anopheles sundaicus sl population dynamics, species complex and

ANOPHELES SUNDAICUS S.L. POPULATION DYNAMICS, SPECIES COMPLEX AND INSECTICIDE SUSCEPTIBILITY IN A

COASTAL AREA OF RAYONG PROVINCE, THAILAND

SUCHADA SUMRUAYPHOL

A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR

THE DEGREE OF DOCTOR OF PHILOSOPHY (TROPICAL MEDICINE)

FACULTY OF GRADUATE STUDIES MAHIDOL UNIVERSITY

2009

COPYRIGHT OF MAHIDOL UNIVERSITY

Copyright by Mahidol University

iii

ACKNOWLEDGEMENTS

I would like to express my sincere gratitude and deepest appreciation to my

major advisor, Associate Professor Chamnarn Apiwathnasorn, Department of Medical

Entomology, Faculty of Tropical Medicine, Mahidol University for his guidance,

valuable advice, kind supervision, encouragement and constructive criticism, which

has enable me to complete this thesis successfully.

I also would like to sincerely thank my co-advisors, Associate Professor

Narumon Komalamisra and Lect. Jiraporn Ruangsittichai, for their kindness and

valuable advice.

I am extremely indebted to my chair and external examiner Professor Jean-

Pierre Dujardin and Associate Professor Yupha Rongsriyam for their genius

supervision and suggestion.

My addition thank to all the staff of the Department of Medical Entomology,

Faculty of Tropical Medicine, Mahidol University, especially thank to Mr. Yuthana

Samang for his helping.

Gratitude is also expressed to all my friends for who made thesis possible.

Finally, I wish to express my extreme gratitude to my father, mother and sister

for their infinite love, warmth and encouragement throughout my life. To them all, I

fully dedicate this thesis, without their invaluable suggestions and warmth support I

could not have accomplished it.

This thesis is supported in part by the Faculty of Tropical Medicine and

Faculty of Graduate Studies, Mahidol University.

Suchada Sumruayphol


Fac. of Grad. Studies, Mahidol Univ. Thesis / iv

ANOPHELES SUNDAICUS S.L. POPULATION DYNAMICS, SPECIES COMPLEX AND INSECTICIDE SUSCEPTIBILITY IN A COASTAL AREA OF RAYONG PROVINCE, THAILAND SUCHADA SUMRUAYPHOL 4737529 TMTM/D Ph.D. (TROPICAL MEDICINE) THESIS ADVISORY COMMITTEE: CHAMNARN APIWATHNASORN, Ph.D. (MEDICAL ENTOMOLOGY), NARUMON KOMALAMISRA, Dr.Med.Sc., JIRAPORN RUANGSITTICHAI, Ph.D. (Biology)

ABSTRACT A longitudinal entomological survey study with laboratory analysis was carried on the coastal

malaria vector, Anopheles sundaicus s.l. The objectives were to conduct a bionomic study with molecular identification, and to determine the insecticide susceptibility of An. sundaicus s.l. in Ban Pak Nam, Muang Rayong District, Rayong Province, Thailand. Knowledge of An. sundaicus s.l., particularly its role in malaria transmission, as well as bionomics, species complex, and insecticide susceptibility, is useful for planning a malaria control program. However, little is currently known due to sparse available data. To obtain such data, mosquitoes were collected monthly May 2007-April 2008 by human landing catch from 1800-2400 hr for 2 consecutive nights, at 3 collection points. A total of 3,048 mosquitoes were captured with 5 species found; An. sundaicus s.l., Culex quinquefasciatus, Culex sitiens, Aedes aegypti, and Aedes albopictus. The most abundant was An. sundaicus s.l. (43.8%). The seasonal abundance of An. sundaicus s.l. fluctuated throughout the year. The highest bite rate (37.6 bites/person/half night) was in September and the lowest (10.2) in January. The half-night biting cycle collection was stepped up, such that the pattern started from early evening (1800-2000 h) and reached a maximum of 6.58±0.82 bites/person/half night at 2400 hr. There was no correlation between rainfall, relative humidity, and mean bite number for the An. sundaicus s.l. in this study. Nested-PCR and real-time PCR methods were used to detect malaria-parasite species in the adult An. sundaicus s.l. The present study was the first to incriminate An. sundaicus (later confirmed as An. epiroticus) as a malaria vector in Thailand for both P. falciparum and P. vivax. Nine of 926 (0.97%) adult females examined were malaria-positive, including 6 P. falciparum and 3 P. vivax. The infective mosquitoes were found in the dry and early rainy seasons (January, February, May, and July). The total estimated EIR was 76.6 positive bites/person/year, i.e. about every 5 days a person may receive an infective bite. The parity rate of An. sundaicus s.l. was very high throughout the year, ranging from 61% in November to 90% in January. Calculated life expectancy was 10 days. The daily probability of survival was quite high (0.905 or 90.5%). The biology of An. sundaicus s.l. was studied in the laboratory. The average number of eggs per female was 83.2, with a mean hatching rate of 82.7%. Owing to difficulties rearing the mosquitoes under laboratory conditions, the average period for larval development, from 1st to 4th instar larvae was 20.4 days. Pupal duration was 2-3 days. The adult male to female ratio was 1:1. The mean emergence rate was 72%. The longevity of An. sundaicus s.l. was 29-54 days with a supply of vitamin and sugar solution. A total of 38 cement tanks were examined for physical and chemical characteristics of larval breeding habitats; An. sundaicus s.l. larvae were presented, together with Aedes and Culex larvae; the density range was 0.97-23.01 larvae per dip in December and May, respectively. Breeding places varied from fresh, brackish, and salt water, typically with full sunlight and mats of green algae on the water surface. Water salinity varied from 9.11-52.42 ppt, with narrow pH range of 8.2-8.7. Conductivity varied from 15.5 to 75.2 microseimen/cm which was double the total dissolved solids (7.7-37.6 g/l). Dissolved oxygen was highest in November (6.27 mg/l) and lowest in March (3.46 mg/l). Water temperature also varied between 24.6-32.76˚C. An. sundaicus s.l. was found susceptible to all insecticides tested. LC50 and LC95 of An. sundaicus s.l. larvae against temephos were 0.004 ppm and 0.01 ppm, respectively. The adult females were also susceptible to 3 insecticides, with 100% mortality at 24 hr post-exposure. The KT50 of An. sundaicus s.l. against 5% malathion, 0.75% permethrin, and 0.05% deltamethrin was 25.8, 15.3, and 15.1 minutes, respectively. PCR was used for the molecular identification of the member species of An. sundaicus complex, by determination of COI, ITS2, and D3 genes; the target mosquitoes were all An. epiroticus. The present study identified potential mosquito vectors of malaria in this defined geographic area. This information coupled with infection rate data can help guide public health policies related to vector control.

KEY WORDS: AN. SUNDAICUS / AN. EPIROTICUS / MALARIA VECTOR / BIONOMICS / SPECIES COMPLEX / INSECTICIDE SUSCEPTIBILITY

185 pages


Fac. of Grad. Studies, Mahidol Univ. Thesis / v

พลวัตประชากร, ชนิดซับซอนและความไวตอยาฆาแมลงของยุง Anopheles sundaicus s.l.ในเขตพื้นที่ชายฝงทะเลจังหวัดระยอง, ประเทศไทย ANOPHELES SUNDAICUS S.L. POPULATION DYNAMICS, SPECIES COMPLEX AND INSECTICIDE SUSCEPTIBILITY IN A COASTAL AREA OF RAYONG PROVINCE, THAILAND

สุชาดา สํารวยผล 4737529 TMTM/D

วป.ด. (อายุรศาสตรเขตรอน)

คณะกรรมการที่ปรึกษาวิทยานิพนธ: ชํานาญ อภิวัฒนศร, Ph.D.,นฤมล โกมลมิศร, Dr.Med.Sc., จิราภรณ เรืองสิทธิชัย, Ph.D

บทคัดยอ การศึกษายุงกนปลองชนิด Anopheles sundaicus s.l. โดยการสํารวจยุงในเขตตําบลปากน้ํา อ.เมืองระยอง จ.ระยอง นํามา

ศึกษาในหองปฏิบัติการ วัตถุประสงคการศึกษา คือเพื่อศกึษาพลวัตประชากร ลักษณะทางโมเลกุลและความไวตอยาฆาแมลงขอยุง An. sundaicus s.l. ปจจุบันขอมูลของยุง An. sundaicus s.l. แถบชายฝงทะเล โดยเฉพาะอยางยิ่งในประเทศไทย มีนอยมาก ประกอบกบับทบาทในการเปนพาหะของโรคยังไมชัดเจน รวมทั้งขอมูลของสถานะ, พลวัตประชากร, ชนิดและความไวตอยาฆาแมลงของยุง An. sundaicus sl มีความจําเปนในการศึกษาเพื่อนํามาใชในการควบคุมมาลาเรียในพื้นที่ชายฝงทะเลในประเทศไทย ในการศึกษานี้จะใชคน 3 คนเปนเหยือ่ลอยุงแลวจับยุงทันทีที่ยุงบินมาเกาะตามตัวของคนทีเ่ปนเหยื่อ จับยุงทุกเดือนๆละ 2 คืน ตั้งแต 18.00-24.00 เปนเวลา 12 เดือน ตั้งแตพฤษภาคม 2550 ถึงเมษายน 2551 จับยุงไดทั้งหมด 3,048 ตัว ประกอบดวย 5 ชนิดคือ An. sundaicus s.l., Culex quinquefasciatus, Culex sitiens, Aedes aegypti และ Aedes albopictus ซึ่ง An. sundaicus s.l. เปนยุงที่พบมากที่สุดคือ 43.8% ในเดือนกันยายนพบคาเฉลีย่สูงสุดไดแก 37.6 ตัวตอคนตอคร่ึงคนื ในเดือนมกราคมพบคาเฉลีย่ต่ําสุดไดแก 10.2 ตัวตอคนตอคร่ึงคืน ในชวงเย็นยุงชนิดนีค้อยๆเพิ่มขึ้นจนสูงสุดในชวง 23:00-24:00 จํานวน 6.58±0.82 ตัวตอคนตอคร่ึงคนื การกระจายตัวของยุงชนิดนี้มีตลอดทั้งปและไมมีความสัมพันธระหวางปริมาณน้ําฝน ความชื้นและคาเฉลีย่จํานวนยุง การศึกษาครั้งนี้ใชวิธีเนสเตททพีซีอาร และวิธีเรียลไทมพีซีอารในการตรวจวัดเชื้อมาลาเรียในยุง An. sundaicus s.l. (An. epiroticus หรือ An. sundaicus species A) และพบ sporozoite ในยุง 9 ตัวจาก 926 ตัว โดยพบเชื้อ Plasmodium falciparum ในยุง 6 ตัวและพบเชือ้ P. vivax 3 ตัว ซึ่งเปนการรายงานการเปนพาหะของมาลาเรียของยุงชนิดนีเ้ปนคร้ังแรกในประเทศไทย พบเชื้อมาลาเรียในเดือนมกราคม กุมภาพันธ พฤษภาคมและกรกฎาคม คาดัชนี EIR ที่ใชประเมินความเสี่ยงของการไดรับเชื้อมาลาเรียจากยุงงชนิดนี้เทากบั 76.6 คือเฉลี่ยทกุ 5 วันคนอาจถูกกัดจากยุงที่มีเชื้อ ในพืน้ที่พบยุงเกามากกวายุงที่เกิดใหมตลอดทั้งป โดยพบวามยีุงที่เคยกินเลือดหรือวางไขมาแลว (parous) รอยละ 61 ถึง 90 ตลอดทั้งป โอกาสของความอยูรอดในแตละวันมีคาสูง (0.905 หรือรอยละ 90.5) การศึกษาชีววิทยาของยุงชนดินี้พบวา จํานวนไขเฉลี่ยในยุงเทากับ 83.2 อัตราการออกจากไขรอยละ 82.7 ระยะเวลาเฉลีย่จากลูกน้ําระยะที่หนึ่งถึงสี่ 20.4 วัน ระยะเวลาดักแด 2-3 วัน สัดสวนตัวผูตอตัวเมียคือ 1:1 อัตราการเกิดเปนตัวเต็มวัยรอยละ 72 ชวงชีวิตของยุงชนิดนี้คอื 29-54 วัน การสํารวจแหลงเพาะพันธุของลูกน้ํายุงชนิดนี้จากบอหมักน้ําปลาเกาจํานวน 38 บอ ซึ่งอยูรวมกับลูกน้ํายุงชนิดอื่นคือลูกน้ํายงุลายและยุงรําคาญ คาเฉลี่ยลูกน้ําจาก 0.97 ถึง 23.01 ตัวตอจวงในเดือนธันวาคมและพฤษภาคมตามลําดับ ลูกน้ํายุง An. sundaicus s.l. สามารถอาศัยในน้ําจืดไปจนถึงน้ําเค็มซึ่งอยูกลางแจงและมีสาหรายสีเขียวลอยเปนแพ คาความเค็มจาก 9.11-52.42 สวนในพันสวน คาความเปนกรดเปนดางของน้ําไมแตกตางกันมากไดแก 8.2 - 8.7 คาการนําไฟฟาที่วัดไดคือ 15.5 ถึง 75.2 ไมโครซีเมนตอเซนติเมตร สวนคาของแข็งที่ละลายน้ําทั้งหมดที่วัดไดคือ 7.7-37.6 กรัมตอลิตร คาออกซิเจนที่ละลายน้ําสูงที่สุดในเดือนพฤศจิกายนคือ 6.27 มิลลิกรัมตอลิตรและต่ําสุดในเดือนมีนาคมคือ 3.46 มิลลิกรัมตอลิตร อุณหภูมิน้ําเฉลี่ย 24.6-32.76 องศาเซลเซียส เมื่อใชวิธพีซีีอารในการแยกแยะชนิดของยุงโดยใชยีน 3 ตัว พบวา COI และ ITS2 สามารถใชในการจําแนกยุงชนิด An. epiroticus กับ An. sundaicus sl ได ยุงชนิดนี้มีความไวตอยาฆาแมลงทุกชนิดโดยคา LC50 ของทีมีฟอสเทากับ 0.004 ppm และคา LC95 เทากับ 0.01 ppm คา KT50 ของ5% malathion, 0.75% permethrin และ 0.05% deltamethrin คือ 25.8 นาที, 15.3 นาที และ 15.1 นาที ตามลําดับ ดังนั้นสามารถใชยาฆาแมลงทั้งหมดนี้ในการควบคุมยุงในพื้นทีศ่ึกษาได

