan red was recorded against M. obesi for

eight weeks. Nile blue-A (0.125%) caused lower mortality and was retained well for

eight weeks in more than 59% termites and it would be recommended for M. obesi

to be used for long biological studies. Sudan red-7B caused comparatively more

mortality and would be used in short term of studies. The decrease in number of dyed

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termite in both cases with the passage of time was due to the mortality of the termites,

but not due the trophallactic transfer of dye. Salihah et al. (1994, 1995, 1996 and

1997) reported that Sudan red-7B at 0.25% concentration was non toxic to H.

indicola and gave prominent pink colour to termite, but its retention period in field

was lesser (42 day) than that of Nile blue-A (1 year and 3 months). Nile blue retained

in R. flavipes and H. formosanus species throughout the 15 days period and did not

cause significant mortality (Su et al., 1991b), Sudan red-7B in termites decreases

immediately after the termites stopped feeding on the stained paper (Su et al.,1983).


prevent subterranean termite attack and to control their infestation. The regular use

of fast-acting termiticides for the management of termites has caused a number of

environment and biological hazards. In addition, to these dilemmas these treatments

are expensive and require specialized techniques. Increasing concern over

environmental contamination and threat to human health by the current termiticides,

particularly chlorinated hydrocarbon has slowly led to search for new materials and

innovative approaches to termites towards more sophisticated biological materials.

Therefore, the present research was the diversion of the attention towards the search

of alternative and environmental friendly methods for termite control.

In our present study different concentrations of seed and leaf extracts of E.

helioscopia, Cannabis sativa and Calotropis procera were tested against M. obesi

and O. lokanandi. The results indicated that aqueous extracts of E. helioscopia, C.

sativa and C. procera contain insecticidal activities. It was also observed that percent

mortality of the two species was concentration depended. Statistical analysis

revealed that O. lokanandi was more susceptible than M. obesi. The mortalities rate

191

in both species were found higher at higher concentration. Many researchers tested

plant extracts for termite control (Table-10.4).

The complex behavioral patterns of social insects such as termites in

conjunction with the cryptic nature of their foraging make them challenging to

manage with conventional insecticides (Sheets et al., 2000). In our research, we

investigated the mortality of workers and soldiers of two termite species i.e., M. obesi

and O. lokanandi against Mercuric Chloride (HgCl2) and Copper Sulphate Table-

10.4. Comparison of different plants extract used against termites

Termites species Reference Plants and plant extract

Coptotermes formosanus Hostettman (1989) Neem and Margosan-O

Coptotermes formosanus Grace and Yates (1992) Neem and Margosan-O

Reticulitermes spertus Park and Shin (2005) Allium sativum L (Garlic)

Insect pest McPartlandC (1997) C. sativa (Bhang)

Mosquito larvae Thomas et al. (2000) C. sativa (Bhang)

Heliothis armigera Parihar and Singh (1992) C. sativa (Bhang)

Nilaparvata lugens Hiremath and Ahn (1997) C. sativa (Bhang)

Heterotermes indicola Manzoor et al. (2011) Ocimum sanctum L

Odontotermes obesus Upadhyay et al. (2010) Capparis deciduas

Aedes aegypti Jalees et al. (1993) Cannabis sativa

Heterotermes indicola Ahmed et al. (2006) Datura alba Nees

Heterotermes indicola Badshah et al. (2004) C. procera (Ak)

Microtermes obesi Sattar this study E. helioscopia(Sun spurge)

Odontotermes lokanandi Sattar this study E.helioscopia(Sun spurge)

Microtermes obesi Sattar this study C. sativa (Bhang)

Odontotermes lokanandi Sattar this study C. sativa (Bhang)

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Microtermes obesi Sattar this study C. procera (Ak)

Odontotermes lokanandi Sattar this study C. procera (Ak)

(CuSo4). The results showed that Mercuric Chloride at high concentration caused

100% mortality in M. obesi, followed by the mortality recorded at medium and low

concentrations, respectively after 12 days; whereas highest mortality (86.90%)

recorded in M. obesi at high concentration of Copper Sulphate, followed by the

mortality observed at medium and low concentrations, respectively. Our results

revealed that Mercuric Chloride at high concentration caused 100% mortality in O.

lokanandi, followed by the mortality recorded at medium and low concentrations,

respectively after 5th day; while Copper Sulphate caused highest (100%) mortality at

high concentration, followed by the mortality recorded at medium and low

concentrations, respectively. During the trial, it was observed that soldiers died

earlier than the workers. This early death of soldiers may be explained on the basis

that they are depended upon workers for their food and as the workers were stressed

due to the effects of chemicals, they may not have fed the soldiers adequately and

the soldiers were thus first to die. Several novel slow-acting pesticides are being

screened for the control of subterranean termites (Hrdy et al., 2004; Rojas et al.,

2004; Kubota et al., 2006; Yeoh and Lee, 2006) because use of slow-acting

insecticides in baiting systems can be distributed within, and kill the entire colony.

Sattar (2000) reported that bait should be palatable and slow-acting toxic so

that the termite foragers carry back to the colony and distribute to their nest mates.

The development of such baits depends on combining phagostimulants with

193

slowacting toxicant. In our study the results showed that the toxicity and palatability

was dose depend and the food consumption by termites was always more in filter

paper treated with phagostimulants than control. The results indicated a decrease in

survival of the termites on urea and yeast as compared with control, whereas, glucose

when compared with control, it did not reduce the survival of M. obesi even at highest

concentration. Maximum survival (84%) was recorded on 4% glucose. It might have

been due the stimulant effect of glucose on termite feeding.

Survival rate decreased with increasing concentrations of sugars. Kanai et al. (1982),

reported that higher concentrations of sugar killed the gut protozoan of termites,

which can lead to reduced survival of termites.

Our study revealed that Microtermes obesi consumed more baits of glucose,

yeast and urea as compared with control. Highest bait consumption (27.19 ± 0.24)

was recorded at 4% glucose, followed by 21.05 ± 0.21 and 15.32±0.54% at 3% yeast

and 3% urea. It might have been due the stimulant effect of all phagostimulants on

termite feeding. Reinhard and Kaib (2001) reported that glucose was feeding

stimulants for R. santonensis. Waller and Curtis (2003) found that baits treated with

the highest concentration of glucose were significantly preferred by subterranean

termites in choice evaluations.

The results showed that the survival of termites on different level of poplar

saw dust extract was found significantly higher. Maximum survival of termites was

recorded at filter paper boiled for 25 minutes, followed by filter paper soaked in

poplar saw dust extract boiled for 20, 15, and 10 minutes, respectively. Lower

survival of termites was recorded at lowest concentration. Termites ate significantly

more boiled filter paper than the control (water) filter paper. The results indicated

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that percent bait consumption recorded more at higher concentrations. Studies

revealed that termites show a preference for certain species of wood (McMahan,

1966; Morales-Ramos and Guadalupe, 2001) and even show higher survivorship on

the preferred wood (McMahan, 1966; Morales-Ramos and

Guadalupe, 2003).

When Microtermes obesi was offered filter paper soaked in distilled water,

poplar saw dust extract, urea and yeast in 5 compartmental choice chambers. The

results revealed that the maximum attraction and percent bait consumption was found

at filter paper soaked in glucose, followed by yeast, poplar saw dust and urea,

whereas the minimum attraction and percent bait consumption was recorded in

control. Researchers reported that various carbohydrates have been suggested to act

as termite phagostimulants (Mishra, 1992; Perrott et al., 2005; Zhou et al., 2008),

galactose has been reported to significantly increase baits consumption by

Reticulitermes spp. (Swoboda, 2004).

Traditionally soil treatments have been conducted, where insecticides of high

repellency and long residual effect have been used for flooding the target area. This

method is costly, inefficient and environmentally unsafe. Use of attractive bait on the

other hand is a safer method of managing of termite populations. Sattar (2000)

reported that bait should be palatable and slow-acting toxicant so that the termite

foragers carry back to the colony and distribute to their nest mates. The development

of such baits depends on combining phagostimulants with slow-acting toxicant.

Our study showed that the food consumption and attraction of the termites

were always more in choice chambers having filter paper soaked in different

195

concentrations of Mercuric Chloride and Copper Sulphate coated with a mixture of

phagostimulants i.e., 4% glucose + 4% yeast + 1% urea + poplar saw dust extract.

Our result indicated that maximum mortalities were observed at highest

concentration after 16th day. The minimum mortality was found in control. This

shows that the level of mortality were depended on concentration. Highest (92.53 ±

0.35) percent mortalities were recorded at Mercuric Chloride, followed by mortality

(90.93 ± 0.13%) at Copper Sulphate after 16 days, which indicated that these

compounds have good slow-acting toxicant characteristics. This confirms the results

of (Brill et al., 1987; Yoshimura et al., 1987; Watson and Lenz, 1990; Sattar, 2000),

who concluded that the compounds of heavy metals were slow-acting toxicants.

Our study was focused to screen out toxicant baits for termite by using

extracts of Euphorbia helioscopia (Sun spurg), Calotropis procera (Ak) and

Cannabis sativa (Bhang) coated with a mixture of phagostimulant. The results

showed that the food consumption and attraction of termites were always more in

choice chambers having filter paper soaked in distilled water than filter paper soaked

in plant extracts. It revealed that the plant extracts had strong repellent activities.

Termites did not attract to the chamber where toxic bait were placed. Plant extracts

were found repellent for subterranean termites (Blaske and Hertel,

2001).

Effective chemical treatments have been used for many years to control infestation

of subterranean termites. The frequent use of fast-acting termiticides to contain

termites has caused a number of biological and environmental risks. Increased

concern over environmental contamination and threat to human health by the current

termiticides has led to search for new innovative approaches to termite control.

196

Interest in the use of slow-acting toxicants to suppress the populations of

subterranean termites has been renewed. The success of slow-acting toxicant bait

depends upon its attraction, palatability, delayed mortality and should be introduced

into the colony‟s gallery system and transferred to unexposed nest-mate by social

grooming or trophallaxis. In our study, we formulated five (05) slowacting toxic baits

viz., Mercuric Chloride, Copper Sulphate, Euphorbia helioscopia (Sun spurg),

Calotropis procera (Ak) and Cannabis sativa (Bhang) to contain subterranean

termites in urban environment. To minimize environmental contamination, termite

colony could be traced by using dye-markers. In our study, two dye-markers i.e., Nile

blue-A and Sudan red-7B were tested againt two termite species i.e., M. obesi and

O. lokanandi. During our experiments, proper concentrations of both dye-markers

were found. Our results showed that these concentrations were not toxic against both

species of termites, and would be persist for a longer period of time in termites

bodies. The success of slow-acting toxic bait depends on the activity of subterranean

termites. In our study, it was found that Subterranean termites were more active in

summer months i.e., June, July, August and September. Maximum termites were

attracted to traps in summer months.