185 หนา


vi

CONTENTS

Page

ACKNOWLEDGEMENTS iii

ABSTRACT (ENGLISH) iv

ABSTRACT (THAI) v

LIST OF TABLES vii

LIST OF FIGURES x

LIST OF ABBREVIATIONS xiii

CHAPTER I INTRODUCTION 1

CHAPTER II OBJECTIVES 5

CHAPTER III LITERATURE REVIEW 6

3.1 Malaria

3.2 Malaria in Thailand

3.3 Rayong and Malaria

3.4 Malaria vectors in Thailand

3.5 An. sundaicus s.l. bionomics

3.6 Identification of An. sundaicus species complex

3.7 Malaria detection in mosquito vectors

3.8 Malaria detection in An. sundaicus s.l.

3.9 Malaria vector control in Thailand and

insecticide susceptibility of An. sundaicus s.l.

6

8

9

13

18

21

36

47

49

CHAPTER IV MATERIALS AND METHODS

4.1 Adult mosquito collection

4.2 Larval collections

4.3 Parity rate detection of An. sundaicus s.l. in study

areas

4.4 Detection of malaria parasite in An. sundaicus s.l.

52

53

54

56

60


vii

CONTENTS (cont.)

4.5 Laboratory biology of An. sundaicus

4.6 Polymerase chain reaction for An. sundaicus

species complex identification

4.7 Insecticide susceptibility test

Page

73

78

81

CHAPTER V RESULTS

5.1 Bionomics of An. epiroticus in Pak-Nam, Rayong

5.1.1 Mosquito density in study area

5.1.1.1 Mosquito composition in

Pak-Nam, Rayong province

between 2007 and 2008

5.1.1.2 Biting cycle of An.

epiroticus

5.1.1.3 Seasonal abundance of

An. epiroticus

5.1.1.4 Larval prevalence

5.1.2 Parity rate of An. epiroticus in study

area

5.1.3 Infection rate of An. epiroticus to

plasmodium parasite

5.1.4 Biology of An. epiroticus in laboratory

5.2 Molecular identification of An. sundaicus s.l.

from Pak-Nam, Rayong

5.2.1 COI sequence results

5.2.2 ITS2 sequence results

5.2.3 D3 sequence results

5.3 Insecticide susceptibility test of An. epiroticus

84

84

84

84

87

89

91

95

97

101

102

104

114

118

121


viii

CONTENTS (cont.)

CHAPTER VI DISCUSSION

6.1 Bionomics of adult and larval of An. epiroticus

6.1.1 Mosquito density

6.1.2 Parity rate of An. epiroticus in Pak-

Nam, Rayong

6.1.3 Malaria infection in An. epiroticus


6.2 Molecular identification of An. epiroticus

6.3 Insecticide susceptibility of An. epiroticus

Page

124

124

124

125

126

128

128

130

CHAPTER VII CONCLUSION 131

REFERENCES 132

APPENDIX 144

BIOGRAPHY 185


ix

LIST OF TABLES

Table Page

1 Malaria cases and deaths, 1990-2007 in Thailand (WHO, 2008) 9

2 Known anopheline vectors and potential vectors of malaria in

Thailand and neighboring countries (Rattanarithikul et al., 2006)

14

3 Distribution of An. dirus species complex in Thailand

(WHO, 1998)

15

4 Formally name of An. maculatus complex found in Thailand 16

5 Adult behavior of An. sundaicus observed in different areas

(Dusfour et al., 2004)

19

6 Summary the 3 forms differentiation of polytene and mitotic

chromosomes in An. sundaicus (Sukowati and Baimai, 1996)

29

7 The number of days in each Plasmodium species for oocyst and

sporozoite dissection (WHO, 1975)

36

8 Primer names, sequence targets and size of the PCR product (bp)

for semi-nested multiplex PCR (Rubio, 1999a)

43

9 Sporozoite rates observed from An. sundaicus in various

locations (Dusfour et al., 2004)

48

10 Insecticide resistance status of An. sundaicus in different

populations (Dusfour et al., 2004a)

50

11 Thai and non-Thai malaria cases in Ban Pak Nam from 2002

through 2008

53

12 Mixture set up for nested PCR reaction 65

13 Characteristics of Plasmodium primers and hybridization probes

set* (Swan et al., 2005)

70

14 Master mix content for real-time PCR detection of human

malaria parasite (Swan et al., 2005) in An. sundaicus (for 10

reactions)

71


x

LIST OF TABLES (cont.)

Table Page

15 Experiment condition for detection malaria parasite using

hybridization probes on the LightCycler®

71

16 Oligonucleotide primers of 3 nucleotide sequences to PCR

amplification of An. sundaicus

79

17 Mixture set up for PCR reaction 79

18 Mosquito composition in Ban Pak Nam since May 2007 to April

2008

85

19 Biting cycle of An. epiroticus collected from May 2007 until

April 2008

87

20 Observed An. epiroticus, malaria cases, and meteorological data

in Pak Nam, Rayong during study period

90

21 Mean value ±SE (range) of larvae and measured water

quality in breeding place of An. epiroticus in

Pak Nam sub-district, Rayong province

92

22 The parity rate of An. epiroticus in the area from May 2007 to

April 2008

96

23 Result of plasmodium detection in An. epiroticus head-thorax

portions

100

24 The biology of An. epiroticus under laboratory condition 101

25 COI gene sequences of An. sundaicus s.l. and An. minimus from

many resources for sequence analysis

105

26 The explanation of 17 COI nucleotide sequences for p-distance

analysis

105

27 The pairwise distance (p-distance) of all 17 COI nucleotide

sequences

106

28 The number of polymorphic sites between An. epiroticus from

this study*

107


xi

LIST OF TABLES (cont.)

Table Page

29 The description of 13 ITS2 nucleotide sequences for p-distance

analysis

115

30 P-distance value of An. epiroticus, An. sundaicus s.l. and An.

minimus

115

31 P-distance of An. epiroticus, An. sundaicus D, and An. minimus 119

32 Bioassay result of temephos against An. epiroticus larvae 122

33 Lethal concentration (LC) and range of An. epiroticus to

temephos

122

34 Susceptibility of An. epiroticus against 3 adulticides by using

diagnostic doses

123

34 Summary the insecticide susceptibility of An. epiroticus in Ban

Pak-Nam, Rayong

123

35 Temperature and relative humidity at Muang Rayong weather

station (weather station code: 478201) from May 2007 to April

2008

151

36 Rainfall (mm) at Muang Rayong from May 2007 to April 2008 152


xii

LIST OF FIGURES

Figure Page

1 Malaria life cycle (CDC, 2009) 7

2 Map of Thailand with Rayong Province 10

3 Map of Rayong Province 11

4 Morbidity rate/100000 pop in Rayong Province from

1999 to 2008

12

5 Malaria cases in 8 districts of Rayong Provice in 2005

(per 100,000)

12

6 Range and optimal range of salinity reported

for breeding sites of An. sundaicus in various countries

(Dusfour et al., 2004a)

20

7 The speckled leg of An. sundaicus (Rattanarithikul et al., 2006) 21

8 Maxillary palpus of An. sundaicus (Rattanarithikul et al., 2006) 21

9 The wing of An. sundaicus (Rattanarithikul et al., 2006) 22

10 Pupa and male genitalia of An. sundaicus (Linton et al., 2001) 23

11 Fourth-instar larva of An. sundaicus (Linton et al., 2001) 24

12 Standard photographic map of the ovarian polytene

chromosomes of An. sundaicus (Sukowati and Baimai, 1996)

26

13 The standard Xa found only in form A (Sukowati and Baimai,

1996)

27

14 Photomicrographs of mitotic karyotypes from larval neuroblast

cells of An. sundaicus (Sukowati and Baimai, 1996)

28

15 Ovarian nurse cell polytene chromosomes of An. sundaicus from

Car Nicobar (Nanda et al., 2004)

30

16 Photomicrographs of mitotic karyotypes from larval neuroblast

cells of An. sundaicus cytotype D (Nanda et al., 2004)

31


xiii

LIST OF FIGURES (cont.)

Figure Page

17 Phylogenetic relationships between 9gene pools of

An. sundaicus complexes from Indonesia (Sukowati et al., 1999)

32

18 Distribution of the four members of the Sundaicus Complex

(Manguin et al., 2008)

35

19 The steps of ELISA for P. falciparum and P. vivax

circumsporozoite proteins detection

37

20 The ssrRNA gene of Plasmodia (Snounou et al., 1993b) 39

21 The Plasmodium ssrRNA gene used (Snounou et al., 1993c) 40

22 The oligonucleotide primers used in the nested PCR

(Singh et al., 1999)

41

23 The oligonucleotide primers used in nested PCR

(Snounou, and Singh, 2002)

42

24 LAMP primer set against P. Berghei (Aonuma et al., 2008) 46

25 HACH portable equipment for checking the water quality 55

26 Materials for ovary dissection of An. sundaicus 57

27 Dissection of female ovaries under stereoscope

(the ovaries were circled)

58

28 Nulliparous female showed a tightly coiled tracheole called

a “skein”

58

29 Parous female showed uncoiled tracheole 59

30 Procedure for DNA extraction 61

31 QIAamp® DNA Mini Kit (QIAGEN, Germany) 62

32 Vortex mixer (Vortex Genie 2) 63

33 Water bath (Optima) 63

34 Heat block (Labnet) for sample heating 64

35 Microcentrifuge (Spectrafuge; Labnet) 64


xiv


Figure Page

36 Genus specific primer used within 2 rounds of PCR: rPLU1,

rPLU5 and rPLU3 and rPLU4 (Snounou and Singh, 2002)

66

37 TGradient Thermocycler (Biometra, Germany) for doing PCR

reaction

67

38 2% agarose gel setting 67

39 Step of loading PCR product into the well 68

40 Gel electrophoresis of PCR product 68

41 Gel Documentation for visualization and photograph the DNA 69

42 LightCycler instrument for Real time PCR 72

43 Multivitamin syrup and sugar solution offered daily for mosquito

adults

74

44 Rearing larvae in plastic tray 74

45 Materials for artificial mating of Anopheles mosquitoes 76

46 Blood fed female mosquitoes for artificial mating 76

47 Male prepared for artificial mating 77

48 Successful mating male was attached to the blood fed anesthetized female

77

49 WHO test kit 83

50 Mosquito fauna in Ban Pak Nam from May 2007- April 2008 86

51 Biting cycle of An. epiroticus from 18:00-24:00 since May 2007

to April 2008

88

52 The seasonality of An. epiroticus within 12 observed months

compared with malaria cases and rainfall

89

53 A-H showed breeding places of An. epiroticus in Ban Pak Nam,

Rayong Province

93

54 Parous (A) and nulliparous (B) of dissected An. epiroticus ovary 96

55 2% agarose gel electrophoresis of An. epiroticus positive

samples with genus Plasmodium

98


xv


Figure Page

56 Result of Real time PCR shows melting curve of species-specific

plasmodium in An. epiroticus

99

57 1.5% agarose gel electrophoresis of COI, ITS2, and D3 of An.

epiroticus

103

58 Multiple alignment of COI sequences of An. sundaicus s.l. 108

59 Phylogenetic analysis of COI sequences using Neighbor-Joining

method of An. sundaicus sequences from various sites whereas

An. minimus was outgroup taxon

113

60 Alignment of the ITS2 sequences for An. sundaicus s.l. 117

61 Phylogenetic tree of ITS2 sequences using NJ and 1,000

bootstraps within MEGA4

118

62 Alignment of the D3 sequences of An. epiroticus in Rayong

(Thailand), An. sundaicus from India, and An. minimus

120

63 Phylogenetic analysis of D3 sequences of An. sundaicus s.l. from

Thailand, (Rayong, Chantaburi, and Phangnga), An. sundaicus

(AY691516) from India and An. minimus as outgroup taxon

121


xvi

LIST OF ABBREVIATIONS

Abbreviations or symbol Term

An. Anopheles

bp base pair

cDNA Complementary DNA

COI cytochrome oxidase I

CS circumsporozoite

Cyt-b Cytochrome b

oC degree Celsius

D3 domain-3

DNA Deoxyribonucleic Acid

dNTP deoxyrinucleotide triphosphate

e.g. exempli gratia – for example

ELISA Enzyme Linked Immunosorbant Assay

FRET Fluorescence resonance energy transfer

et al. et all - and other

h hour(s)

i.e. id est - that is

IRS Indoor Residual Spraying

ITN Insecticide Treated Nets

ITS2 internal transcribed spacer 2

kDa kilodalton

km kilometer

LAMP Loop-mediated isothermal amplification

min minute(s)

µl microliter(s)


xvii

LIST OF ABBREVIATIONS (cont.)