Slow-acting toxic baits can produce good results in summer months.

197

SUMMARY

Ecological studies on subterranean termites were conducted in Islamabad

during 2010 to 2012. In order to monitor and determine infested areas, 1200 stakes

of poplar wood were driven into the soil at different localities and were checked at

fortnightly intervals. Out of 1200 poplar wooden stakes only 65 were found infested

by Odontotermes lokanandi and Microtermes obesi. Later the infested stakes were

replaced by “NIFA-TERMAPs”.

During the trial, it was observed that some of the traps always harbour the

same single species; and some times a single trap had a mixed population of two

species. When such traps were opened there observed an antagonistic behaviour that

termites attacked each other till death of the weaker and fewer numbers. Incidence

of O. lokanandi was found dominant in Islamabad, as the frequency of capturing O.

lokanandi was much higher than that of M. obesi.

Mean number of M. obesi per trap was found higher than O. lokanandi. These

variations were due to difference in body size of individuals of both species.

Individuals of M. obesi are smaller in size than that of O. lokanandi so more

individuals were counted in 1 gm sample. Both termite species were also found

different in case of yield per trap and number per 1.0 gm sample. This variation

shows that the termite population in the colony of M. obesi is high than O. lokanandi

so more termites come to the foraging point. Results revealed that the foragers of

both species captured throughout the trial were predominantly workers.

178

198

Positive and significant correlation was found among atmospheric

temperature, precipitation and population of both subterranean termite species i.e.,

M. obesi and O. lokanandi; however, the correlation was found non significant and

negative between relative humidity and foraging activities of both termite species.

Workers and soldiers of Microtermes obesi and O. lokanandi were forcefed

on different concentrations of dye-markers viz., Nile blue-A and Sudan red-7B.

Results showed that Nile blue-A at high concentration caused 100% mortality in M.

obesi after 15 days, followed by mortality observed at medium and low

concentrations, respectively. When M. obesi was treated with Sudan red-7B, 100%

mean mortality was found on 9th day at high concentration, followed by medium and

low concentrations, respectively. 100% mortality in O. lokanandi at high

concentration of Nile blue-A was recorded after 5th day of the trial, followed by

mortality at medium and low concentrations, respectively. Similarly, when O.

lokanandi was force-fed on different concentrations of Sudan red-7B; 100% mean

mortality was recorded at high concentration, after 5 days, followed by mortality at

medium and low concentrations, respectively.

Experiments were conducted to screen out the best relative humidity for

staining of termites. The relative humidities used were 100% using H2O, 92% using

Na2Co3 and 76% using NaCl. Microtermes obesi and O. lokanandi were force-fed on

bait containing Nile blue-A and Sudan red-7B. Three concentrations viz, 0.5%,

0.25% and 0.125% of each dye were tested against both species. The results showed

that M. obesi gained Nile blue colour in 100% Relative Humidity after 10 days at all

concentrations; while at 92 and 76% relative humidities, termites gained slight colour

only at 0.5% concentration after 7 and 10 days. The results indicated that maximum

199

blue O. lokanadi were observed at 0.25% concentration after 4 dys, but this

concentration was found toxic to O. lokanadi. The results revealed that M. obesi did

not get any colour under all levels of the relative humidity (100, 92 and 76%) after 4

days, when they were treated against Sudan red-7B at 0.125, 0.25 and 0.5%

concentrations. Almost the same situation was observed in O. lokanandi.

Retention time of Nile blue and Sudan red was recorded against M. obesi for

eight weeks. Nile blue-A (0.125%) caused lower mortality and was retained well for

eight months in more than 59% termites and it is recommended for use against M.

obesi and thus may be used for periodically long studies. Sudan red-7B caused

comparatively more mortality and can be used in short term studies. The decrease in

number of dyed termite in both cases with the passage of time was due to mortality

of the termites but not due the trophallactic transfer of dye.

Insecticidal activity of leaf and seed crude extracts of Euphorbia helioscopia,

Cannabis sativa and Calotropis procera were evaluated against the workers and

soldiers of Microtermes obesi and O. lokanandi. Different

concentrations i.e., 50, 33 and 25% of the aqueous extracts were tested against both

species. The mortality of M. obesi and O. lokanandi were recorded against leaf and

seed aqueous extract of E. helioscopia, C. sativa and C. procera for 11 (eleven) and

07 (seven) days, respectively. Results revealed that after eleven days of force feeding

of M. obesi, all extracts showed moderate toxic effect, however, 100% mortality was

observed on 11th day; while 100% mortality of O. lokanandi was noted on 7th day of

the trial.

200

Two synthetic chemicals Mercuric Chloride and Copper Sulphate were also

tested against termites. Copper Sulphate at high concentration (0.1% level) caused

86.90% mean mortality in M. obesi on 12th day; whereas 100% mean mortality was

achieved on 12th day at high concentration (0.1% level) by using Mercuric Chloride.

100% mean mortality in O. lokanandi was achieved on 5th day at high concentration

of Copper Sulphate and Mercuric Chloride.

Different compounds were tested to find out better phagostimulant,

acceptable and palatable to Microtermes obesi. The results revealed that all tested

compounds attracted termites. These termites survived for a long period on tested

compounds as compared to control. Maximum percent survival was recorded on 4%

glucose, followed by 1% yeast and 4% urea; while 74.67 ± 0.67% survival was

recorded in control. The highest bait consumption recorded was 4% glucose,

followed by 3% yeast, 3% urea and control. The results showed that maximum

survival of termites was recorded at filter paper soaked in saw dust extract boiled for

25 minutes; while low survival was found at filter paper soaked in saw dust extract

boiled for 0 minute. Similarly, maximum percent bait consumption was found at

filter paper soaked in poplar saw dust extract, which was boiled for 25 minutes;

whereas lower consumption was observed in case of 0 minute bioling.

The present study was focused to formulate palatable and slow-acting

toxicant baits for an effective control of termites. Five compounds including

Mercuric Chloride, Copper Sulphate, extracts of Calotropis procera, Euphorbia

helioscopia and Cannabis sativa each at three different concentrations were tested

against Microtermes obesi to screen potential slow-acting toxicants. The results

showed that after 16 days, maximum percent mortality (92.53%) was observed incase

201

of bait of Mercuric Chloride, followed by bait of Copper Sulphate. Baits of

Euphorbia helioscopia, Calotropis procera and Cannabis sativa showed moderate

toxicity (less than 60%) for Microtermes obesi. The result revealed that highest

percent bait consumption was observed in case of bait of Mercuric Chloride,

followed by baits of Copper Sulphate, E. helioscopia, C. procera and C. sativa,

respectively.

RECOMENDATIONS

1. Subterranean termites were observed more active in summer months i.e.,

June, July, August and September. Maximum termites were attracted to traps in

summer months. Slow-acting toxic baits can produce good results in summer

months.

202

2. Nile blue-A caused lower mortality in termites and was retained well for

maximum period of time. It is recommended for use against Microtermes obesi and

O. lokanandi for long-term. Sudan red-7B caused comparatively more mortality in

termites and can be used in short-term of studies.

3. Phagostimulants i.e., glucose, urea, yeast and saw dust extracts were

evaluated. The results revealed that all tested compounds attracted termites.

Termites consumed significantly high baits by feeding them on aforementioned

compounds as compared to control. These compounds are recommended to

formulate slow-acting toxic baits.

4. Baits of Mercuric Chloride and Copper Sulphate were found palatable and

slow-acting, thus these baits are recommended for control of subterranean termites

in urban and rural environment.

183

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APPENDICES

Appendix 1:- ANOVA of mean weight of Microtermes obesi

Source DF SS MS F P

Treat. 28 3.499 0.125 0.844 . 0.698 ns

Rep. 23 741.095 32.222 217.671 0.000 ***

Error 644 95.330 0.148

Total 695 839.924

Coefficient of Variation = 40.298%

Appendix 2:- ANOVA of mean weight of Odontotermes lokanandi

Source DF SS MS F P

Treat. 45 15.995 0.355 4.186 0.000 ***

Rep. 23 427.719 18.597 218.989 0.000 ***

Error 1035 87.892 0.085

Total 1103 531.607 Coefficient

of Variation = 46.370%

Appendix 3:- ANOVA of mean number of M. obesi in one gram sample

Source DF SS MS F P

Treat. 28 8136.172 290.578 0.848 0.686 ns

Rep. 5 3394.667 678.933 1.982 0.085 ns

Error 140 47969.000 342.636



Appendix 4:- ANOVA of mean number of O. lokanandi in one gram sample

Source DF SS MS F P

Treat. 45 3184.467 70.766 1.170 0.229 ns

272

Rep. 5 1044.946 208.989 3.457 0.005 **

Error 225 13603.554 60.460



Appendix 5:- ANOVA of % workers of M. obesi

Source DF SS MS F P

Treat. 28 254.881 9.103 0.760 0.803 ns

Rep. 7 202.284 28.898 2.414 .022 *

Error 196 2346.096 11.970



Appendix 6:- ANOVA of % workers of O. lokanandi

Source DF SS MS F P

Treat. 45 698.738 15.528 1.380 0.062 ns

Rep. 7 332.578 47.511 4.221 0 .000 ***

Error 315 3545.259 11.255

Total 367 4576.575


273

)

Appendix 7:- Correlation of Temperature and termites (M. obesi and O. lokanandi

M .obesi O. lokanandi

Number = 24

Covariance = 92344.12 Correlation =

0.694

Intercept = -16587.37

Slope = 1456.798

Standard Error = 322.264

Student's T value = 4.521

Probability = 0.000

Number = 24


0.645

Intercept = -9434.64

Slope = 1061.548



Probability = 0.001

Appendix 8:- Correlation Humadity and termites (M. obesi and O. lokanandi)

M. obesi O. lokanandi

Number = 24

Covariance = 14652.68 Correlation

= 0.070

Intercept = 8820.56

Slope = 92.700



Probability = 0.746

Number = 24


0.010

Intercept = 11287.06

Slope = 9.958



Probability = 0.962

Appendix 9:- Correlation of Precipitation and termites (M. obesi and O. lokanandi)

M. obesi O. lokanandi

Number = 24


= 0.608

Intercept = 5987.87

Slope = 133.171



Probability = 0.002

Number = 24


= 0.557

Intercept = 5968.38

Slope = 89.506



Probability = 0.004

Appendix 10:- ANOVA Day1 Mortality(%) in M. obesi by using Nile blue (H2O)

Source DF SS MS F P

Treat. 2 0.056 0.028 0.25 0.790 ns

Rep. 2 0.722 0.361 3.25 0.145 ns

Error 4 0.444 0.111

274

- Mortality (%) in M. obesi by using lue (H2O)

Total 8 1.222


Appendix 11: ANOVA Day2 Nile b

Source DF SS MS F P

Treat. 2 0.722 0.361 1 0.444 ns

Rep. 2 0.056 0.028 0.077 0.927 ns

Error 4 1.444 0.361

Total 8 2.222


Appendix 12:- ANOVA Day3 Mortality (%) in M. obesi by using Nile blue (H2O)

Source DF SS MS F P

Treat. 2 0.902 0.451 0.915 0.471 ns

Rep. 2 3.616 1.808 3.669 0.125 ns

Error 4 1.971 0.492

Total 8 6.490

Coefficient of Variation =12.189%


Source DF SS MS F P

Treat. 2 0.738 0.369 1.296 0.368 ns

Rep. 2 0.054 0.027 0.095 0.911 ns

Error 4 0.139 0.284

Total 8 1.931


Appendix 14:-ANOVA Day5 Mortality (%) in M. obesi by using Nile blue (H2O)

Source DF SS MS F P

Treat. 2 1.540 0.770 1.055 0.428 ns

Rep. 2 2.778 1.388 1.904 0.262 ns

Error 4 2.917 0.729

Total 8 7.235

275

- M. obesi by using lue (H2O)


Appendix 15:-ANOVA Day6 Mortality(%) in M. obesi by using Nile blue (H2O)

Source DF SS MS F P

Treat. 2 50.357 25.178 46.253 0.002 **

Rep. 2 1.277 0.638 1.173 0.397 ns

Error 4 2.177 0.544

Total 8 53.811



Source DF SS MS F P

Treat. 2 144.677 72.338 209.151 0.000 ***

Rep. 2 0.424 0.212 0.613 0.586 ns

Error 4 1.383 0.346

Total 8 146.485


Appendix 17: ANOVA Day8 Mortality (%) in Nile b

Source DF SS MS F P

Treat. 2 171.564 85.782 115.204 0.000 ***

Rep. 2 1.204 0.602 0.808 0.507 ns

Error 4 2.978 0.745

Total 8 175.746



Source DF SS MS F P

Treat. 2 241.440 120.720 106.642 0.000 ***

Rep. 2 0.458 0.229 0.202 0.825 ns

Error 4 4.528 1.132

Total 8 246.426


276


Appendix19:-ANOVA Day10 Mortality (%) in M. obesi by using Nile blue (H2O)

Source DF SS MS F P

Treat. 2 494.655 247.328 1024.838 0.000 ***

Rep. 2 0.820 0.410 1.699 0.292 ns

Error 4 0.965 0.241

Total 8 496.441


Appendix 20:-ANOVA Day11Mortality (%) in M. obesi by using Nile blue (H2O)

Source DF SS MS F P

Treat. 2 753.167 376.583 541.617 0.000 ***

Rep. 2 0.872 0.436 0.627 0.579 ns

Error 4 2.782 0.695

Total 8 756.819



Source DF SS MS F P

Treat. 2 2410.487 1205.244 2103.841 0.000 ***

Rep. 2 0.181 0.090 0.157 0.859 ns

Error 4 2.292 0.573

Total 8 2412.959


Appendix 22:- ANOVA Day13Mortality (%) in M. obesi by using Nile blue (H2O)

Source DF SS MS F P

Treat. 2 2558.602 1279.301 1396.465 0.000 ***

Rep. 2 1.106 0.553 0.604 0.590 ns

Error 4 3.664 0.916

Total 8 2563.373


Appendix 23: ANOVA Day14 Nile b

Source DF SS MS F P

Treat. 2 2783.323 1391.661 1774.550 0.000 ***

Rep. 2 0.358 0.178 0.228 0.806 ns

Error 4 3.137 0.784

Total 8 2786.817


277

- M. obesi by using lue (H2O)


Source DF SS MS F P

Treat. 2 2921.457 1460.728 7851.502 0.000 ***

Rep. 2 0.882 0.441 2.371 0.209 ns

Error 4 0.744 0.186

Total 8 2923.083


Appendix25:-ANOVA Day1 Mortality (%) in O. lokanandi by using Nile blue(H2O)

Source DF SS MS F P

Treat. 2 0.224 0.112 0.169 0.849 ns

Rep. 2 1.155 0.578 0.874 0.4841 ns

Error 4 2.642 0.661

Total 8 4.022


Appendix 26:- ANOVA Day2 Mortality (%) in O. lokanandi by using Nile blue (H2O)

Source DF SS MS F P

Treat. 2 3.788 1.894 26.840 0.005 **

Rep. 2 1.0413 0.521 7.378 0.046 *

Error 4 0.282 0.071

Total 8 5.112



Source DF SS MS F P

Treat. 2 298.678 149.339 541.880 0.000 ***

Rep. 2 1.143 0.571 2.073 0.241 ns

Error 4 1.102 0.276

Total 8 300.924



Source DF SS MS F P

Treat. 2 1945.302 972.651 1311.980 0.000 ***

Rep. 2 5.764 2.882 3.887 0.115 ns

Error 4 2.965 0.741

278


Total 8 1954.032


279

Appendix 29:-ANOVA Day5 Mortality (%) in O. lokanandi by using Nile blue (H2O)

Source DF SS MS F P

Treat. 2 2393.525 1196.763 826.589 0.000 ***

Rep. 2 0.009 0.005 0.003 0.997 ns

Error 4 5.791 1.448

Total 8 2399.326


Appendix 30:- ANOVA Day1 Mortality (%) in M. obesi by using Sudan red (H2O)

Source DF SS MS F P

Treat. 2 0.163 0.082 0.121 0.889 ns

Rep. 2 0.167 0.083 0.123 0.887 ns

Error 4 2.700 0.675

Total 8 3.030


Appendix 31:-ANOVA Day2 Mortality (%) in M. obesi by using Sudan red (H2O)

Source DF SS MS F P

Treat. 2 0.170 0.085 0.125 0.886 ns

Rep. 2 0.173 0.087 0.128 0.884 ns

Error 4 2.717 0.679

Total 8 3.060



Source DF SS MS F P

Treat. 2 0.403 0.202 0.324 0.741 ns

Rep. 2 1.587 0.793 1.273 0.374 ns

Error 4 2.494 0.623

Total 8 4.484


Appendix 33:-ANOVA Day4 Mortality (%) in M. obesi by using Sudan red (H2O)

Source DF SS MS F P

Treat. 2 9.087 4.544 9.641 0.029 *

Rep. 2 2.176 1.088 2.309 0.216 ns

280

Mortality (%) in

Error 4 1.885 0.471

Total 8 13.148


Appendix 34:- ANOVA Day5Mortality (%) in M. obesi by using Sudan red (H2O)

Source DF SS MS F P

Treat. 2 23.390 11.695 16.395 0.012 *

Rep. 2 1.094 0.547 0.767 0.522 ns

Error 4 2.853 0.713

Total 8 27.337


Appendix 35:-ANOVA Day6 M. obesi by using Sudan red (H2O)

Source DF SS MS F P

Treat. 2 90.391 45.195 178.662 0.000 ***

Rep. 2 1.263 0.632 2.497 0.198 ns

Error 4 1.012 0.253

Total 8 92.666


Appendix36:- ANOVA Day7 Mortality (%)in M. obesi by using Sudan red (H2O)

Source DF SS MS F P

Treat. 2 186.657 93.329 366.075 0.000 ***

Rep. 2 7.296 3.648 14.310 0.015 *

Error 4 1.020 0.255

Total 8 194.974


Appendix37:- ANOVA Day8 Mortality (%) in M. obesi by using Sudan red (H2O)

Source DF SS MS F P

Treat. 2 241.715 120.858 145.599 0.000 ***

Rep. 2 0.267 0.133 0.161 0.857 ns

Error 4 3.320 0.830

Total 8 245.302


281


Source DF SS MS F P

Treat. 2 261.426 130.713 134.170 0.000 ***

Rep. 2 1.708 0.854 0.877 0.483 ns

Error 4 3.897 0.974

Total 8 267.032


Appendix 39:-ANOVA Day1 Mortality (%) O. lokanandi by using Sudan red (H2O)

Source DF SS MS F P

Treat. 2 0.058 0.029 0.4 0.694 ns

Rep. 2 0.034 0.017 0.234 0.802 ns

Error 4 0.289 0.072

Total 8 0.381


Appendix 40:- ANOVA Day2 Mortality (%) in O. lokanandi by using Sudan red (H2O)

Source DF SS MS F P

Treat. 2 8.889 4.444 1.257 0.999 ns

Rep. 2 0.643 0.322 0.909 0.473 ns

Error 4 1.415 0.354

Total 8 2.058


Appendix 41:- ANOVA Day3 Mortalty (%) O. lokanandi by using Sudan red (H2O)