Abbreviations or symbol Term

Mabs monoclonal antibodies

ml milliliter(s)

mg milligram(s)

µM micromolar

mM millimolar

mm millimeter(s)

MOPH Ministry of Public Health

PBS phosphate buffered saline

PCR Polymerase Chain Reaction

P. falciparum, Pf Plasmodium falciparum

P. malariae, Pm Plasmodium malariae

P. ovale, Po Plasmodium ovale

P. vivax, Pv Plasmodium vivax

RNA Ribonucleic acid

rRNA Ribosomal Ribonucleic acid

rpm Round per minute

sec. second (s)

s.l. sensu lato

SnM-PCR semi-nested multiplex polymerase chain

reaction

sp. species

ssrRNA small subunit ribosomal RNA

SSU Small Sub Unit

UN United Nation

WHO World Health Organization

% percent


Fac. of Grad. Studies, Mahidol Univ. Ph.D. (Tropical Medicine) / 1

CHAPTER I

INTRODUCTION

Malaria is the world's most deadly tropical parasitic disease which kills more

people than any other communicable disease with nearly a million deaths annually

(WHO, 2008). It is endemic in 109 countries with approximately 3.3 billion people

living in the risk areas. There are 247 million cases of malaria worldwide (WHO,

2008), half of these in children under 5 years old (Marten and Hall, 2000).

Malaria risk is often associated with forest areas. For example in the Southeast

Asia, while forest cover represents 18% of the territory of the eight malaria endemic

countries, these areas suffer between 31% to 87% of the malaria cases (WHO, 2006).

Also in Thailand malaria is forest-related and most prevalent along the international

borders, especially on the Thai–Myanmar and Thai-Cambodia border where present

population movement.

Two-third of the Earth’s surface is covered by the oceans, one-third is land and

the coastal zone. Although, the coastal zone covers less than 15% of the Earth’s land

surface, this is place that abundant people live and work. Coastal areas around the

world offer particularly favorable conditions for malaria transmission. Efficient vector

species grow in brackish water or wetland habitats, and coastal areas are often

attractive to a variety of human activities. Many important cities and towns are

located in coastal areas (WHO, 2006). Thailand has approximately 2,600 km along

the coasts of the Gulf of Thailand and Andaman Sea, which nearly 11 million people

live in the coastal Provinces.

Malaria is a major public health problem in Thailand and continued to decrease

over the past two decades and have disappeared from most of the major cities (MOPH,

2003). Malaria control requires an integrated approach comprising prevention

including treatment with effective antimalarial drug and vector control (WHO, 2006).

The purposes of vector control, it is important to understand the life-cycle and natural

history of mosquitoes, correct identify mosquito vector to species, understand


Suchada Sumruayphol Introduction / 2

mosquito behavior; and know the susceptibility of mosquitoes to insecticides (WHO,

2005).

Malaria is transmitted by anopheline female mosquitoes. Anopheles

mosquitoes as a general rule breed in clean, freshwater pools may be stagnant or slow

flowing. In addition some coastal areas, malaria is transmitted by brackish-water

breeding mosquitoes. In Asia, the most important is An. sundaicus s.l., more rarely,

An. subpictus (WHO, 2005). Approximately 420 anophelines species have been

identified (Subbarao, 1998). There are only about 70 species that transmit human

malaria in the world (WHO, 1998a), which differ in their transmission potential

(Kiszewski et al., 2004). There are 6 species have been informed to be malaria

vectors. In Thailand, the 3 species are the primary vectors which are An. dirus, An.

minimus and An. maculatus (Green et al., 1991). The other 3 species that are An.

aconitus, An. sundaicus and An. pseudowillimori are incriminated as the secondary

malaria vectors (Rattanarithikul, 2006). An. dirus is a well understood vector for

malaria in deep forest areas, whereas An. minimus and An. maculatus s.l. exhibits its

influence in forested-fringe areas especially along international Thailand borders. On

the contrary, An. sundaicus s.l. is less focus for studying the malaria transmission in

Thailand owning to it is not related to the hilly forested areas, whereas, it related to the

coastal, mangrove wetland zones. It has been incriminated as secondary vector of

malaria in Thailand (Gould et al., 1966; Harinasuta et al., 1974).

The capacity study of mosquito vector must study several aspects. The vector

capacity of human malaria can be summarized as followed (Apiwathnasorn, 2002).

Susceptibility: The mosquito must be susceptible to the parasites.

Longevity: The mosquito must live long enough to transmit the parasites.

Host preference: The mosquito must be anthropophilic.

Density: The mosquito must bite man repeatedly in sufficient numbers.

Environmental factors: such as temperature, humidity, rainfall influences vector’s

biology

Although, malaria is still a potential fatal disease in some hilly-forested areas

in Thailand but continues to be endemic in some costal areas. In Thailand, the coastal

malaria continually occurs from the past. As reported by MOPH in recent year, there

were malaria cases in coastal Provinces such as Trat with 310 cases, Chonburi with



134 cases, Phangnga with 635 cases and Rayong with 174 cases (MOPH, 2008).

Unlike in along the international Thailand border where malaria vectors have been

much more studied, very little is known about the vector to malaria transmission in the

coastal areas. Therefore, there are need the knowledge of An. sundaicus s.l., coastal

malaria vector, in coastal area of Thailand. Rayong Province has 100 km long

coastline although the north is hilly, the Province consists mostly of low coastal plains.

The epidemiological importance of An. sundaicus s.l. mosquitoes in coastal

community in Rayong Province, few observations have been carried out regarding

their knowledge.

Pak Nam sub district is located in the southern part of Muang Rayong district,

Rayong Province, at which nearby the coast. Pak Nam has a population of 4,368, with

a population density of 436.8 persons per km2. This is crowded community and low

hygiene. This community has serious environmental conditions owning to intense

labor migrations from Myanmar, Cambodia and Lao PDR both legal and illegal to this

area, urbanization and fishery-industrial activities. The main activity is fishery and

fish sauce production. This community has various leave concrete ponds.

This site has been used insecticide for control of mosquito which is 10%

permethrin, 2% bifenthrin for adulticide and temephos (abate) for larvicide.

According to the report of VBDC 3.3 Rayong, malaria was first reported in the area in

2002. Afterward, its outbreak was considered in 2005 with 15 cases. Consequent,

malaria cases were reported every year until now. The highest cases were in 2007

with 40 cases consisting 26 Thai cases and 14 not Thai cases (Personal

communication). Moreover, there was no the primary malaria vector in the area.

There was only one Anopheles mosquito presented in community which was An.

sundaicus s.l. from human landing catch. The knowledge of An. sundaicus s.l. on the

coastal areas of Southeast Asia is poorly known due to a little of available data

(Dusfour et al., 2004a).

Although An. sundaicus s.l. is incriminated to be an important malaria vector

in the coast of Thailand, its role in malaria transmission is not clear. Currently, there

is insufficient information on its status, bionomics, specie complex, malaria infection

or insecticide susceptibility and make problem on development of vector control in

coastal zone of Thailand. Therefore, the study of all aspects of An. sundaicus s.l. is


Suchada Sumruayphol Introduction / 4

very crucial for understanding the potential of An. sundaicus s.l. to transmit malaria

and very useful for further control of malaria vectors in coastal areas in Thailand.



CHAPTER II

OBJECTIVES

General objectives

1. To determine the vector status of An. sundaicus s.l. in the study area by

- investigate population density of An. sundaicus s.l. both adult and larval stage

on coastal community from May 2007 to May 2008 at Pak Nam, Muang Rayong

district, Rayong Province, Thailand including the breeding place characteristics

- study biting behavior of An. sundaicus s.l. in study areas

- investigate seasonal abundance of An. sundaicus s.l. in study area

- investigate parity rate of An. sundaicus s.l.

- determine the infection rate of An. sundaicus s.l. with malaria parasite

- study biology of An. sundaicus s.l. in laboratory

2. To identify the An. sundaicus s.l. population in study area by molecular assessment

3. To examine the insecticide susceptibility of An. sundaicus s.l.

Specific objective

To conduct the bionomic knowledge of An. sundaicus s.l. in coastal areas of

Rayong Province where this mosquito presents

This information acquired through this research will enhance the effective

malaria control in various areas particularly in coastal region in Thailand.


Suchada Sumruayphol Literature Review / 6

CHAPTER III

LITERATURE REVIEW

3.1 Malaria Malaria is disease cause by Plasmodium protozoa. Beside Plasmodium species

infected various animals, there are only four species are crucial for human infection.

These are Plasmodium vivax, P. falciparum, P. malariae and P. ovale (Sinden and

Gills, 2002).

Malaria is transmitted by biting of female Anopheles mosquito infected with

sporozoites in its salivary grand.

The malaria parasites require two hosts (Figure 1).

For human host, the infective female Anopheles mosquito injects sporozoites

into human host during feed on blood. Sporozoites invade liver cells and become

schizonts that rapture and release merozoites. The parasites undergo asexual

multiplication in erythrocytes. Merozoites infect red blood cells. The trophozoites

mature to schizonts which rapture and release merozoites. Some parasites become

gametocytes.

For mosquito host, after female Anopheles mosquito feeds on human blood

containing male (microgametocytes) and female (macrogametocytes) gametocytes.

The microgametocytes enter macrogametocytes and become zygotes in stomach of

mosquito. The zygotes develop into ookinetes and oocytes in the midgut wall. The

oocytes mature, rapture and release sporozoites to salivary glands which ready to

inject to human host by mosquito blood meal (CDC, 2009)

Malaria is endemic in 109 countries with approximately 3.3 billion people

living in the risk areas. There are 247 million cases of malaria worldwide with nearly a

million deaths each year (WHO, 2008). About 59 % occur in Africa, 38 % in Asia,

and 3 % in the Americas (WHO/UNICEF, 2005). In Southeast Asia, 2006, the most

seriously affected country was Myanmar followed by Vietnam, Cambodia, Philippines,



Thailand and Lao PDR with 475,297, 91,350, 89,109, 35,110, 30,293, and 20,418

cases, respectively (WHO, 2008).

Figure 1 Malaria life cycle (CDC, 2009)



3.2 Malaria in Thailand Malaria remains one of the most important infectious diseases in Thailand,

although there was reduction of cases and deaths (Chareonviriyaphap, 2000) mainly

due to an improved health care system with prompt treatment of cases, information

campaigns, and effective vector control (MOPH, 1993). Moreover, the deforestation

was associated with reduction of malaria cases (Rosenberg et al., 1990). Malaria has

occurred along the national borders of Myanmar, Cambodia, and Malaysia

(Chareonviriyaphap, 2000). In these areas, there are extensive human migration i.e.

Burmese refugees (Kondrashin et al., 1991).

There were first deaths from malaria since 1912 with 38,000 in Thailand.

Consequently the control program was initiated by drug distribution and DDT house

spraying reduced death rates to 22.8 per 100,000 populations in 1963 (Chaiphet,

1999). The epidemiological data showed a downward trend in total cases from

273,880 in 1990 to 82,743 in 1995 afterward in 1998 it was again increased with

131,055 cases and gradually declined every year until now (WHO, 2008). In addition

to, foreigner cases (mostly Burmese) have been increased from 48,000 in 1991 to

66,000 in 1997 and increased to some 79,490 during 1997-1999 (MOPH, 2003).