Source DF SS MS F P

Treat. 2 32.007 16.003 41.529 0.002 **

Rep. 2 1.438 0.719 1.866 0.268 ns

Error 4 1.541 0.385

Total 8 34.986



Source DF SS MS F P

Treat. 2 519.687 259.844 260.695 0.000 ***

Rep. 2 3.737 1.869 1.875 0.266 ns

Error 4 3.987 0.997

282

Mortality (%) in

Total 8 527.412



Source DF SS MS F P

Treat. 2 5350.225 2675.113 637.783 0.000 ***

Rep. 2 2.665 1.333 0.318 0.745 ns

Error 4 16.778 4.194

Total 8 5369.668


Appendix 44:-ANOVA % number of dyed M. obesi after day 5 by using Sudan red

Source DF SS MS F P

Treat. 2 1.095 0.548 0.639 0.5873 ns

Data 3 3.428 1.143 1.333 0.4094 ns

Error 3 2.571 0.857

Total 8 6


Appendix45: ANOVA % number of dyed M. obesi after day 10 by using Sudan red

Source DF SS MS F P

Treat. 2 2.667 1.333 2.667 0.2727 ns

Data 4 5 1.25 2.5 0.3056 ns

Error 2 1 0.5

Total 8 6


Appendix46:-ANOVA % number of dyed M. obesi after day 15 by using Sudan red

Source DF SS MS F P

Treat. 2 0.333 0.167 0.333 0.7500 ns

Data 4 5 1.25 2.5 0.3056 ns

Error 2 1 0.5

Total 8 6


283

ANOVA M. obesi

Appendix47:- % number of dyed after day 20 by using Sudan red

Source DF SS MS F P

Treat. 2 0.667 0.333 0.222 0.8128 ns

Data 3 1.5 0.5 0.333 0.8045 ns

Error 3 4.5 1.5

Total 8 6



Source DF SS MS F P

Treat. 2 0.667 0.333 0.25 0.7936 ns

Data 3 2 0.667 0.5 0.7082 ns

Error 3 4 1.333

Total 8 6



Source DF SS MS F P

Treat. 2 0.5 0.25 0.125 0.888 ns

Data 4 2 0.5 0.25 0.888 ns

Error 2 4 2

Total 8 6



Source DF SS MS F P

Treat. 2 1.167 0.583 0.467 0.682 ns

Data 4 3.5 0.875 0.7 0.659 ns

Error 2 2.5 1.25

Total 8 6


284

ANOVA % number of dyed M. obesi


Source DF SS MS F P

Treat. 2 0.5 0.25 0.188 0.838 ns

Data 3 2 0.667 0.5 0.708 ns

Error 3 4 1.333

Total 8 6



Source DF SS MS F P

Treat. 2 0.5 0.25 0.25 0.800 ns

Data 4 4 1 1 0.556 ns

Error 2 2 1

Total 8 6

Coefficient of Variation = 50%

Appendix53:- after day 50 by using Sudan red

Source DF SS MS F P

Trea. 2 1.25 0.625 0.469 0.665 ns

Data 3 2 0.667 0.5 0.708 ns

Error 3 4 1.333

Total 8 6



Source DF SS MS F P

Treat. 2 0.917 0.458 0.611 0.587 ns

Data 2 3 1.5 2 0.250 ns

Error 4 3 0.75

Total 8 6



Source DF SS MS F P

Treat. 2 0.375 0.187 0.208 0.819 ns

285


Data 1 1.5 1.5 1.667 0.253 ns

Error 5 4.5 0.9

Total 8 6


Appendix56:-ANOVA % number of dyed M. obesi after day 5 by using Nile blue

Source DF SS MS F P

Treat. 2 0.25 0.125 0.119 0.890 ns

Data 1 0.75 0.75 0.714 0.437 ns

Error 5 5.25 1.05

Total 8 6



Source DF SS MS F P

Treat. 2 0.464 0.232 0.162 0.857 ns

Data 3 1.714 0.571 0.4 0.764 ns

Error 3 4.285 1.428

Total 8 6



Source DF SS MS F P

Treat. 2 0.262 0.131 0.367 0.720 ns

Data 3 4.929 1.643 4.6 0.121 ns

Error 3 1.071 0.357

Total 8 6


Appendix59:- after day 20 by using Nile blue

Source DF SS MS F P

Treat. 2 0.524 0.262 0.167 0.857 ns

Data 4 2.857 0.714 0.454 0.773 ns

Error 2 3.143 1.571

Total 8 6

286



Appendix 60:-ANOVA % number of dyed M. obesi after day 25 by using Nile blue

Source DF SS MS F P

Treat. 2 0.833 0.417 0.417 0.692 ns

Data 3 3 1 1 0.500 ns

Error 3 3 1

Total 8 6

Coefficient of Variation =50%


Source DF SS MS F P

Treat. 2 2 1 2 0.447 ns

Data 5 5.5 1.1 2.2 0.469 ns

Error 1 0.5 0.5

Total 8 6



Source DF SS MS F P

Treat. 2 0.25 0.125 0.25 0.800 ns

Data 4 5 1.25 2.5 0.306 ns

Error 2 1 0.5

Total 8 6



Source DF SS MS F P

Treat. 2 0.667 0.333 0.167 0.867 ns

Data 5 4 0.8 0.4 0.825 ns

Error 1 2 2

Total 8 6


287



Source DF SS MS F P

Treat. 2 2.167 1.083 2.167 0.433 ns

Data 5 5.5 1.1 2.2 0.469 ns

Error 1 0.5 0.5

Total 8 6


288


Appendix 65:- after day 50 by using Nile blue

Source DF SS MS F P

Treat. 2 0.5 0.25 0.25 0.794 ns

Data 3 3 1 1 0.500 ns

Error 3 3 1

Total 8 6


Appendix 66:-ANOVA % number of dyed M.obesi after day 55 by using Nile blue

Source DF SS MS F P

Treat. 2 0.25 0.125 0.25 0.817 ns

Data 5 5.5 1.1 2.2 0.469 ns

Error 1 0.5 0.5

Total 8 6



Source DF SS MS F P

Treat. 2 1.2 0.6 0.6 0.625 ns

Data 4 4 1 1 0.556 ns

Error 2 2 1

Total 8 6


Appendix 68:-ANOVA Day1Mortality (%) in M.obesi by using Leaf extraxt of E.

helioscopia

Source DF SS MS F P

Treat. 2 0.925 0.462 0.25 0.790 ns

Rep. 2 0.925 0.462 0.25 0.790 ns

Error 4 7.398 1.850

Total 8 9.248


Appendix69:-ANOVA Day2 Mortality (%) in M. obesi by using Leaf extract of E.

helioscopia

289

Source DF SS MS F P

Treat. 2 41.522 20.761 6.1697 0.059 ns

Rep. 2 0.961 0.481 0.143 0.871 ns

Error 4 13.460 3.365

Total 8 55.944


Appendix70:-ANOVA Day3 Mortality (%) in M. obesi by using leaf extract of E.

helioscopia

Source DF SS MS F P

Treat. 2 78.193 39.096 23.799 0.006 **

Rep. 2 23.833 11.916 7.254 0.047 *

Error 4 6.571 1.643

Total 8 108.597


Appendix 71:- ANOVA Day4 Mortality (%) in M. obesi by using leaf extract of E.

helioscopia

Source DF SS MS F P

Treat. 2 55.958 27.979 55.022 0.001 **

Rep. 2 32.691 16.345 32.144 0.003 **

Error 4 2.034 0.509

Total 8 90.683


Appendix 72:- ANOVA Day5Mortality (%) in M. obesi by using leaf extract of E.

helioscopia

Source DF SS MS F P

Treat. 2 64.894 32.447 9.079 0.0326 *

Rep. 2 35.121 17.561 4.914 0.0837 ns

Error 4 14.296 3.574

Total 8 114.311



helioscopia

Source DF SS MS F P

Treat. 2 91.428 45.714 6.946 0.050 *

Rep. 2 3.084 1.542 0.234 0.801 ns

Error 4 26.326 6.581

290

Total 8 120.837


Appendix 74:- ANOVA Day7 Mortality (%) M. obesi by using leaf extreact of E.

helioscopia

Source DF SS MS F P

Treat. 2 65.136 32.568 6.426 0.056 ns

Rep. 2 6.466 3.233 0.638 0.575 ns

Error 4 20.274 5.068

Total 8 91.876



helioscopia

Source DF SS MS F P

Treat 2 97.530 48.765 16.095 0.012 *

Rep. 2 21.362 10.681 3.525 0.131 ns

Error 4 12.119 3.030

Total 8 131.011



helioscopia

Source DF SS MS F P

Treat. 2 152.290 76.145 9.941 0.028 *

Rep. 2 40.054 20.027 2.615 0.188 ns

Error 4 30.639 7.660

Total 8 222.983


Appendix 77:- ANOVA Day10 Mortality (%) in M. obesi by using leaf extract of

E. helioscopia

Source DF SS MS F P

Treat. 2 375.487 187.743 15.694 0.013 *

Rep. 2 58.342 29.170 2.439 0.203 ns

Error 4 47.849 11.962

Total 8 481.678


291


E. helioscopia

Source DF SS MS F P

Treat. 2 308.170 154.085 18.568 0.009 **

Rep. 2 54.890 27.445 3.307 0.142 ns

Error 4 33.193 8.298

Total 8 396.254


Appendix 79:- ANOVA Day1 Mortality (%) in M. obesi by using seed extract of E.