However, the malaria cases and deaths in Thailand from 1990 to 2007 were shown in

Table 1 (WHO, 2008). P. falciparum and P. vivax are predominant malaria species

found in Thailand (Snounou et al, 1993). P. vivax was increased compared to P.

falciparum from less than 20 % in 1965 to more than 50 % in 2002 owing to P.

falciparum is easy to treatment by drug and effective control of drug-resistance

(Sattabongkot et al, 2004). Another reason was changing in vector potential, i.e.,

changing in abundance of vectors that have a high potential to transmit P. vivax

(Sattabongkot et al, 2004). The rapid increase of An. barbirostris/campestris group in

Sa Kaeo Province (eastern Thailand) was the good example (Limrat et al, 2001;

Apiwathnasorn, 2002).



Table 1 Malaria cases and deaths, 1990-2007 in Thailand (WHO, 2008)

Year Malaria cases Malaria deaths 1990 273,880 1,287 1991 198,380 - 1992 168,370 - 1993 115,220 - 1994 102,119 - 1995 82,743 - 1996 87,622 826 1997 97,540 764 1998 131,055 688 1999 125,379 740 2000 81,692 625 2001 63,528 424 2002 44,555 325 2003 37,355 325 2004 26,690 230 2005 29,782 71 2006 30,293 113 2007 30,889 38

. Mefloquine, primaquine, quinine, tetracycline, and artemeter/artesunate have

been currently used for falciparum malaria treatment, whereas chloroquine and

primaquine have been used for treatment of P. vivax, despite increasing the resistance

of chloroquine in the country reported (Chareonviriyaphap, 2000).

3.3 Rayong and Malaria Rayong Province is located on the eastern part of Thailand (Figure 2).

An area occupies approximately 3,552 km2. The north neighbors to Amphur Nong

Yai, Amphur Bo Thong and Amphur Sri Raja of Chonburi. The south is adjacent to

the Gulf of Thailand. The east is nearby to Amphur Na Yai Arm and Amphur Kaeng

Hang Maew of Chanthaburi and Amphur Bang Lamung of Chonburi. The total

number of Rayong population was 548,657 (male: 2737388, female: 274919) on 2003.

Rayong Province comprises of mostly coastal plains, slope, hilly land and mountains

with 100 km long coastline. It has two major rivers, namely Rayong and Pra Sae



River. The former flows through Amphur Pluak Daeng, Amphur Ban Kai, Amphur

Muang to the sea at Tambol Pak Nam, whereas the latter flows through king Amphur

Khao Chamao, Amphur Klaeng to the sea at Tambol Pak Nam Pra Sae.

The distance from Bangkok is 179 km (Rayong, 2009). The Province is subdivided

into six districts are Mueang Rayong, Ban Chang, Klaeng, Wang Chan, Ban Khai,

Pluak Daeng and two minor districts are Khao Chamao and Nikhom Patthana (Figure

3).



Figure 2 Map of Thailand with Rayong Province

Figure 3 Map of Rayong Province

Despite malaria has been occurred in some hilly-forested areas in Thailand but

continues to be endemic in some costal areas. In Thailand, the coastal malaria

continually occurs from the past. As reported by MOPH in recent year, there were

malaria cases in coastal Provinces such as Trat with 310 cases, Chonburi with 134

cases, Phangnga with 635 cases and Rayong with 174 cases (MOPH, 2008).

Malaria remains an important vector borne disease in Rayong Province. In the

last 10 years, the highest morbidity rate was reported in 1999 (per 100,000 people). It

extremely reduced until 2005, the lowest morbidity rate presented with 15.77

cases/100000 people. Consequently, morbidity rate gradually increased in 2006 to

2007 (MOPH, 2008) (Figure 4). According to 2005, Pruakdaeng district presented the

highest morbidity cases (29.14/100000 people). Including Muang Rayong district,

malaria cases occurred 6.69/100000 people (Figure 5).



0

20

40

60

80

100

120

140

160

180

Year

Mor

bidi

ty ra

te/1

0000

0 po

p

1999 2000 2001 2002 2003 2004 2005 2006 2007 2008

Figure 4 Morbidity rate/100000 pop in Rayong Province from 1999 to 2008

05

101520253035

Pruakdaeng

Wangchan

Kaochamao

BanchangBankai

KlangM uang

Nik iompattana

Districts of Rayong province

Mor

bidi

ty ra

te/1

0000

0 po

p

Figure 5 Malaria cases in 8 districts of Rayong Provice in 2005 (per 100,000)



Ban Pak-Nam, Muang Rayong, has been found malaria cases since the past.

According to the preliminary study, found that there was first malaria outbreak in

January to February, 2005 with 14 cases in this area both Thai and Cambodian.

Consequent, malaria cases were reported every year until now. The highest cases

were in 2007 with 40 cases consisting 26 Thai cases and 14 non Thai cases (Personal

communication). Moreover, there was no the primary malaria vector in the area.

There was only one Anopheles mosquito presented in community which was An.

sundaicus from human landing catch. The knowledge of An. sundaicus s.l. on the

coastal areas of Southeast Asia is poorly known due to a little of available data

(Dusfour, Harbach, and Manguin, 2004). This site has been used insecticide for

control of mosquito which is 10% permethrin, 2% bifenthrin for adulticide and

temephos (abate) for larvicide (personal communication).

Although An. sundaicus s.l. is incriminated to be an important malaria vector

in the coast of Thailand, its role in malaria transmission in Thailand is not clear.

Currently, there is insufficient information on its status, bionomics, specie complex,

and malaria infection or insecticide susceptibility and make problem on development

of targeted control measures in coastal zone in Thailand. Therefore, the study of all

aspects of An. sundaicus s.l. is very crucial for understanding the potential of An.

sundaicus s.l. to transmit malaria and very useful for further malaria control at coastal

zone in Thailand.

3.4 Malaria vectors in Thailand 73 species of genus Anopheles were reported in Thailand (Rattanarithikul et

al., 2006). There are 6 species have been informed to be malaria vectors. The 3

species are the primary vectors which are An. dirus, An. minimus and An. maculatus

(Green et al., 1991). The other 3 species that are An. aconitus, An. sundaicus and An.

pseudowillimori are incriminated as the secondary vectors of malaria (Rattanarithikul

et al., 2006). However, Rattanarithikul et al (2006) recognized anopheline vectors and

potential vectors of malaria both Thailand and neighboring countries as in Table 2.



Table 2 Known anopheline vectors and potential vectors of malaria in Thailand and

neighboring countries (Rattanarithikul et al., 2006)

Disease/vector Vector in Thailand Vector elsewhere

An. aconitus X X

An. annularis (X) X

An. campestris (X) -

An. culicifacies - X

An. dirus X X

An. baimaii (dirus D) X -

An. hodgkimi (X) -

An. karwari [X] -

An. kochi (X) X

An. maculatus [X] X

An. minimus X X

An. nivipes (X) -

An. philippinensis [X] X

An. pseudowillmori X -

An. stephensi - X

An. subpictus - X

An. epiroticus [X] X

An. tessellatus [X] X

An. sawadwongporni (X) -

An. vagus (X) X

An. willmori - X

Barbirostris Group [X] X

Hyrcanus Group (X) X

Umbrosus Group - X

X : sporozoites in the salivary glands (X) : ELISA [X] : oocysts - : No evidence



Primary vectors of Thailand

An. dirus complex

The An. dirus species complex belongs to the Leucosphyrus group subgenus

Cellia and series Neomyzomyia. It is widely distribution of species complex in

Thailand particularly in forest areas as WHO reported in 1998 (WHO, 1998) (Table

3). There are at least 7 species of An. dirus complex but 5 species were found in

Thailand (Rattanarithikul et al., 2006).

Table 3 Distribution of An. dirus species complex in Thailand (WHO, 1998)

Species Location in Thailand

A It occurs throughout Thailand exclude in the south.

B, C It is sympatric in southern Thailand. Both have variation to occur north-

south geography.

D It is commonly found in the north-western side of Thailand. It occurs with

species A along Thai-Myanmar border.

F It is found in Thai-Myanmar border.

An. dirus complexes are forested mosquitoes which are anthropophilic. These

species are mainly exophagic and exophilic. They breed in stagnant and shaded water

in forest, orchards and plantations (Green et al., 1991) animal hoof prints, small

muddy pools, cavities of trees and rocks were also found (Apiwathnasorn, 2002). An. minimus complex

An. minimus complex Theobald belongs to subgenus Cellia and series

Myzomyia. There are three species in An. minimus complex. It comprises two

formally named An. minimus (species A) and An. harrisoni (species C) and one

informally named An. minimus species E. Thailand presents An. minimus A, An.

minimus C and An. minimus D also found. An. minimus A is almost found throughout

Thailand, An. minimus C occurs along Thai-Myanmar border (Garros et al., 2006). In

Thailand both An. minimus A and C were reported as predominantly zoophilic and

feed on human more outdoors than indoors. They prefer to rest outdoor (WHO, 1998).

These are forest-fringe species. They prefer to breed in partial shaded slow moving



streams of sunlit habitats (Meek, 1995) i.e. foothill stream, springs, seepages,

irrigation ditches, rice-fields (Apiwathnasorn, 2002). An. maculatus complex

An. maculatus complex Theobald belongs to Theobaldi group of series

Neocellia. The Maculatus group complex includes 8 species which are A, B, C, D, G,

H, I and J. Six species were found throughout Thailand (A, B, C, G, H and I) and

formally name were shown in the Table 4.

Table 4 Formally name of An. maculatus complex found in Thailand

An. maculatus complex Formally name

Species A An. sawadwongporni

Species B An. maculatus s.s.

Species C An. dravidicus

Species G An. notanandai

Species H An. willmori

Species I An. pseudowillmori

These An. maculatus complex species are found in hill and forest edge areas. All

species were predominantly zoophilic and prefer to bite human outdoors. Their

breeding place were sunlight i.e. seepages, pools formed in stream (Apiwathnasorn,

2002). An. maculatus was regarded as major malaria vector only in southern Thailand

(Rattanarithikul et al., 1996).



Secondary vectors of Thailand

An. aconitus, An. pseudowillimori and An. sundaicus are the secondary vectors

of Thailand. An. aconitus

An. aconitus Donitz belongs to subgenus Cellia series Myzomyia. It was

incriminated as malaria vector in Thailand by (Gould et al., 1967) since they found

one female presented oocysts and sporozoites of P. vivax and on 1987 (Kondrashin,

and Rashid, 1987) reported that it was secondary vector or local important vector in

Thailand. It is presented across Thailand especially in central plain (rice-fields) and

also foothills therefore it was considered to be the important malaria vector (Junkum et

al., 2004). An. aconitus was zoophilic rather than anthropophilic and more likely bite

human outdoors than indoors (Junkum et al., 2007). An. aconitus larvae prefer

sunlight areas for habitats i.e. rice-fields (Rattanarithikul, and Panthusiri, 1994).

An. pseudowillimori

An. pseudowillimori Theobald is a member of Maculatus group. In Thailand, it

has been incriminated the malaria vector in the north and the northwestern border with

Myanmar of the country (Green et al., 1991).

An. sundaicus complex

Anopheles sundaicus Rodenwald is a member of series Pyretophorus

(Pseudomyzomyia) subgenus Cellia Theobald, group Ludlowae, genus Anopheles,

subfamily Anophelinae, Family Culicidae, suborder Nematocera, order Diptera and

Class Insecta (WHO, 1998). Status of An. sundaicus is varied depending upon

geographical location. It has been incriminated as secondary vector of malaria in

Thailand (Gould et al., 1966; Harinasuta et al., 1974) but in India (Rao, 1984)

Myanmar, Vietnam (Nguyen et al., 1993) and Indonesia (Miyagi et al., 1994) it has

been the primary vector of malaria (Kondrashin, and Rashid 1987). In 2005, An.

sundaicus species A of Southeat Asia was formally named as An. epiroticus (Linton et

al., 2005). It is generally a salt water breeder. The major breeding places are swamps

and pits along coastal areas containing brackish water. Rao reported that the most

appropriate breeding places are sea and fresh water combination or brackish water

(Rao, 1984) but in Thailand there is less information about An. sundaicus. Moreover,

no sporozoites were discovered in this mosquito species but there was only oocysts



positive detection in Thailand (Scanlon et al., 1968). Additional, An. sundaicus was

regarded as a potential malaria vector since it presented nearby Thai tourist areas

(Chowanadisai et al., 1989).

3.5 An. sundaicus s.l. bionomics

Adult bionomics

The bionomics of An. sundaicus complexes are depending upon the

geographical location. It was strongly anthropophilic and exophagic in Java island

(Indonesia) (Chow, 1970), Vietnam (Nguyen et al., 1993) and Sarawak (Malaysia)

(Linton et al., 2001). In contrasting, in Car Nicobar island, India, An. sundaicus was

zoophilic, exophagic and exophilic (Kumari et al., 1993). However, the behavior of

An. sundaicus was summarized (Dusfour et al.,2004a) from distinct location as in

Table 5.



Table 5 Adult behavior of An. sundaicus observed in different areas (Dusfour et al.,

2004a)

Country Trophic preference Resting preference Biting preference

Cambodia Exophagy Anthropophily

Cambodia Exophagy/endophagy Endophily Zoophily/anthropophily

India (Nicobar/Andaman) Endophagy Endophily Anthropophily

India (Andaman/Orissa) Endophily Anthropophily

India (Nicobar/Andaman) Exophagy Exophily Zoophily/anthropophily

India (Nicobar) Zoophily

India (Nicobar) Endophagy Endophily

India (West Bengal) Zoophily/anthropophily

Indonesia (northern Sumatra) Exophagy/endophagy Exophily

Indonesia (western Java) Endophagy Endophily

Indonesia (central Java) Endophagy Endophily

Indonesia (Sulawesi) Endophagy Endophily Anthropophily

Malaysia Exophagy Exophily Zoophily

Malaysia Exophagy Anthropophily

Malaysia (Sarawak) Endophagy

Thailand Exophagy Zoophily

Vietnam Endophily Anthropophily

Vietnam Exophily

Larval bionomics

An. sundaicus complexes require sunlit breeding places which prefer brackish

water, floating algae and vegetation in coastal areas and on islands (Dusfour et al.,

2004a). An. sundaicus larvae were reported in brackish water at many sites e.g. Car

Nicobar island of India (Kumari et al., 1993), Vietnam (Nguyen, Quy, Thi, and

Nguyen, 1993), Malaysia Borneo (Linton et al., 2001), Sarawak of Malaysia (Chang et

al., 2001) and Thailand (Dusfour et al., 2004). They were also presented in the inland

ponds of fresh water habitats from Sumatra Indonesia (Sukowati and Baimai, 1996),

Car Nicobar island of India (Das et al., 1997) and Sarawak Malaysia (Linton et al.,

2001). In Andaman and Nicobar islands of India, it breeds in both brackish water as



well as fresh water (Alam et al., 2006). In conclusion, An. epiroticus has only been

collected in brackish water habitats, whereas An. sundaicus s.s., species D and species

E have been collected in both brackish and fresh water sites (Dusfour et al., 2007a).

The wide variety of salinity was observed from 0% to 11% as shown in Figure 6.

Figure 6 Range and optimal range of salinity reported for breeding sites of

An. sundaicus in various countries (Dusfour et al., 2004a)



3.6 Identification of An. sundaicus species complex The accurate identification of Anopheline vectors is vital for both basic and

applied research. It is for further appropriate malaria vector control. Two problems

for Anopheline identification are strong morphological similarity between species and

morphological variation within species (Krzywinski, and Besansky 2003). Therefore,

there need combination approaches between morphological, molecular, distribution

and bionomic data to resolve these problems (Krzywinski, and Besansky 2003).

In term of An. sundaicus, it was suspected to be sibling species since 1968 by

Reid hence it occurred in both salt and fresh water habitats including the

differentiation of behavior and ecology of adults (Reid, 1968). There were many

identification methods for An. sundaicus.

Morphological identification based on adult female (Rattanarithikul et al.,

2006)

Adult:

An. sundaicus adult is distinguished from other members of Pyretophorus

series in having speckled legs (different from An. indifinitus, An. subpictus and An.

vagus).

Figure 7 The speckled leg of An. sundaicus (Rattanarithikul et al., 2006)

The maxillary palpus presents 3 pale bands. The subapical dark band is equal to or

greater than half length of apical pale band.

Figure 8 Maxillary palpus of An. sundaicus (Rattanarithikul et al., 2006)



Like all of the subgenus Cellia, An. sundaicus has 4 or more dark marks on its wing

involving both costa and veins R-R1, accessory sector pale (ASP) spot present on costa

and/or subcosta, and/or R1.

Figure 9 The wing of An. sundaicus (Rattanarithikul et al., 2006)

Pupae: Pupae have seta 6-IV branched (different from An. litoralis, An. ludlowae and

An. vagus), paddle marginal spicules with hooked tips (different from An. limosus, An.

litoralis and An. vagus), refractile index of paddle not more than 0.75 (different from

An. ludlowae and An. subpictus) and seta 1-P less than two-thirds paddle length

(different from An. parangensis). An. sundaicus pupae are indistinguishable from An.

indefinites pupae.

Larvae:

Larvae are more easily distinguished than pupae: seta 3-C half or more length

of seta 2-C (different from An. limosus and An. vagus), setae 9,10,12-P,M with simple

branches (different from An. parangensis), seta 6-IV-VI branched at base (different

from An. ludlowae), seta 3-C less than three-fourths length of seta 2-C and seta 1-P

usually with more than eight branches arising at base (different from An. subpictus and

An. indefinites).



Figure 10 Pupa and male genitalia of An. sundaicus. Pupa: (A) left side of cephalothorax, dorsal to

right; (B) dorsal (left) and ventral (right) aspects of metathorax and abdomen. C, Male genitalia, aspects

as indicated. Ae, aedeagus; C1, claspette; CT, cephalothorax; Gc, gonocoxite; Gs, gonostylus; LAe,

leaflets of aedeagus; Pa, paddle; Ppr, paraproct; Pr, proctiger; I-VIII, abdominal segments I-VIII; 0-14,

setal numbers for specified areas, e.g., seta 3-I. Scales in mm (Linton et al., 2001)



Figure11. Fourth-instar larva of An. sundaicus. A, Head, dorsal (left) and ventral (right) aspects of left

side; B, thorax and abdominal segments I-VI, dorsal (left) and ventral (right) aspects of left side; C,

abdominal segments VII-X, left side; D, palmate seta 1-IV showing the structure of leaflets. A, antenna;

C, cranium; M, mesothorax; P, prothorax; S, spiracular lobe; Sa, saddle; T, metathorax; I-VIII, X,

abdominal segments I-VIII, X; 0-15, setal numbers for specified areas, e.g. seta 5-C Scales in mm

(Linton et al., 2001)



Cytogenetic study

The inversion of chromosomes has been led into the speciation process.

Chromosome inversions between species complex make various polytene

chromosome patterns but polytene chromosomes are not presented in all stage or both

sexes of mosquito and some species complexes have same banding patterns.

Moreover, banding pattern analysis is time-consuming and expertise requirement

(Krzywinski and Besansky, 2003).

According to cytogenetic study, there were 4 cytogenetic forms, A, B, C and

D, in Thailand, Indonesia and India.

Cytogenetic work were first time carried out by Sukowati and Baimai (1996)

between the population of An. sundaicus from Thailand (Phang Nga and Trat) and

Indonesia (Sumatra and Java) based on differences in ovarian polytene chromosomes

and mitotic karyotypes in larval neuroblast cells. An. sundaicus populations were

distinguished into 3 forms as A, B and C. Form A was widely distribution in Thailand

and Indonesia, whereas form B was found in North Sumatra and Central Java that it

was strongly linked to fresh water. Form C presented only in North Sumatra. The

chromosomes had 3 synapsed elements: a short pair of sex chromosomes

(chromosome 1) and two longer pairs of autosomes (chromosome 2 and 3) which were

labeled as chromosome arms 2R/2, 2L/3, 3R/4 and 3L/5 and they were divided into 50

zones (Figure 12). All samples showed the same chromosomal arrangements to

standard, except for the banding patterns at the up of chromosome X and zone 19 of

chromosome arm 2R/2 (Figure 13). Mitotic karyotypes from An. sundaicus used

larval neuroblast cells were shown in Figure 14. They differed in both polytene

chromosome and heterochromatic variations in Y-chromosomes as shown in Table 6.



Figure 12 Standard photographic map of the ovarian polytene chromosomes of An.

sundaicus (Sukowati and Baimai, 1996)



Figure 13 The standard Xa found only in form A (a) and the different banding pattern

of Xb presenting in form B and C (b); The standard banding patterns (2Ra) (c) and the

loosely diffuse area (2Rb) of zone 19 of chromosome arm 2R (d). Chromosome 2Rb

occurred only form C (Sukowati and Baimai, 1996)



Figure 14 Photomicrographs of mitotic karyotypes from larval neuroblast cells of

An. sundaicus. Form A, male (a) and female (b), Form B, male (c) and female (d).

Form C, male (e) and female (f) (Sukowati and Baimai, 1996)



Table 6 Summary the 3 forms differentiation of polytene and mitotic chromosomes in

An. sundaicus (Sukowati and Baimai, 1996)

Forms Polytene banding patterns Mitotic chromosomes

Xa Xb 2Ra 2Rb Y ch

A + - + - Y1 normal

B - + + - Y2 normal

C - + - + Y1 large

Note: +: present, -: absence, ch: pericentromeric heterochromatic blocks in autosome

2.

More recently, cytotype D was found on Car Nicobar Island, India both in

fresh and brackish water breeding places (Nanda et al., 2004). They found that

ovarian polytene chromosomes had X chromosome of Xa type as reported in case of

species A and chromosome arm (2b) similar to that in species C. This combination

suggested the existence of a new cytogenetic variant which was namely cytotype D

(Figure 15). They also examined male and female mitotic karyotypes as shown in

Figure 16.



Figure 15 Ovarian nurse cell polytene chromosomes of An. sundaicus from Car

Nicobar. A: X chromosome showing standard (Xa) arrangement. b: Centromeric

portion of chromosome arm 2 with diffuse area in zone 19. An arrow: centromeric end

of the chromosome (Nanda et al., 2004)



Figure 16 Photomicrographs of mitotic karyotypes from larval neuroblast cells of

An. sundaicus cytotype D, a: male, b: female, an arrow: the additional block of

heterochromatin in the centromeric region of autosome 2 (Nanda et al., 2004)



Biochemical methods

Sukowati and coworkers (Sukowati et al., 1999) studied electrophoresis of

isoenzymes in 3 forms of An. sundaicus in Indonesia confirmed previous study

(Sukowati and Baimai, 1996). A total of 12 enzyme systems containing 15 loci

presented allelic variations within and among population of An. sundaicus complexes.

Phylogenetic dendograms showed form A fall into one cluster related to form C,

which form B belonged to more distinct cluster as shown in Figure 17.

Figure 17 Phylogenetic relationships between 9gene pools of An. sundaicus

complexes from Indonesia (Sukowati et al., 1999)

These data correlated with cytological study for 3 species of An. sundaicus

complex in Indonesia. An. sundaicus complexes were found sympatric at Asahan in

north Sumatra. They concluded that species A was the most widely distribution

presenting in coastal areas of Thailand, Sumatra and Java. Species B was found

together with species A at central Java and north Sumatra. Species C was only found

in northeastern Sumatra, where it was sympatric with species A and B (Sukowati et

al., 1999).



DNA-Based method

The malaria vectors in Thailand as well as Southeast Asia are species coplexes

which are morphologically similar. Therefore, there are development molecular

technique for differentiate these sibling species.

The first evidence for An. sundaicus molecular identification was examined by

using one mitochondrial DNA (partial cytochrome oxidase I (COI)) gene and partial

nuclear internal transcript spacer-2 (ITS-2) of 28s rRNA gene (Linton et al., 2001).

The 472 bp and 619 bp for COI and ITS-2 were amplified for molecular study. They

stated that 13 ITS-2 sequences showed a single haplotype and closest similarity to An.

gambiae sequences at 74%. For COI sequences showed higher levels of intraspecific

variation than ITS-2 sequences with 4 haplotypes. They presented 5 polymorphic sites

(3 were parsimony informative sites, 2 were singleton polymorphic sites) and did not

affect protein coding sequence in any cases (Linton et al., 2001).

Consequently, 2 mitochondrial DNA (COI and Cytochrome b: Cyt-b) were

amplified by multiplex PCR and were compared between An. sundaicus populations

from Vietnam, Thailand and Malaysia Borneo (Dusfour et al., 2004b). Two distinct

species were identified as An. sundaicus s.s. on Malaysian Borneo and An. sundaicus

species A on coastal areas of Vietnam and Thailand.

Recently, An. sundaicus species A was formally named as An. epiroticus by

Linton and coworkers (Linton et al., 2005). They used COI, Cyt-b and ITS-2

sequences. They compared specimens of An. sundaicus from Cambodia, peninsular

Malaysia, Thaialnd and Vietnam. According to sequence data, An. sundaicus species

complex was divided into 2 species which were An. sundaicus s.s. on Borneo from

previous study (Linton et al., 2001) and An. sundaicus species A on the continent.