helioscopia

Source DF SS MS F P

Treat. 2 0.943 0.472 0.400 0.694 ns

Rep. 2 6.440 3.219 2.731 0.179 ns

Error 4 4.715 1.179

Total 8 12.098



helioscopia

Source DF SS MS F P

Treat. 2 12.693 6.347 2.397 0.206 ns

Rep. 2 6.311 3.156 1.192 0 .393 ns

Error 4 10.590 2.648

Total 8 29.595



helioscopia

Source DF SS MS F P

Treat. 2 42.535 21.268 12.687 0.019 *

Rep. 2 37.575 18.787 11.207 0.023 *

Error 4 6.705 1.676

Total 8 86.815


292


helioscopia

Source DF SS MS F P

Treat. 2 87.173 43.586 51.740 0.001 **

Rep. 2 39.490 19.745 23.439 0.006 **

Error 4 3.370 0.842

Total 8 130.033



helioscopia

Source DF SS MS F P

Treat. 2 162.347 81.173 53.032 0.001 **

Rep. 2 11.172 5.586 3.650 0.125 ns

Error 4 6.123 1.531

Total 8 179.642


Appendix 84:- ANOVA Day6 Mortality (%) in M. obesi by using seed extract of

E. helioscopia

Source DF SS MS F P

Treat. 2 639.351 319.675 140.436 0.000 ***

Rep. 2 19.198 9.598 4.217 0.103 ns

Error 4 9.105 2.276

Total 8 667.653



E. helioscopia

Source DF SS MS F P

Treat. 2 391.614 195.807 281.712 0.000 ***

Rep. 2 29.101 14.550 20.934 0.008 **

Error 4 2.780 0.695

Total 8 423.495



helioscopia

293

Source DF SS MS F P

Treat. 2 165.294 82.647 14.376 0.015 *

Rep. 2 25.959 12.980 2.258 0.221 ns

Error 4 22.996 5.749

Total 8 214.250



E. helioscopia

Source DF SS MS F P

Treat. 2 130.110 65.055 12.894 0.018 *

Rep. 2 20.480 10.240 2.029 0.246 ns

Error 4 20.182 5.045

Total 8 170.772



E. helioscopia

Source DF SS MS F P

Treat. 2 119.234 59.617 42.759 0.002 **

Rep. 2 64.870 32.435 23.264 0.006 **

Error 4 5.577 1.394

Total 8 189.681


Appendix 89:- ANOVA Day11 Mortality (%) in M. obesi by using Seed extract of

E. helioscopia

Source DF SS MS F P

Treat. 2 67.900 33.950 3.635 0.126 ns

Rep. 2 18.682 9.341 1 0.444 ns

Error 4 37.363 9.341

Total 8 123.945


Appendix 90:-ANOVA Day1 Mortality (%) in O lokanandi by using leaf extract of

E. helioscopia

Source DF SS MS F P

294

Treat. 2 11.745 5.873 3.707 0.123 ns

Rep. 2 0.527 0.264 0.166 0.852 ns

Error 4 6.337 1.584

Total 8 18.610


Appendix 91:ANOVADay2 Mortality (%) in O. lokanandi by using leaf extract of

E. helioscopia

Source DF SS MS F P

Treat. 2 35.766 17.883 34.797 0.003 **

Rep. 2 3.300 1.650 3.211 0.147 ns

Error 4 2.056 0.514

Total 8 41.122


Appendix 92:ANOVA Day3 Mortality (%) in O. lokanandi by using leaf extract of

E. helioscopia

Source DF SS MS F P

Treat. 2 84.756 42.378 7.105 0.048 *

Rep. 2 2.397 1.199 0.201 0.826 ns

Error 4 23.859 5.965

Total 8 111.013



E. helioscopia

Source DF SS MS F P

Treat. 2 125.358 62.679 18.033 0.010 **

Rep. 2 4.916 2.458 0.707 0.546 ns

Error 4 13.903 3.476

Total 8 144.177



E. helioscopia

Source DF SS MS F P

Treat. 2 297.579 148.789 10.515 0.026 *

295

Rep. 2 3.426 1.713 0.121 0.889 ns

Error 4 56.599 14.149

Total 8 357.605



E. helioscopia

Source DF SS MS F P

Treat. 2 539.665 269.832 17.916 0.010 *

Rep. 2 51.470 25.735 1.709 0.291 ns

Error 4 60.246 15.061

Total 8 651.381



E. helioscopia

Source DF SS MS F P

Treat. 2 702.885 351.443 11.001 0.024 *

Rep. 2 107.876 53.938 1.688 0.294 ns

Error 4 127.784 31.946

Total 8 938.545


Appendix 97:- ANOVA Day1 Mortality (%) in O. lokanadi by using seed extract of

E. helioscopia

Source DF SS MS F P

Treat. 2 58.615 29.308 8.956 0.033 *

Rep. 2 67.035 33.517 10.242 0.027 *

Error 4 13.090 3.273

Total 8 138.741



E. helioscopia

Source DF SS MS F P

Treat. 2 408.879 204.439 59.049 0.001 **

Rep. 2 67.235 33.618 9.709 0.029 *

296

Error 4 13.849 3.462

Total 8 489.963



E. helioscopia

Source DF SS MS F P

Treat. 2 447.118 223.559 36.106 0.003 **

Rep. 2 56.427 28.213 4.557 0.093 ns

Error 4 24.767 6.192

Total 8 528.311


Appendix 100:-ANOVA Day4 Mortality (%) in O. lokanadi by using seed extract

of E. helioscopia

Source DF SS MS F P

Treat. 2 876.361 438.180 98.221 0.000 ***

Rep. 2 108.254 54.127 12.133 0.020 *

Error 4 17.845 4.461

Total 8 1002.459


Appendix 101-ANOVA Day5 Mortality (%) in O. lokanadi by using seed extract of

E. helioscopia

Source DF SS MS F P

Treat. 2 587.355 293.678 29.198 0.004 **

Rep. 2 210.304 105.152 10.455 0.026 *

Error 4 40.232 10.058

Total 8 837.891


Appendix 102-ANOVA Day6 Mortality (%) in O. lokanadi by using seed extract of E.

helioscopia

Source DF SS MS F P

Treat. 2 1167.742 583.871 15.180 0.014 *

Rep. 2 249.192 124.596 3.239 0.146 ns

Error 4 153.849 38.462

Total 8 1570.783


297

Appendix 103:-ANOVA Day1 Mortality (%) in M. obesi by using leaf extract of C. sativa

Source DF SS MS F P

Treat. 2 3.672 1.836 1.569 0.314 ns

Rep. 2 10.799 5.399 4.614 0.091 ns

Error 4 4.681 1.170

Total 8 19.152


Appendix104:-ANOVA Day2 Mortality (%) in M. obesi by using leaf extract of C. sativa

Source DF SS MS F P

Treat. 2 12.862 6.431 5.168 0.078 ns

Rep. 2 22.674 11.337 9.109 0.032 *

Error 4 4.978 1.245

Total 8 40.514



Source DF SS MS F P

Treat. 2 19.608 9.804 4.603 0.092 ns

Rep. 2 20.48 10.241 4.808 0.086 ns

Error 4 8.520 2.130

Total 8 48.612



Source DF SS MS F P

Treat. 2 54.542 27.271 8.269 0.038 *

Rep. 2 36.908 18.454 5.596 0.069 ns

Error 4 13.192 3.298

Total 8 104.643


Appendix107:-ANOVA Day5 Mortality (%) in M. obesi by using leaf extract of C. sativa

Source DF SS MS F P

Treat. 2 43.656 21.828 9.376 0.031 *

Rep. 2 72.682 36.341 15.610 0.013 *

Error 4 9.312 2.328

298

Total 8 125.650


Appendix108:- ANOVA Day6 Mortality (%) in M. obesi by using leaf extract of C. sativa

Source DF SS MS F P

Treat. 2 118.291 59.146 8.558 0.036 *

Rep. 2 20.613 10.306 1.491 0.328 ns

Error 4 27.644 6.911

Total 8 166.548


Appendix 109:- ANOVA Day7 Mortality (%) in M. obesi by using leaf extract of C.

sativa

Source DF SS MS F P

Treat. 2 111.693 55.847 10.913 0.024 *

Rep. 2 31.792 15.896 3.106 0.153 ns

Error 4 20.471 5.118

Total 8 163.956


Appendix 110:- ANOVA Day8 Mortality (%) in M. obesi by using leaf extract of C. sativa

Source DF SS MS F P

Treat. 2 114.632 57.315 19.286 0.009 **

Rep. 2 38.495 19.247 6.477 0.056 ns

Error 4 11.888 2.972

Total 8 165.015


Appendix 111:- ANOVA Day9 Mortality (%) in M. obesi by using leaf extract of C. sativa

Source DF SS MS F P

Treat. 2 144.863 72.432 36.814 0.003 **

Rep. 2 17.608 8.804 4.475 0.095 ns

Error 4 7.870 1.968

Total 8 170.342


299

Appendix112-ANOVA Day10 Mortality (%) in M. obesi by using leaf extract of C. sativa

Source DF SS MS F P

Treat. 2 149.122 74.561 14.079 0.016 *

Rep. 2 4.068 2.034 0.384 0.704 ns

Error 4 21.183 5.296

Total 8 174.374

Coefficient of Variation

Appendix 113:-ANOVA Day11Mortality (%) in M. obesi by using leaf extract of C. sativa

Source DF SS MS F P

Treat. 2 37.913 18.956 7.556 0.044 *

Rep. 2 1.705 0.853 0.340 0.731 ns

Error 4 10.036 2.508

Total 8 49.654


Appendix114:-ANOVA Day1 Mortality (%) in M. obesi by using seed extract of C. sativa

Source DF SS MS F P

Treat. 2 0.942 0.471 0.188 0.836 ns

Rep. 2 10.458 5.229 2.082 0.240 ns

Error 4 10.047 2.512

Total 8 21.448


Appendix115:- ANOVA Day2 Mortality (%) in M. obesi by using seed extract of C. sativa

Source DF SS MS F P

Treat. 2 11.344 5.672 1.999 0.250 ns

Rep. 2 9.546 4.773 1.683 0.295 ns

Error 4 11.346 2.837

Total 8 32.236


Appendix 116:-ANOVA Day3 Mortality (%) in M. obesi by using seed extract of C. sativa

Source DF SS MS F P

Treat. 2 18.360 9.180 4.769 0.087 ns

Rep. 2 24.666 12.333 6.408 0.057 ns

Error 4 7.698 1.925

300

Total 8 50.725


Appendix117:-ANOVA Day4 Mortality (%) in M. obesi by using seed extract of C. sativa

Source DF SS MS F P

Treat. 2 16.558 8.279 0.965 0.455 ns

Rep. 2 81.873 40.937 4.771 0.087 ns

Error 4 34.321 8.580

Total 8 132.753


Appendix118- ANOVA Day5 Mortality (%) in M. obesi by using of seed extract of

C. sativa

Source DF SS MS F P

Treat. 2 122.537 61.268 3.816 0.118 ns

Rep. 2 17.967 8.983 0.560 0.611 ns

Error 4 64.219 16.055

Total 8 204.723


Appendix 119:- ANOVA Day6 Mortality (%) in M. obesi by using seed extract of C.sativa

Source DF SS MS F P

Treat. 2 128.900 64.450 11.661 0.021 *

Rep. 2 81.275 40.638 7.353 0.046 *

Error 4 22.108 5.527

Total 8 232.284


Appendix 120:ANOVA Day7 Mortality (%) in M.obesi by using seed extract of C. sativa

Source DF SS MS F P

Treat. 2 125.183 62.592 17.965 0.010 *

Rep. 2 18.773 9.387 2.694 0.182 ns

Error 4 13.937 3.484

Total 8 157.893


Appendix121:- ANOVA Day8 Mortality (%) in M. obesi by using seed extract of C. sativa

Source DF SS MS F P

301

Treat 2 131.178 65.589 19.114 0.009 **

Rep 2 30.162 15.081 4.395 0.098 ns

Error 4 13.726 3.432

Total 8 175.066


Appendix122:-ANOVA Day9 Mortality (%) in M. obesi by using of seed extract of

C. sativa

Source DF SS MS F P

Treat. 2 267.297 133.648 9.160 0.032 *

Rep. 2 16.554 8.277 0.567 0.607 ns

Error 4 58.364 14.591

Total 8 342.215


Appendix123:- ANOVA Day10 Mortality (%) in M. obesi by using of seed extract

of C. sativa

Source DF SS MS F P

Treat. 2 135.192 67.596 24.676 0.006 **

Rep. 2 25.553 12.776 4.664 0.090 ns

Error 4 10.957 2.739

Total 8 171.702


Appendix124:- ANOVA Day11Mortality (%) in M. obesi by using seed extract of C.sativa