The “epiroticus” in modern Greek is “from the mainland”. Therefore, An. epiroticus

was named for reflect its primary distribution in Southeast Asia (Linton et al., 2005).

Afterward, molecular identification of An. sundaicus cytotype D from India

was examined by Alam and coworkers (Alam et al., 2006). This study confirmed

cytogenetic type D of Nanda and others (Nanda et al., 2004) by using nucleotide

sequences. They studied the differentiation of ITS-2 and Domain-3 (D3) of 28s rRNA

within 4 Islands; Teressa, Nancowry, Car Nicobar and Katchal Islaand. There were no

differences between ITS-2 and D3 of all populations and between fresh and brackish



water of An. sundaicus but different from An. sundaicus species A and An. sundaicus

s.s. They concluded that there was only one member of An. sundaicus complex exists

on Andaman and Nicobar Islands which was An. sundaicus species D.

In the recent year, An. sundaicus from Sumatra and Java Island of Indonesia

was distinguished by using mtDNA (COI and Cyt-b) and was named as An. sundaicus

species E (Dusfour et al., 2007b).

Additionally, Dusfour and coworkers (Dusfour et al., 2007a) developed

multiplex PCR with mtDNA (COI and Cyt-b gene) to identify 3 species complexes of

An. sundaicus and gave distributional information. An. sundaicus s.s. presented in

northern Borneo of Malaysia. An. epiroticus (An. sundaicus species A) occurred in

coastal areas from southern Vietnam, Cambodia, Thailand and southward to

peninsular Malaysia. An. sundaicus species D was located on Nicobar and Andaman

Island of India. An. sundaicus species E was found on Sumatra and Java Island of

Indonesia. The distribution of An. sundaicus complex was shown in Figure 18.



Figure 18 Distribution of the four members of the Sundaicus Complex (Manguin et

al., 2008)



3.7 Malaria detection in mosquito vectors A principal component in malaria vector control is the sensitive detection of

Plasmodium species in the mosquito vectors. The infection status of malaria vectors

are usually estimated by the presence of Plasmodium sporozoites in the salivary glands

(Bass et al., 2008).

There are several methods to determining the Plasmodium parasite in mosquito

vectors. In recent year the numbers of novel methods have been available for malaria

detection in mosquito vectors.

Dissecting Plasmodium-infected mosquitoes

The traditional technique for determining both sporozoites (infective mosquito)

and oocysts (infected mosquito) is dissection of salivary glands and midgut of

mosquito vectors and visualized by light microscopy. However, this method requires

expert microscopist, time consuming, cannot detect malaria species, and sometime

needs large numbers and fresh of specimens (Wilson et al., 1998).

Dissection of Anopheles midgut and salivary grand for determining the oocysts

and sporozoites should be carried out 5-7 days and 8-21 days, respectively after

malaria infection. The days for dissection are variable depending on parasite species

(Table 7) (WHO, 1975).

Table 7 The number of days in each Plasmodium species for oocyst and sporozoite

dissection (WHO, 1975)

Plasmodium Species Days to oocysts Days to sporozoites P. vivax 4-7 8-10 P. ovale 3-4 10-15 P. falciparum 3-7 10-18 P. malariae 3-11 15-21



Immunological method

In the last decade Enzyme-linked immunosorbent assays (ELISA) using

monoclonal antibodies to circumsporozoite (CS) proteins of sporozoites were widely

used to determine sporozoite rates and quantified the sporozoite numbers in Anopheles

mosquitoes (Beier and Koros, 1991).

Three monoclonal antibodies (Mabs) to circumsporozoite (CS) proteins (P.

falciparum, P. vivax-210, and P. vivax-247) were developed to detect the malaria-

infected mosquitoes (Wirtz et al., 1985; Wirtz et al., 1987; Wirtz et al., 1992). The

sensitivity and specificity of the ELISA are based on Mabs used. It can detect CS

proteins in the oocysts, haemolymph, and sporozoites in the salivary glands. This

method cannot use to incriminate Anopheles as malaria vectors since it may detect CS

in other organs not only in salivary glands. In the ELISA, fresh, frozen, or dried

mosquitoes can be used (Beier and Koros, 1991). The ELISA step is shown in the

Figure 19.

Figure 19 The procedure of ELISA for P. falciparum and P. vivax circumsporozoite

proteins detection



Although, this method is widely used and has proven to be robust and cheap,

there are several disadvantages. Firstly, there have been overestimates the infection

rates of positive salivary glands (Bass et al., 2008). Secondly, mosquitoes often stored

for later analysis by either drying on silica gel or keeping in ethanol. In term of drying

on silica gel is not inhibitory to ELISA but the latter approach causes the specimens

are not suitable for ELISA testing. Thirdly, although monoclonal antibodies have

been produced for all four Plasmodium but each assay must be run separately and

make many times for detecting one or two of the species. Finally the ELISA may be

insensitive to very low-level infections (Arez et al., 2000).

Molecular techniques

Polymerase chain reaction (PCR)

The molecular method offers the higher sensitivity and specificity especially,

amplification of DNA by polymerase chain reaction (PCR) technique. Although this

method is more expensive but it is more reliable than microscopy for malaria detection

in mosquito vectors due to the underestimation of microscopy and can identify malaria

parasite into species level (Snounou et al., 1993a; Wilson et al., 1998). Wilson et al

(1998) reported that PCR had 3 times more sensitive than microscopy in the detection

of sporozoites in mosquito salivary glands. This meant that if using microscopy alone,

the sporozoite result may be occur false negative.

DNA sequences of the small subunit ribosomal RNA (ssrRNA) gene have to

specific to each species or shared by all Plasmodium genus, but not found in other

organisms. These differences have been adapted to a nested PCR protocol that can be

used for the detection of any malaria parasite and for the identification of the four

Plasmodium species that infect in both humans and Anopheles mosquitoes (Snounou,

and Singh 2002).

Many primers were designed to species specific regions in the sequences

encoding the small subunit ribosomal RNA (ssrRNA) to detect only P. falciparum

(Tassanakajon et al., 1993) or all four Plasmodium species (Snounou et al., 1993b) in

mosquito vectors. All these primers can be used to amplify the target region. Owing

to they are specific to parasite species, and not found in the genomes of insect hosts

(Snounou and Singh, 2002). The oligonucleotide primers are

5’CGCTACATATGCTAGTTGCCAGAC3’ and 5’CGTGTACCATACATCCTA



CCAAC3’ for P. falciparum detection (Tassanakajon, Boonsaeng, Wilairat, and

Panyim, 1993). The primers for all 4 Plasmodium species are shown in Figure 20.

Figure 20 The ssrRNA gene of Plasmodia. Blank areas: sequences conserved in all

species. Stippled areas: sequences unique to each species. An arrow: the position of

primers used for PCR amplification. A broken line: the fragment amplified (Snounou

et al., 1993b)



Nested polymerase chain reaction (Nested PCR)

Snounou and coworkers (1993) developed nested PCR method to enhance

sensitivity using two rounds of PCR which is now widely used as “gold standard” for

PCR based Plasmodium detection. In the first PCR (nest 1) amplification, Genus-

specific primers (rPLU5 and rPLU6) were used. The PCR products of nest 1

amplification were then used as DNA template for four separate second PCR (nest 2)

amplifications with primers specific for each 4 malaria parasites. The 10

oligonucleotide primers were shown in Figure 21. The Plasmodium infection of

mosquito vectors was detected with this method. They detected malaria parasite in An.

gambiae from Guinea Bissau. They found 12 out of 75 were infected with P.

falciparum and P. malariae (Snounou et al., 1993a).

Although, this is high sensitive but it requires separate PCR reaction for each

species and further detect PCR products by gel electrophoresis then take time

consuming.

Figure 21 The Plasmodium ssrRNA gene used. The species-specific primers were

designed to hybridize to ssrRNA genes present in the Plasmodium genome (Snounou

et al., 1993c)



Consequently, Singh and others (1999) developed nested PCR technique by

initially screened using Plasmodium genus-specific primers in both nest 1 and 2

amplifications and only positive samples will be subjected to species identification by

further nest 2 amplifications. They used Plasmodium genus-specific primers for the

nest 1 and nest 2 amplifications and reused only the nest 1 PCR products of positive

samples for species-specific nest 2 amplifications. They used the genus-specific

primer rPLU1 and rPLU5 instead of rPLU6 since this allowed PCR products of nest 1

amplifications to be used as DNA templates with the genus-specific primers rPLU3

and rPLU4 in nest 2 amplifications and only positive samples will be subjected to

further each species primers (Figure 22) (Singh et al., 1999).

Figure 22 The oligonucleotide primers used in the nested PCR (Singh et al., 1999)



Afterward, Snounou and Singh improved and summarized the protocols of

nested PCR analysis of Plasmodium parasites in the text book “Malaria and methods

and protocols” which slightly changed the primers for detection of P. ovale (Snounou,

and Singh, 2002) as Figure 23.

Figure 23 The oligonucleotide primers used in nested PCR (Snounou, and Singh,

2002)



Semi-nested multiplex PCR

A semi-nested multiplex polymerase chain reaction (SnM-PCR) based on the

amplification of the sequences of the 18S small subunit ribosomal RNA (ssrRNA)

gene (Rubio, 1999a). This technique was simultaneously developed by Rubio et al

(Rubio et al., 1999a; Rubio et al., 1999b; Rubio et al., 2002). Two round of PCR

amplification was carried out. The first PCR amplification with a universal reverse

primer (UNR) and forward prime specific for the genus Plasmodium (PLF) was

performed. The second amplification was carried out with the same Plasmodium

forward primer (PLF) and four specific reverse primers (FAR, VIR, OVR, MAR) for

P. falciparum, P. vivax, P. ovale, and P. malariae, respectively (Table 8). This

method was optimized to detect malaria parasite in wild-caught Anopheles in Bolivia

(Lardeuxa, 2008).

Table 8 Primer names, sequence targets and size of the PCR product (bp) for semi-

nested multiplex PCR (Rubio, 1999a)

Primer

Sequence (5’-3’) Specificity Size of PCR product (bp)

UNR

GACGGTATCTGATCGTCTTC Universal

PLF AGTGTGTATCAATCGAGTTTC Plasmodium

P. falciparum = 787 P. vivax = 783 P. ovale = 794 P. malariae = 821

FAR

AGTTCCCCTAGAATAGTTACA P. falciparum 395

VIR

AGGACTTCCAAGCCGAAGC P. vivax 499

OVR GCATAAGGAATGCAAAGAACAG P. ovale

436

MAR

GCCCTCCAATTGCCTTCTG P. malariae 269



Real time polymerase chain reaction (Real time PCR)

The real-time PCR technique is used for monitor the progress of PCR reaction

in real time and quantify PCR product in the same time. It is based on the detection of

the fluorescence produced by a reporter molecule which increases in PCR reaction

proceeds. It occurs from the accumulation of the PCR product with each cycle of

amplification. These fluorescent reporter molecules include dyes that bind to the

double-stranded DNA (e.g. SYBR® Green) or sequence specific probes (e.g. FRET

Hybridization Probes, Molecular Beacons or TaqMan® Probes). It can initiate with

small amounts of nucleic acid and quantify the end product accurately. Additionally,

this method no needs to detect PCR product after PCR reaction which saves both the

resources and the time. Real-time PCR assays are now easy to perform, have high

sensitivity and specificity (PREMIER Biosoft International, 2009).

This technique uses a fluorescent detection system to measure the amount of

PCR product at each and every cycle. The product yield is plotted against the cycle

number. The result is an amplification plot, a curve that shows the accumulation of all

PCR reaction. Tracking the entire reaction allows quantification of PCR product

during the exponential phase of the PCR product at every cycle. Quantification of

amplification product in the exponential phase, as opposed to the endpoint of PCR

reaction, underpins the precious of the real-time technique (Higuchi, Dollinger, Walsh,

and Griffith, 1992). The fluorescence emitted by the reporter molecule manifolds as

the PCR product accumulates with each cycle of amplification. There are two

detection molecules in real-time PCR techniques which are non- specific detection

using DNA binding dyes (i.e. SYBR® Green) and specific detection using target

specific probes (i.e. FRET Hybridization Probes, TaqMan® Probes, Molecular

Beacons, Scorpion® Primers).

It has been developed and studied for detection of the malaria parasite in

human by many researchers (Schoone et al., 2000; Rougemont et al., 2004; Farcas et

al., 2004; Perandin et al., 2004; Swan et al., 2005; Vo et al., 2007).

In recent year, Bass et al used this technique to detect Plasmodium in Anopheles

mosquitoes and found that this method was the most sensitive when compare with

nested PCR and single PCR (Bass et al., 2008).



Loop-mediated isothermal amplification (LAMP)

LAMP has been developed by Notomi et al (2000). This is a novel DNA

amplification method with the distinguished feature that the reaction can run under

isothermal conditions (Notomi et al., 2000). It requires only one enzyme, Bst DNA

polymerase, which can synthesize a new strand of DNA while simultaneously

displacing the former complementary strand thereby enabling DNA amplification at a

single temperature. Four primers (FIP, BIP, F3, and B3), F3 and B3 for the formation

of a stem-loop structure while FIP and BIP designed complementary to the inner

sequence of the stem-loop structure, are employed for amplification of the target

sequence. It provides high specific. The detection of amplified DNA is easy since

the amplification from stem-loop structures leads to the accumulation of large amounts

of products (Aonuma et al., 2008).

Recently, this technique was applied for detection the rodent malaria parasite

(P. berghei) in An. stephensi. The primer set was shown in Figure 24. They found

that it can detect both oocysts and sporozoites from whole mosquito bodies. It can

identify as few as only one oocyst in midgut of mosquito. They concluded that it can

be use as malaria detection in mosquito vectors since its reliability (Aonuma et al.,

2008).



Figure 24 LAMP primer set against P. berghei. A: Partial sequence of P. berghei and

location of primers, FIP (F1c-F2), BIP (B1c-B2), F3, and B3. Arrows: sequences of

primers and directions of extensions. Numbers on the left indicate the nucleotide

position. B: Sequence of primers for LAMP reaction (Aonuma et al., 2008)



3.8 Malaria detection in An. sundaicus s.l.

Malaria transmission and vector capacity information of An. sundaicus has

been poorly known in many areas (Dusfour et al., 2004a).

In Thailand, It was first reported in 1966 with null sporozoite positive

detection by means of dissection method (Gould et al., 1966). This was only one

report for malaria infection of An. sundaicus in Thailand.

In southern Vietnam, there was no sporozoite positive whereas the biting rate

was high with 12.78 bites/man/hour (Coosemans et al., 1998). Whereas, in Mekong

delta, Vietnam presented high density of An. sundaicus (up to 190 bites/man/night) but

low malaria cases reported (Trung et al., 2004). They extensively surveyed of 11,002

An. sundaicus and specimens were no infected with Plasmodium by ELISA method

(Trung et al., 2004).

In Malaysia, in the year 1971, An. sundaicus was found in high number but it

was not the cause of malaria transmission in coastal Malaysia (Huehne, 1971).

Afterward, low rate of natural Plasmodium infection of An. sundaicus was reported

which was 0.04% from 13,493 female dissection (Sandosham and Thomas, 1983).

Although, An. sundaicus is the important malaria vector and still high endemic

in Car Nicobar island of India but Kumari and Sharma (1994) found that there were no

sporozoite or oocyst positive detection from 863 female dissection (Kumari and

Sharma, 1994).

The malaria cases were very low in Central Java of Indonesia but An.

sundaicus occurred in high amount (Sundaraman et al., 1957).

The extremely increase of mosquito density cause the reducing of malaria

transmission due to development of Plasmodium into sporozoites cannot be

completed. This can take place wherever that An. sundaicus occurs in high density

(Coosemans et al., 1992).

Moreover, there was little information about sporozoite rate in An. sundaicus.

However, Dousfour et al (2004) summarized data for sporozoite detection of An.

sundaicus in various sites as shown in Table 9.



Table 9 Sporozoite rates observed from An. sundaicus in various locations

(Dusfour et al., 2004)

Country Year Sporozoite rate (%) Bangladesh 1952 4.3

Cambodia 1977 0.4

India (Calcutta) 1936 3.6

India 1948 2.7

Indonesia (Sulawesi) 1953 0.04

Indonesia (Sulawesi) 1973 0.07

Indonesia (Java) 1952-56 0.04–0.3

Indonesia (Flores) 1991 4.2

Malaysia (Sabah Province) 1957 1.65

Thailand 1966 0

Vietnam (Go Cong Province) 1961 2.9

Vietnam (Go Cong Province) 1971 4.4

Vietnam (Ho Chi Minh Province) 1968 1.03

Vietnam (Mekong Delta) 1968 0.18

Vietnam (Tra Vinh Province) 1975 2.7

Vietnam (Bac Lieu Province) 1998 0



3.9 Malaria vector control in Thailand and insecticide susceptibility of

An. sundaicus s.l.

The main malaria vector control in Thailand has been mainly based on

insecticide such as Indoor Residual Spraying (IRS) and Insecticide Treated Nets (ITN)

(Van Bortel et al., 2008). Other methods are environmental management, biological

control, larviciding, personal protection, etc. IRS is useful to control vectors which

are endophilic. ITN are appropriate for areas where vectors occur in high biting rate at

bedtime. In both control methods, the vectors must be susceptible to the insecticide

used (Mabaso et al., 2004).

Insecticides are classified as four main classes according to their chemical

structures (Marquardt, 2005).

Organochlorines

Most of these compounds are inhibitors of the function of nervous

system (e.g. DDT, BHC, dieldrin). These insecticides are long lasting in environment,

wildlife, and human.

Organophosphates

This class acts by binding the acetylcholinesterase at nerve junction.

This enzyme cannot remove acetylcholine from nerve junction and nerves continue

uncontrolled manner leading to paralysis and death of insect. These insecticides are

temephos, chlorpyrifos, malathion, fenitrothion, and pirimiphos methyl etc.

Carbamates

This group is identical mode of action with organophosphates (e.g.

carbaryl)

Pyrethroids

These insecticides active components from pyrethrum flower called

“pyrethrins”. They are identical mode of action with DDT but safety (knock down

agent) to use in aerosol since lack of persistence and can be use in extremely low

quantities. These insecticides are permethrin, lambda-cyhalothrin, and deltamrthrin.

Other insecticides are insect growth regulators (IGR), chitin synthesis

inhibitors, neonicotinoids and chloronicotinyls, and bacteria (Marquardt, 2005).

After long time using insecticides, some vectors have developed resistant to

these insecticides result in problematic for control them. Hence, resistant status of



insect vectors is crucial for vector control strategies. It is basic requirements in

selecting the appropriate insecticides for vector borne disease control (WHO, 1998b).

Before 1985, no insecticide resistance in malaria vector was reported in

Thailand. Between 1990 and 1997, Anopheles dirus s.l. and An. minimus s.l. were

resistant to DDT and An. minimus s.l. was resistant to permethrin (0.25%) in a

population from northern Thailand (Chareonviriyaphpap et al., 1999).

One of the most important An. sundaicus control strategy was DDT spray

inside houses (Meek, 1995). Consequence, it rapidly developed resistance to DDT

(Table 10) in various locations. DDT was used for malaria control program in

Thailand since 1949. However, the insecticide resistance status of An. sundaicus was

summarized by Dusfour et al., 2004a. Other insecticides were used in areas where

DDT resistance occurred, but few records report whether An. sundaicus has developed

resistance in Thailand.

Table 10 Insecticide resistance status of An. sundaicus in different populations

(Dusfour et al., 2004a)

Date Country Resistance status

1954

1956

1962

1973

1976

1978

1979

1989

1985

1985

1987

1994

1997

2000

Indonesia

Indonesia (Java)

Malaysia (Sabah)

Indonesia (Sulawesi)

Indonesia

India

Singapore

India (Kamorta Island)

Vietnam

Indonesia (Java)

Indonesia (central Java)

Vietnam

India

Indonesia

DDT resistant

DDT resistant

Dieldrin resistant, DDT susceptible

DDT susceptible

DDT and dieldrin resistant

DDT resistant

Susceptible to 3 organochlorines,

5 organophosphates, and 1 pyrethroid

DDT susceptible, temephos Susceptible

DDT resistant, others susceptible

DDT susceptible

DDT resistant

DDT resistant, others susceptible

DDT susceptible

DDT resistant



According to Tsunami disaster along Thailand’s Andaman coast in December

26th, 2004, the environmental change especially many inland fresh water became

brackish from the sea water. This phenomenon caused An. sundaicus rapidly

increased in the affected areas. On 2006, insecticide susceptibility test of An.

epiroticus from Tsunami-affected areas in Thailand were carried out (Komalamisra et

al., 2006). They reported that An. epiroticus was susceptible to all insecticides which

were 5% malathion, 0.75% permethrin, 0.05% deltamethrin, and 4% DDT with LT50

at 44.7, 10.4, 9.7, and 26.3 minutes, respectively and revealed 100% mortality at 24

hour after exposure (Komalamisra et al., 2006).

Recently, Bortel et al (2008) reported An. epiroticus populations were

susceptible to DDT and permethrin (0.75%) in Thailand whereas, in Cambodia, they

were resistant to etofenprox (0.5%) and cyflutrin (0.15%) and DDT resistance was

observed only in Vietnam. The KT50 for pyrethroids (permethrin, alpha-cypermethrin,

and lambda-cyhalothrin) varied between 7 and 28 minutes but no correlation with

mortality and the KT50 for DDT ranged from 21 to 44 minutes within An. epiroticus

from these three countries (Van Bortel et al., 2008).

At the study site, the control of An. sundaicus and other mosquitoes,

insecticides have been used (Ban Pak Nam, Rayong Province) which are 10%

permethrin, 2% bifenthrin for adulticide and temephos (abate) for larvicide.

The biological control of An. sundaicus larvae with bacteria in Thailand

(Samet Island, Rayong Province) was also estimated by (Chowanadisai et al., 1989).

They found that Bacillus sphaericus 2362 at 2 ml/m2 in a flowable concentrate

formula with 2x1010 spores/gram can reduce 90% larval density for at least 8 days

(Chowanadisai et al., 1989).


Suchada Sumruayphol Materials and Methods / 52

CHAPTER IV

MATERIALS AND METHODS

Study site

This study was carried out at Ban Pak Nam, Mueang Rayong District, Rayong

Province. Rayong Province is located in eastern part of Thailand. Ban Pak Nam is

located in the southern part of Muang Rayong District, Rayong Province, at which

nearby the Gulf of Thailand coast. It has a population of 4,368, with a population

density of 436.8 persons per km2. It is crowded and low hygienic community. This

community has legal and illegal labor migrants from Myanmar, Cambodia, and Lao

PDR to this area for urbanization and fishery-industrial activities. The area presents

fishery activity as well as fish sauce production. There are a lot of discarded fish

sauce ponds, canals in this area. According to the preliminary survey there were An.

sundaicus in this area. Malaria was first reported in the area in 2002. Afterward, its

outbreak was considered in 2005 with 14 cases. Consequently, malaria cases were

reported every year until now. The highest cases were in 2007 with 40 cases

consisting 26 Thai cases and 14 non-Thai cases (Personal communication).

A total of 85 malaria cases were reported in Pak-Nam, Rayong by VBDC 3.3

Rayong since 2002 through 2008 which were 61 Thai cases (71.8%) and 24 non-Thai

cases (28.2%). P. falciparum accounted for 3 cases (4%) and P. vivax for 82 cases

(96%). All P. falciparum cases were non-Thai. Thai patients were only P. vivax

infection. The data were shown in Table 11.



Table 11 Thai and non-Thai malaria cases in Ban Pak Nam from 2002 through 2008

Year Thai malaria cases Non-Thai malaria cases

No. of cases P. vivax P. falciparum No. of cases P. vivax P. falciparum

2002 1 1 0 0 0 0

2003 11 11 0 4 3 1

2004 2 2 0 4 2 2

2005 14 14 0 0 0 0

2006 6 6 0 2 2 0

2007 26 26 0 14 14 0

2008 1 1 0 0 0 0

Total 61 61 0 24 21 3

Malaria was first reported in the area at 2002. Afterward, its outbreak was

considered in 2005 with 14 cases. Consequent, malaria cases were reported every year

until now. The highest cases were in 2007 with 40 cases consisting 26 Thai cases and

14 not Thai cases (Personal communication).

4.1 Adult mosquito collection

This study was reviewed and approved by the Human Ethics Committee and

Animal Care and Use Committee from the Faculty of Tropical Medicine, Mahidol

University. The ethical approval number of human ethics was MUTM 2009-005-01

and FTM-ACUC 2009-001 for animal ethics approval. Mosquito collection was

conducted for study the populatin density, biting behavior, and seasonal abundance of

An. sundaicus including the composition of mosquito in the areas in Ban Pak Nam,

Muang district, Rayong Province. Mosquitoes were collected monthly from May

2007 to April 2008. Human landing catch method with two consecutive nights and 3

points at outdoor was performed from 1800-2400 hour. Collections were made hourly

for 45 minutes and 15 minutes for rest period. All mosquitoes were collected. The

time of collection were also recorded. The living adult specimens from human landing

catch were confirmed species by using microscopic examination of morphological

characters by using “Illustrated keys to the mosquitoes of Thailand. IV. Anopheles”

(Rattanarithikul et al., 2006) which was shown in Appendix A. The biology of

collected anopheles was studied by set up isofemale colonies in laboratory.