Source DF SS MS F P

Treat. 2 119.153 59.576 9.094 0.033 *

Rep. 2 45.962 22.981 3.508 0.132 ns

Error 4 26.206 6.551

Total 8 191.321


Appendix125:- ANOVA Day1 Mortality (%) in O. lokanandi by using leaf extract

of C. sativa

Source DF SS MS F P

Treat. 2 62.526 31.263 20.698 0.008 **

Rep. 2 19.845 9.922 6.569 0.055 ns

Error 4 6.042 1.51

Total 8 88.413

302


Appendix 126:- ANOVA Day2 Mortality (%) in O. lokanandi by using leaf extract

of C. sativa

Source DF SS MS F P

Treat. 2 76.084 38.042 13.652 0.016 *

Rep. 2 7.266 3.633 1.304 0.367 ns

Error 4 11.146 2.786

Total 8 94.496



of C. sativa

Source DF SS MS F P

Treat. 2 214.882 107.441 16.134 0.012 *

Rep. 2 52.215 26.108 3.921 0.114 ns

Error 4 26.636 6.659

Total 8 293.735



of C. sativa

Source DF SS MS F P

Treat. 2 340.564 170.282 40.805 0.002 **

Rep. 2 72.275 36.138 8.660 0.035 *

Error 4 16.692 4.173

Total 8 429.532


Appendix 129:ANOVA Day5 Mortality (%) in O. lokanandi by using leaf extract

of C. sativa

Source DF SS MS F P

Treat. 2 518.890 259.445 21.969 0.007 **

Rep. 2 118.167 59.083 5.003 0.082 ns

Error 4 47.239 11.810

Total 8 684.296


303

:- Mortality (%) in

Appendix 130 ANOVA Day6 O. lokanandi by using leaf extract of C. sativa

Source DF SS MS F P

Treat. 2 1764.118 882.059 35.279 0.003 **

Rep. 2 86.510 43.255 1.730 0.288 ns

Error 4 100.009 25.002

Total 8 1950.637



of C. sativa

Source DF SS MS F P

Treat. 2 532.383 266.192 32.355 0.003 **

Rep. 2 60.735 30.367 3.691 0.124 ns

Error 4 32.909 8.227

Total 8 626.027


Apeendix 132:ANOVA Day1 Mortality (%) in O. lokanand by using seed extract of

C. sativa

Source DF SS MS F P

Treat. 2 51.175 25.588 11.789 0.021 *

Rep. 2 13.457 6.728 3.099 0.154 ns

Error 4 8.682 2.170

Total 8 73.314


Apeendix 133:- ANOVA Day2 Mortality (%) in O. lokanand by using seed extract

of C. sativa

Source DF SS MS F P

Treat. 2 58.800 29.400 10.001 0.028 *

Rep. 2 5.342 2.671 0.909 0.473 ns

Error 4 11.758 2.939

Total 8 75.900



of C. sativa

Source DF SS MS F P

304

:- Mortality (%) in

Treat. 2 265.263 132.631 156.871 0.000 ***

Rep. 2 143.835 71.918 85.061 0.001 ***

Error 4 3.382 0.845

Total 8 412.480


Apeendix 135 ANOVA Day4 O. lokanand by using seed extract of C. sativa

Source DF SS MS F P

Treat. 2 245.047 122.523 66.921 0.001 ***

Rep. 2 167.049 83.524 45.620 0.002 **

Error 4 7.324 1.831

Total 8 419.419



of C. sativa

Source DF SS MS F P

Treat. 2 579.271 289.636 16.588 0.012 *

Rep. 2 48.635 24.318 1.393 0.348 ns

Error 4 69.841 17.460

Total 8 697.748


Apeendix 137:-ANOVA Day6 Mortality (%) in O. lokanand by using seed extract

of C. sativa

Source DF SS MS F P

Treat. 2 419.942 209.971 37.554 0.003 **

Rep. 2 87.244 43.622 7.802 0.042 *

Error 4 22.365 5.591

Total 8 529.551



of C. sativa

Source DF SS MS F P

305

:- Mortality (%) in

Treat. 2 613.152 306.576 17.505 0.011 *

Rep. 2 29.641 14.821 0.846 0.494 ns

Error 4 70.056 17.514

Total 8 712.849


Appendix139:-ANOVA Day1 Mortality (%) in M. obesi by using leaf extract of C.

procera

Source DF SS MS F P

Treat 2 2.829 1.415 1.999 0.250 ns

Rep 2 2.562 1.281 1.811 0.275 ns

Error 4 2.830 0.707

Total 8 8.221


Appendix 140 ANOVA Day2 M. obesi by using leaf extract of

C. procera

Source DF SS MS F P

Treat. 2 2.954 1.477 1.999 0.250 ns

Rep. 2 2.382 1.191 1.612 0.307 ns

Error 4 2.955 0.739

Total 8 8.291



C. procera

Source DF SS MS F P

Treat. 2 39.008 19.504 5.346 0.074 ns

Rep. 2 17.608 8.804 2.413 0.205 ns

Error 4 14.593 3.648

Total 8 71.209



306

:- Mortality (%) in

C. procera

Source DF SS MS F P

Treat. 2 15.252 7.626 1.870 0.267 ns

Rep. 2 31.888 15.944 3.909 0.115 ns

Error 4 16.316 4.079

Total 8 63.456



C. procera

Source DF SS MS F P

Treat. 2 114.929 57.464 9.990 0.028 *

Rep. 2 51.597 25.798 4.485 0.095 ns

Error 4 23.008 5.752

Total 8 189.533



C. procera

Source DF SS MS F P

Treat. 2 178.906 89.453 21.615 0.007 **

Rep. 2 3.198 1.599 0.386 0.702 ns

Error 4 16.554 4.1385

Total 8 198.659


307

:- Mortality (%) in M. obesi

Appendix 145 ANOVA Day7 by using leaf extract of

C. procera

Source DF SS MS F P

Treat. 2 225.728 112.864 114.369 0.000 ***

Rep. 2 48.075 24.038 24.358 0.006 **

Error 4 3.947 0.987

Total 8 277.751



C. procera

Source DF SS MS F P

Treat. 2 119.886 59.943 24.271 0.006 **

Rep. 2 8.134 4.0669 1.647 0.301 ns

Error 4 9.879 2.470

Total 8 137.899



C. procera

Source DF SS MS F P

Treat. 2 93.039 46.519 8.985 0.033 *

Rep. 2 12.120 6.060 1.170 0.398 ns

Error 4 20.709 5.177

Total 8 125.867


Appendix 148:-ANOVA Day10 Mortality (%) in M. obesi by using leaf extract of

C. procera

Source DF SS MS F P

Treat. 2 98.960 49.480 14.244 0.015 *

Rep. 2 12.747 6.373 1.835 0.272 ns

Error 4 13.895 3.474

Total 8 125.601

308

:- M. obesi


Appendix 149:-ANOVA Day11 Mortality (%) in M. obesi by using leaf extract of

C. procera

Source DF SS MS F P

Treat. 2 35.28 17.64 16.774 0.011 *

Rep. 2 2.103 1.052 1 0.444 ns

Error 4 4.207 1.052

Total 8 41.590


Appendix 150 ANOVA Day1 by using seed extract of

C. procera

Source DF SS MS F P

Treat. 2 0.925 0.462 0.4 0.694 ns

Rep. 2 14.365 7.182 6.213 0.059 ns

Error 4 4.624 1.156

Total 8 19.914



C. procera

Source DF SS MS F P

Treat. 2 7.157 3.579 2.839 0.171 ns

Rep. 2 14.743 7.371 5.848 0.065 ns

Error 4 5.042 1.261

Total 8 26.942



C. procera

Source DF SS MS F P

Treat. 2 4.342 2.171 1.609 0.307 ns

Rep. 2 10.268 5.134 3.805 0.119 ns

Error 4 5.398 1.349

Total 8 20.008

309

:- Mortality (%) in M. obesi



C. procera

Source DF SS MS F P

Treat. 2 73.128 36.564 8.588 0.036 *

Rep. 2 34.535 17.268 4.056 0.109 ns

Error 4 17.029 4.257

Total 8 124.692



C. procera

Source DF SS MS F P

Treat. 2 184.138 92.069 172.777 0.000 ***

Rep. 2 10.058 5.029 9.437 0.031 *

Error 4 2.132 0.533

Total 8 196.327


Appendix 155 ANOVA Day6 Mortality (%) in by using seed extract of

C. procera

Source DF SS MS F P

Treat. 2 74.541 37.271 7.848 0.041 *

Rep. 2 34.985 17.493 3.684 0.124 ns

Error 4 18.996 4.749

Total 8 128.522



C. procera

Source DF SS MS F P

Treat. 2 92.209 46.104 8.988 0.033 *

Rep. 2 6.329 3.164 0.617 0.584 ns

Error 4 20.518 5.129

Total 8 119.055

310

:- M. obesi



C. procera

Source DF SS MS F P

Treat. 2 177.436 88.718 9.942 0.028 *

Rep. 2 1.288 0.644 0.072 0.932 ns

Error 4 35.695 8.924

Total 8 214.420



C. procera

Source DF SS MS F P

Treat. 2 164.594 82.297 16.670 0.012 *

Rep. 2 5.506 2.753 0.558 0.611 ns

Error 4 19.747 4.937

Total 8 189.847



C. procera

Source DF SS MS F P

Treat. 2 53.552 26.776 7.252 0.047 *

Rep. 2 7.802 3.901 1.057 0.428 ns

Error 4 14.769 3.692

Total 8 76.123


311

:-

Appendix 160 ANOVA Day1Mortality (%) in O. lokanandi by using leaf extract of

C. procera

Source DF SS MS F P

Treat. 2 40.958 20.479 41.432 0.002 **

Rep. 2 24.877 12.438 25.164 0.005 **

Error 4 1.977 0.494

Total 8 67.812


Appendix 161:ANOVA Day2 Mortality (%) in O. lokanandi by using leaf extract

of C. procera

Source DF SS MS F P

Treat. 2 28.610 14.304 2.843 0.171 ns

Rep. 2 4.688 2.344 0.466 0.658 ns

Error 4 20.130 5.032

Total 8 53.427



C. procera

Source DF SS MS F P

Treat. 2 43.396 21.698 2.646 0.185 ns

Rep. 2 1.328 0.664 0.081 0.924 ns

Error 4 32.800 8.200

Total 8 77.525



C. procera

Source DF SS MS F P

Treat. 2 139.879 69.939 33.981 0.003 **

Rep. 2 28.183 14.091 6.847 0.051 ns

Error 4 8.233 2.058

Total 8 176.294


312


C. procera

Source DF SS MS F P

Treat. 2 169.330 84.665 16.139 0.012 *

Rep. 2 32.521 16.261 3.099 0.154 ns

Error 4 20.984 5.246

Total 8 222.836



C. procera

Source DF SS MS F P

Treat. 2 262.110 131.055 28.751 0.004 **

Rep. 2 28.571 14.285 3.134 0.152 ns

Error 4 18.233 4.558

Total 8 308.914



C. procera

Source DF SS MS F P

Treat. 2 237.133 118.567 21.409 0.007 **

Rep. 2 22.775 11.388 2.056 0.243 ns

Error 4 22.153 5.538

Total 8 282.061


Appendix 167:ANOVA Day1 Mortality (%) in O. lokanandi by using Seed extract

of C. procera

Source DF SS MS F P

Treat. 2 48.089 24.045 13 0.018 *

Rep. 2 0.924 0.462 0.25 0.790 ns

Error 4 7.398 1.850

Total 8 56.413



of C. procera

313

Source DF SS MS F P

Treat. 2 246.226 123.113 21.791 0.007 **

Rep. 2 9.481 4.741 0.840 0.496 ns

Error 4 22.599 5.650

Total 8 278.306



of C. procera

Source DF SS MS F P

Treat. 2 356.590 178.295 36.241 0.003 **

Rep. 2 6.137 3.068 0.624 0.581 ns

Error 4 19.679 4.920

Total 8 382.405



of C. procera

Source DF SS MS F P

Treat. 2 204.511 102.256 19.093 0.009 **

Rep. 2 46.982 23.491 4.386 0.098 ns

Error 4 21.423 5.356

Total 8 272.915



of C. procera

Source DF SS MS F P

Treat. 2 414.778 207.388 115.459 0.000 ***

Rep. 2 95.445 47.723 26.569 0.005 **

Error 4 7.185 1.796

Total 8 517.408


314


of C. procera

Source DF SS MS F P

Treat. 2 811.810 405.905 21.765 0.007 **

Rep. 2 164.398 82.199 4.408 0.097 ns

Error 4 74.599 18.650

Total 8 1050.808


Appendix 173: ANOVA Day7 Mortality (%) in O. lokanandi by using Seed extract

of C. procera

Source DF SS MS F P

Treat. 2 156.291 78.146 59.326 0.001 **

Rep. 2 2.634 1.317 1 0.444 ns

Error 4 5.269 1.317

Total 8 164.195


Appendix 174:- ANOVA Day1 Mortality (%) in M. obesi by using CuSo4

Source DF SS MS F P

Treat. 2 2.843 1.421 0.408 0.689 ns Rep.

2 7.736 3.868 1.110 0.414 ns

Error 4 13.941 3.485

Total 8 24.520


315

:- obesi by using

Appendix 175 ANOVA Day2 Mortality (%) in M. CuSo4

Source DF SS MS F P

Treat. 2 0.961 0.481 0.248 0.792 ns

Rep. 2 0.961 0.481 0.248 0.792 ns

Error 4 7.761 1.940

Total 8 9.684



Source DF SS MS F P

Treat. 2 4.062 2.031 1.002 0.444 ns

Rep. 2 10.597 5.299 2.614 0.188 ns

Error 4 8.109 2.027

Total 8 22.768



Source DF SS MS F P

Treat. 2 7.716 3.858 1.733 0.287 ns

Rep. 2 27.129 13.564 6.092 0.061 ns

Error 4 8.906 2.226

Total 8 43.751



Source DF SS MS F P

Treat. 2 273.229 136.614 237.323 0.000 ***

Rep. 2 9.350 4.675 8.121 0.039 *

Error 4 2.303 0.576

Total 8 284.882



Source DF SS MS F P

Treat. 2 525.520 262.760 69.582 0.001 ***

Rep. 2 3.776 1.888 0.5 0.640 ns

Error 4 15.105 3.776

Total 8 544.401

316

:- Mortality (%) in obesi by using



Source DF SS MS F P

Treat. 2 1421.160 710.580 298.346 0.000 ***

Rep. 2 10.035 5.018 2.107 0.237 ns

Error 4 9.527 2.382

Total 8 1440.722


Appendix 181 ANOVA Day8 M. CuSo4

Source DF SS MS F P

Treat. 2 1322.971 661.485 88.660 0.001 ***

Rep. 2 1.115 0.558 0.075 0.929 ns

Error 4 29.844 7.461

Total 8 1353.930



Source DF SS MS F P

Treat. 2 1448.642 724.321 64.730 0.001 ***

Rep. 2 0.973 0.486 0.043 0.958 ns

Error 4 44.759 11.190

Total 8 1494.374



Source DF SS MS F P

Treat. 2 718.998 359.499 53.796 0.001 **

Rep. 2 15.309 7.654 1.145 0.404 ns

Error 4 26.730 6.683

Total 8 761.037



Source DF SS MS F P

Treat. 2 1037.077 518.539 48.184 0.002 **

Rep. 2 1.389 0.694 0.065 0.939 ns

317


Error 4 43.046 10.762

Total 8 1081.512


Appendix 185:- ANOVA Day12 Mortality (%) in M. obesi bu using CuSo4

Source DF SS MS F P

Treat. 2 1005.711 502.855 169.500 0.000 ***

Rep. 2 7.605 3.803 1.282 0.371 ns

Error 4 11.867 2.967

Total 8 1025.183


Appendix 186:- ANOVA Day1 Mortality (%) in M. obesi by using HgCl2

Source DF SS MS F P

Treat. 2 0.903 0.451 0.239 0.798 ns

Rep. 2 11.489 5.744 3.040 0.158 ns

Error 4 7.558 1.889

Total 8 19.950


Appendix 187 ANOVA Day2 M. HgCl2

Source DF SS MS F P

Treat. 2 12.113 6.056 3.266 0.144 ns

Rep. 2 0.548 0.274 0.148 0.867 ns

Error 4 7.417 1.854

Total 8 20.078



Source DF SS MS F P

Treat. 2 1.032 0.516 0.122 0.889 ns

Rep. 2 3.197 1.598 0.377 0.708 ns

Error 4 16.975 4.244

Total 8 21.204



318


Source DF SS MS F P

Treat. 2 13.088 6.544 1.850 0.269 ns

Rep. 2 3.363 1.682 0.475 0.653 ns

Error 4 14.149 3.537

Total 8 30.600



Source DF SS MS F P

Treat. 2 288.1852 144.092 37.731 0.003 **

Rep. 2 0.043 0.022 0.006 0.994 ns

Error 4 15.276 3.819

Total 8 303.503



Source DF SS MS F P

Treat. 2 743.117 371.559 54.807 0.001 **

Rep. 2 7.106 3.553 0.524 0.628 ns

Error 4 27.117 6.779

Total 8 777.341


Appendix 192- ANOVA Day7 Mortality (%) in M. obesi by using HgCl2

Source DF SS MS F P

Treat. 2 659.645 329.822 83.688 0.001 ***

Rep. 2 26.638 13.319 3.379 0.138 ns

Error 4 15.764 3.941

Total 8 702.047


Appendix 193 ANOVA Day8 M. HgCl2

Source DF SS MS F P

Treat. 2 1074.787 537.393 146.089 0.000 ***

Rep. 2 4.755 2.377 0.646 0.571 ns

Error 4 14.714 3.679

Total 8 1094.256


319



Source DF SS MS F P

Treat. 2 1100.041 550.020 1157.423 0.000 ***

Rep. 2 10.760 5.380 11.321 0.023 *

Error 4 1.901 0.475

Total 8 1112.701



Source DF SS MS F P

Treat. 2 1173.885 586.943 279.202 0.000 ***

Rep. 2 6.066 3.033 1.44 0.338 ns

Error 4 8.409 2.102

Total 8 1188.360



Source DF SS MS F P

Treat. 2 1428.773 714.386 379.412 0.000 ***

Rep. 2 13.374 6.687 3.552 0.129 ns

Error 4 7.531 1.883

Total 8 1449.679



Source DF SS MS F P

Treat. 2 1992.733 996.367 474.344 0.000 ***

Rep. 2 12.512 6.256 2.978 0.161 ns

Error 4 8.402 2.101

Total 8 2013.647


Appendix 198:- ANOVA Day1 Mortality (%) in O. lokanandi by using CuSo4

Source DF SS MS F P

Treat. 2 4.109 2.054 1.582 0.312 ns

Rep. 2 2.896 1.448 1.115 0.412 ns

320


Error 4 5.194 1.299

Total 8 12.199


321


Source DF SS MS F P

Treat. 2 24.757 12.378 0.862 0.489 ns

Rep. 2 12.357 6.179 0.430 0.677 ns

Error 4 57.471 14.368

Total 8 94.585



Source DF SS MS F P

Treat. 2 258.222 129.111 169.889 0.000 ***

Rep. 2 28.049 14.025 18.454 0.009 **

Error 4 3.040 0.760

Total 8 289.311



Source DF SS MS F P

Treat. 2 708.293 354.147 72.675 0.001 ***

Rep. 2 2.311 1.156 0.237 0.799 ns

Error 4 19.492 4.873

Total 8 730.0964



Source DF SS MS F P

Treat. 2 1806.290 903.145 163.793 0.000 ***

Rep. 2 18.591 9.295 1.686 0.294 ns

Error 4 22.056 5.514

Total 8 1846.936


Appendix 203:- ANOVA Day1Mortality (%) in O. lokanandi by using HgCl2

Source DF SS MS F P

Treat. 2 1.008 0.504 0.4 0.694 ns

Rep. 2 15.015 7.508 5.957 0.063 ns

Error 4 5.041 1.260

Total 8 21.064


322

:- ANOVA

Appendix 204:- ANOVA Day2 Mortality (%) in O. lokanandi by using HgCl2

Source DF SS MS F P

Treat. 2 3.286 1.643 0.974 0.452 ns

Rep. 2 7.834 3.917 2.321 0.214 ns

Error 4 6.750 1.688

Total 8 17.871



Source DF SS MS F P

Treat. 2 318.500 159.250 216.232 0.000 ***

Rep. 2 43.283 21.642 29.385 0.004 **

Error 4 2.946 0.736

Total 8 364.730



Source DF SS MS F P

Treat. 2 2259.030 1129.515 233.306 0.000 ***

Rep. 2 1.364 0.682 0.141 0.873 ns

Error 4 19.365 4.841

Total 8 2279.759



Source DF SS MS F P

Treat. 2 3259.840 1629.920 117.302 0.000 ***

Rep. 2 4.881 2.440 0.176 0.845 ns

Error 4 55.581 13.895

Total 8 3320.302


Appendix 208:- ANOVA Survival (%) of M. obesi at Phagostimulant 0.1%

Source DF SS MS F P

Treat. 3 1881.000 627.000 209.000 0.000 ***

Rep. 2 0.667 0.333 0.111 0.897 ns

Error 6 18.000 3.000

323

Total 11 1899.667


Appendix 209:- ANOVA Survival (%) of M. obesi at Phagostimulant 1%

Source DF SS MS F P

Treat. 3 2570.333 856.778 225.468 0.000 ***

Rep. 3 10.333 3.444 0.906 0.500 ns

Error 5 19.000 3.800

Total 11 2599.666


Appendix 210:- ANOVA Survival (%) of M. obesi at Phagostimulant 2%

Source DF SS MS F P

Treat. 3 1317.000 439.000 199.545 0.000 ***

Rep. 3 10.333 3.444 1.566 0.308 ns

Error 5 11.000 2.200

Total 11 1338.333


Appendix 211 Survival (%) M. obesi at Phagostimulant 3%

Source DF SS MS F P

Treat. 3 1180.000 393.333 64.130 0.000 ***

Rep. 3 17.333 5.778 0.942 0.486 ns

Error 5 30.667 6.133

Total 11 1228.000


Appendix 212:- ANOVA Survival (%) M. obesi at Phagostimulant 4%

Source DF SS MS F P

Treat. 3 2499.000 833.000 543.261 0.000 ***

Rep. 3 24.333 8.111 5.289 0.052 ns

Error 5 7.667 1.533

Total 11 2531.000



Source DF SS MS F P

324

:- ANOVA

Treat. 3 7339.333 2446.444 470.470 0.000 ***

Rep. 3 6.000 2.000 0.385 0.769 ns

Error 5 26.000 5.2

Total 11 7371.333



Source DF SS MS F P

Treat. 3 13892.000 4630.667 1929.444 0.000 ***

Rep. 3 1.333 0.444 0.185 0.902 ns

Error 5 12.000 2.4

Total 11 13905 .333



Source DF SS MS F P

Treat. 3 17182.333 5727.444 12886.75 0.000 ***

Rep. 2 2.667 1.333 3.00 0.125 ns

Error 6 2.667 0.444

Total 11 17187.667


Appendix 216:-ANOVA % bait consumption by M. obesi at Phagostimulant 0.1%

Source DF SS MS F P

Treat. 3 70.410 23.470 27.774 0.001 ***

Rep. 2 2.314 1.157 1.369 0.324 ns

Error 6 5.070 0.845

Total 11 77.795


325

:- ANOVA

Appendix 217 % bait consumption by M. obesi at Phagostimulant 1%

Source DF SS MS F P

Treat. 3 111.787 37.262 30.011 0.001 ***

Rep. 2 1.179 0.590 0.475 0.644 ns

Error 6 7.449 1.242

Total 11 120.416


Appendix 218:- ANOVA % bait consumption by M. obesi at Phagostimulant 2%

Source DF SS MS F P

Treat. 3 228.067 76.022 134.968 0.000 ***

Rep. 2 0.158 0.079 0.140 0.872 ns

Error 6 3.380 0.563

Total 11 231.605



Source DF SS MS F P

Treat. 3 635.311 211.771 183.784 0.000 ***

Rep. 2 3.950 1.975 1.714 0.258 ns

Error 6 6.914 1.152

Total 11 646.175



Source DF SS MS F P

Treat. 3 926.772 308.924 923.824 0.000 ***

Rep. 2 0.480 0.240 0.718 0.525 ns

Error 6 2.006 0.334

Total 11 929.259


326

:- ANOVA


Source DF SS MS F P

Treat. 3 369.198 123.066 234.919 0.000 ***

Rep. 2 1.349 0.674 1.287 0.343 ns

Error 6 3.143 0.524

Total 11 373.689



Source DF SS MS F P

Treat. 3 590.413 196.804 228.700 0.000 ***

Rep. 2 1.265 0.632 0.735 0.518 ns

Error 6 5.163 0.861

Total 11 596.841


Appendix 223 %bait consumption by M. obesi at Phagostimulant 7%

Source DF SS MS F P

Treat. 3 358.7883 119.596 73.067 0.000 ***

Rep. 2 0.628 0.314 0.192 0.830 ns

Error 6 9.821 1.637

Total 11 369.237


Appendix 224:-ANOVA % consumption of bait (combination of phagostimulants)

by M. obesi

Source DF SS MS F P

Treat. 10 869.706 86.970 182.118 0.000 ***

Rep. 2 1.221 0.610 1.279 0.300 ns

Error 20 9.551 0.478

Total 32 880.479


Appendix 225:- ANOVA % bait consumption (Sawdust extract) by M. obesi

Source DF SS MS F P

Treat. 6 785.483 130.914 184.336 0.000 ***

Rep. 2 1.103 0.552 0.777 0.482 ns

Error 12 8.522 0.710

Total 20 795.109


327

Appendix226:-ANOVA Mortality (%) in M. obesi in bait containing CuSo4 in Day 4

Source DF SS MS F P

Treat. 3 426.387 142.129 450.408 0.000 ***

Rep. 2 0.24 0.12 0.380 0.699 ns

Error 6 1.893 0.316

Total 11 428.52


Appendix 227:- ANOVA Mortality (%) in M. obesi in bait containing CuSo4 in Day 8

Source DF SS MS F P

Treat. 3 1454.333 484.778 1983.182 0.000 ***

Rep. 2 1.307 0.653 2.673 0.148 ns

Error 6 1.467 0.244

Total 11 1457.107


Appendix 228:- ANOVA Mortality (%) in M. obesi in bait containing CuSo4 in Day 12

Source DF SS MS F P

Treat. 3 5414.546 1804.849 4562.820 0.000 ***

Rep. 2 0.187 0.093 0.236 0.797 ns

Error 6 2.373 0.396

Total 11 5417.107


Appendix 229:- ANOVA Mortality (%) in M. obesi in bait containing CuSo4 i Day 16

Source DF SS MS F P

Treat. 3 8378.28 2792.76 12321.000 0.000 ***

Rep. 2 0.987 0.493 2.176 0.195 ns

Error 6 1.36 0.227

Total 11 8380.627


Appendix 230:- ANOVA Mortality (%) in M. obesi in bait containing HgCl2 in Day 4

Source DF SS MS F P

Treat. 3 1348.84 449.613 1348.84 0.000 ***

Rep. 2 0.027 0.013 0.04 0.961 ns

Error 6 2 0.333

Total 11 1350.867


328

:- ANOVA


Source DF SS MS F P

Treat. 3 4497.32 1499.107 5621.65 0.000 ***

Rep. 2 2.987 1.493 5.6 0.042 *

Error 6 1.6 0.267

Total 11 4501.907



Source DF SS MS F P

Treat. 3 9200.053 3066.684 12545.527 0.000 ***

Rep. 2 0.027 0.013 0.055 0.947 ns

Error 6 1.467 0.244

Total 11 9201.547



Source DF SS MS F P

Treat. 3 11331.347 3777.116 11037.026 0.000 ***

Rep. 2 0.08 0.04 0.117 0.892 ns

Error 6 2.053 0.342

Total 11 11333.48


Appendix234:-ANOVA Mortality (%) in M. obesi in bait of E. helioscopia in Day4

Source DF SS MS F P

Treat. 3 14.547 4.849 24.795 0.001 ***

Rep. 2 0.107 0.053 0.273 0.770 ns

Error 6 1.173 0.196

Total 11 15.827


329

Appendix 235:-ANOVA Mortality (%) in M. obesi in bait of E. helioscopia in Day8

Source DF SS MS F P

Treat. 3 87.933 29.311 1648.75 0.000 ***

Rep. 2 2.027 1.013 57.00 0.000 ***

Error 6 0.107 0.018

Total 11 90.067



Source DF SS MS F P

Treat. 3 681.747 227.248 751.926 0.000 ***

Rep. 2 0.32 0.16 0.529 0.614 ns

Error 6 1.813 0.302

Total 11 683.88



Source DF SS MS F P

Treat. 3 1435.413 478.471 1045.204 0.000 ***

Rep. 2 0.027 0.013 0.029 0.971 ns

Error 6 2.747 0.458

Total 11 1438.187


Appendix 238:- ANOVA Mortality (%) in M. obesi in bait of C. sativa bait in Day 4

Source DF SS MS F P

Treat. 3 18.707 6.236 14.768 0.003 **

Rep. 2 0.24 0.12 0.284 0.762 ns

Error 6 2.533 0.422

Total 11 21.48


Appendix239:-ANOVA Mortality (%) in M. obesi in bait of C. sativa bait in Day 8

Source DF SS MS F P

Treat. 3 129.013 43.004 132.548 0.000 ***

Rep. 2 0.72 0.36 1.110 0.389 ns

Error 6 1.947 0.324

330

Total 11 131.68



Source DF SS MS F P

Treat. 3 423.307 141.102 273.690 0.000 ***

Rep. 2 0.107 0.053 0.103 0.903 ns

Error 6 3.093 0.516

Total 11 426.507



Source DF SS MS F P

Treat. 3 1426.387 475.462 618.375 0.000 ***

Rep. 2 3.92 1.96 2.549 0.158 ns

Error 6 4.613 0.769

Total 11 1434.92


Appendix 242: ANOVA Mortality (%) in M. obesi in bait of C. procera in Day 4

Source DF SS MS F P

Treat. 3 22.24 7.413 16.848 0.002 **

Rep. 2 0.56 0.28 0.636 0.561 ns

Error 6 2.64 0.44

Total 11 25.44


Appendix 243:- ANOVA Mortality (%) in M. obesi in bait of C. procera in Day 8

Source DF SS MS F P

Treat. 3 221.12 73.707 290.947 0.000 ***

Rep. 2 1.787 0.893 3.526 0.097 ns

Error 6 1.52 0.253

Total 11 224.427



Source DF SS MS F P

331

Treat. 3 872.747 290.916 564.276 0.000 ***

Rep. 2 0.96 0.48 0.931 0.444 ns

Error 6 3.093 0.516

Total 11 876.8



Source DF SS MS F P

Treat 3 2019.893 673.298 4094.378 0.000 ***

Rep 2 2.107 1.053 6.405 0.033 *

Error 6 0.987 0.164

Total 11 2022.987


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