Meteorological data collections of monthly temperature, relative humidity and rainfall

were arranged by local district weather station.

4.2 Larval collections

Mosquito larval collection was carried out by dipping method for studying

breeding habitats of An. sundaicus monthly from May 2007 through April 2008. The

larval density and abundance of An. sundaicus breeding places were carried out. A

total of 38 cement ponds within 4 houses were selected for larval collection. All 4

sites were selected nearby the patient houses which were 12° 39' 29.4" N;101° 15'

59.5"E, 12° 39' 24.7" N;101° 16' 22.8"E, 12° 39' 28.2" N;101° 16' 05.5"E and 12° 39'

25.0" N;101° 16' 23.1"E. Water quality was estimated as well by HACH portable

equipment (Figure 25). The physical and chemical characters of An. sundaicus

breeding places were examined. Total dissolved solid (TDS), and temperature were

measured for physical character whereas, pH, conductivity, salinity, and dissolved

oxygen (DO) were checked for chemical character. The collected An. sundaicus

larvae were reared under laboratory conditions until emerge into adult for

morphological identification, molecular identification, and biological study in

laboratory.



Figure 25 HACH portable equipment for checking the water quality



4.3 Parity rate detection of An. sundaicus s.l. in study areas

Parity is used to determine the age structure of a population, and it can also be

used to ascertain the net reproductivity of a colony (Githeko et al., 1993). Parous

mosquitoes are those that have taken a blood meal and oviposited at least once.

Nulliparous mosquitoes have never oviposited. The simplest technique for

determining parity is to examine the tracheoles within the ovaries (Kardos and

Bellamy 1961). The female ovaries were dissected by mean of Detinova (1962). As

the ovaries expand after the primary blood meal, the tracheoles that are associated with

them are permanently distended (Hoc and Charlwood 1990). Therefore, in nulliparous

females the tracheoles are tightly wound coils called skeins. Parous females will have

tracheoles that have distended. In younger females there may be a mixture of both

skeins and extended tracheoles; if any distended tracheoles are present, it can assume

that the female is parous and has fed and laid eggs at least one time.

Every An. sundaicus female were dissected for ovaries in the drop of distilled

water under a stereo microscope by gently pulling the last two abdominal segments

with the tip needle while securing the mosquito thorax with another tip needle. After

allowing the ovaries to dry out, examine ovaries under a compound microscope. The

parity rate can be used to calculate the daily survival rate and the expectation of life of

An. sundaicus populations in study area, as described by Service (1993). The survival

rate is the most important factor of the vector capacity. The parity rate was shown by

percentage.

Materials (Figure 26)

Microscope slides

Distilled water for dissecting solution

Forceps

Pins mounted on small wooden sticks

Compound Microscope

Procedure

1. Gently anesthetize adult females by ether.

2. Place a drop of distilled water on a clean microscope slide.

3. Under the stereoscope, gently grasp female by the thorax with pin, and place ventral

side up with her abdomen in the water.



4. While viewing the specimen under the stereoscope, take a tip pin and gently remove

the 7th and 8th abdominal segments of the female by grasping them and pulling away

slowly.

5. Locate the ovaries; they will appear as a pair of white oval objects attached to the

removed segments (Figure 27). Dissect away the accessory tissues and isolate the

ovaries.

6. Transfer ovaries to a new slide and allow to air dry.

7. View under a compound microscope. Locate the tracheoles and determine if the

specimen is nulliparous (Figure 28) or parous (Figures 29).

Figure 26 Materials for ovary dissection of An. sundaicus



Figure 27 Dissection of female ovaries under stereoscope (the ovaries were circled)

Figure 28 Nulliparous female showed a tightly coiled tracheole called a “skein”



Figures 29 Parous female showed uncoiled tracheole



4.4 Detection of malaria parasite in An. sundaicus s.l.

After morphological identification, only female of An. sundaicus were divided

into two parts which were head-thorax portion and abdomen portion (after ovary

dissection for examine the parity rate). The DNA extraction procedure was shown in

Figure 30. Both parts of all parous An. sundaicus were extracted DNA by QIAamp®

DNA Mini Kit (QIAGEN, Germany) (Figure 31) following the manufacturer's

instructions with slightly modification. The extracted DNA was subjected to Nested

Polymerase Chain Reaction (Nested-PCR) (Snounou and Singh, 2002) for detecting

genus plasmodium. The genus positive samples were further examined plasmodium

species by real time PCR (Swan et al., 2005).

DNA extraction for detection of malaria parasite in An. sundaicus s.l.

Ninety µl of tissue lysis buffer (ATL) was added to each 1.5 ml

microcentrifuge tubes containing An. sundaicus samples, after homogenized, 10 µl

Proteinase K were added and mixed by vortex (Figure 31). The samples were

incubated at 56 °C overnight by water bath (Figure 33). Five µl of Rnase A (50 µg/µl)

was added and incubated at 40 °C for 30 minutes. One hundred µl of lysis buffer (AL)

was added and briefly mix by vortex. The samples were incubated at 70 °C for 10

minutes by heat block (Figure 34). After briefly centrifuge, 100 µl of absolute ethanol

was added into samples then vortex and centrifuge. The solution was carefully

pipetted into QIAamp spin column. The cap was closed and centrifuged for 1 min at

8,000 rpm. The column was placed into a new 2 ml collection tube, and discarded the

tube containing the filtrate. Five hundreds µl of washing buffer (AW1) was added

then centrifuge for 1 min at 8,000 rpm and changed to AW2, then centrifuge 14,000

rpm for 3 minutes. DNA was eluted by adding 50 µl of the elution buffer (AE).



Figure 30 Procedure for DNA extraction



Figure 31 QIAamp® DNA Mini Kit (QIAGEN, Germany)



Figure 32 Vortex mixer (Vortex Genie 2)

Figure 33 Water bath (Optima)



Figure 34 Heat block (Labnet) for sample heating

Figure 35 Microcentrifuge (Spectrafuge; Labnet)



Nested Polymerase Chain Reaction (Nested-PCR) for detecting genus

malaria parasite (Snounou and Singh, 2002)

Two rounds of DNA amplification were carried out with DNA extract of An

sundaicus both head-thorax and abdomen portions as described above. In the first

amplification (Nest 1), a pair of oligonucleotide primers (rPLU1 and rPLU5) was used

to amplify a 1.6–1.7 kb fragment of the plasmodium ssrRNA genes. The product of

this first amplification was used as the DNA template for a second amplification (Nest

2), by using rPLU3 and rPLU4 primers to amplify genus-specific nucleotide

approximately 235 bp. These primers were shown in Figure 36. All PCR reactions

were performed by the TGradient Thermocycler (Biometra, Germany) as Figure 37.

The final volume of each PCR reaction was 20 µl (18µl master mix and 2 µl DNA

template) with slightly modification. The positive control samples were An. dirus

mosquito samples and human blood samples.

Table 12 Mixture set up for nested PCR reaction

PCR Reagents Stock

concentration Final concentration

Quantify for 20 µl of

reaction mixture variable

Distilled water - - 12.95 µl

PCR buffer+KCl 10X 1X 2 µl

dNTPmix 10 mM 0.125 mM (125 µM ) 0.25 µl

Primer rPLU1/ rPLU3 10 µM 250 nM (0.25 µM) 0.5 µl

Primer rPLU5/ rPLU4 10 µM 250 nM (0.25 µM) 0.5 µl

MgCl2 25 mM 2 mM 1.6 µl

Taq DNA Polymerase 5 U/µl 1 U/µl 0.2 µl

Total volume - - 18 µl

The cycle of PCR amplification reactions are as follow;

Step 1: 95 ˚C for 5 minutes.

Step 2: 94 ˚C for 1 minute.

Step 3: 58 ˚C and 64 ˚C for Nest 1 and Nest 2, respectively for 1 minute


Step 5: Repeat steps 2–4 for a total of 25 cycles (Nest 1) or 30 cycles (Nest 2)

Step 6: The reaction is completed by reducing the temperature to 4 ˚C



The PCR products were examined by 2% agarose gel electrophoresis. One

gram of SeaKem® LE agarose (USA) was melted in 50 ml of 1XTBE buffer. The

melted agarose mixture should be allowed to cool to approximately 55 ˚C before

pouring into the gel cast, and then polymerized for at least 30 minutes before removing

the combs (Figure 38). Five µl of each PCR product was loaded into the well

simultaneously with 1 µl of 6X DNA loading dye (Fermentas, Canada) as Figure 39.

Then electrophoresis was carried out for one hour at 100 Volt (Figure 40), following

staining with ethidium bromide about 20 minutes and destaining by tab water around 1

hour. DNA was visualized and photographed by Gel Documentation model G: Box

HR (Syngene, UK) using GeneSnap program (Figure 41). The positive PCR products

presented approximately 235 bp band. These positive samples were further

determined the plasmodium species by Real-time PCR (Swan et al., 2005).

Figure 36 Genus specific primer used within 2 rounds of PCR: rPLU1, rPLU5 and

rPLU3 and rPLU4 (Snounou and Singh, 2002)



Figure 37 TGradient Thermocycler (Biometra, Germany) for doing PCR reaction

Figure 38 2% agarose gel setting



Figure 39 Step of loading PCR product into the well

Figure 40 Gel electrophoresis of PCR product



Figure 41 Gel Documentation for visualization and photograph the DNA



Real-time PCR for detecting species malaria parasite (Swan et al., 2005)

The genus positive samples were determined the plasmodium species by Real-

time PCR (Swan et al., 2005). A genus specific primer set corresponding to the 18S

rRNA gene and FRET Hybridization probes for P. falciparum over a region

containing base pair mismatches allowing for differentiation of the other Plasmodium

species by use of melting curve analysis as reported by Swan and co-workers (Swan et

al., 2005). The primers and hybridization probes and melting temperatures were listed

in Table 13.

Table 13 Characteristics of Plasmodium primers and hybridization probes set* (Swan

et al., 2005)

Primers

and

Probes

Nucleotide Sequence (5’-3’)

Parasite

targeted

Melting

temperature

(°C)

PF1

PF2

CATTYGTATTCAGATGTC

TTCTTTTAACTTTCTCGC

Plasmodium

Species

PF3

PF4

GATACCGTCGTAATCTTAACCTAACcTAT-FL

LC RED640 GACTAGGTGTTGGATGAAAGTG-PO

LC RED640 GACTAGGTGTTGGATGAtAGaG-PO

LC RED640 GACTAGGctTGTTGGATGAAAGTG-PO

LC RED640 GACTAGGTtTTGGATGAAAGTG-PO

P. falciparum

P. malariae

P. vivax

P. ovale

60.0 ± 2.0

57.0 ± 1.0

51.8 to 55.5

49.5 ± 1.0

Y = C/T * Lower case letters indicated basepair mismatches in LC RED640 hybridization probe for detection of P. malariae, P. vivax and P. ovale respectively, that result in the appropriate temperature shift when hybridization with LC RED640 probe for P. falciparum

All PCR reactions were performed using the LightCycler FastStart DNA

Master Hybridization Probes kit (Roche Applied Science, Germany). The final

volume of each LightCycler reaction was 20 µl (15 µl PCR master mix and 5 µl

mosquito sample). The content of each LightCycler reaction was shown in Table 13.



Table 14 Master mix content for real-time PCR detection of human malaria parasite

(Swan et al., 2005) in An. sundaicus (for 10 reactions)

Ingredient Stock Mix Volume (µl)

Water

MgCl2

FS DNA MHP

Primers-Primer 1

Primers-Primer 2

Probe (Fluor)-Probe 1

Probe (RED640)- Probe 2

25 mM

10 X

25 µM

50 µM

20 µM

20 µM

4 mM

1 X

0.5 µM

1 µM

0.2 µM

0.4 µM

84

32

20

4

4

2

4.0

Total volume 150

The amplification and detection were carried out using the LightCycler System

(Figure 42) contained 4 cycle programs: Initial heating, PCR amplification, mealting

curve analysis and cooling of the instrument. The protocol was shown in Table 14.

Table 15 Experiment condition for detection malaria parasite using hybridization

probes on the LightCycler®

Step Type Temp°C Time

(sec)

Cycles Ramp time Acquisition

Initial Heating none 95 600 1 - no

PCR

Amplification

Quantification

95

55

72

10

15

15

45

20°C/sec

20°C/sec

20°C/sec

no

single

no

Melting Curve Melting Curve 95

59

45

85

0

20

20

0

1

0.2°C/sec

0.2°C/sec

no

no

no

continues

Cool Down none 40 10 1 no

Fluorescence was monitored during every PCR cycle at the annealing step and

during the post-PCR temperature.



Figure 42 LightCycler instrument for Real time PCR



4.5 Laboratory biology of An. sundaicus s.l.

Considering the importance of An. sundaicus as an efficient malaria vector,

colonization was attempted in laboratory. An. sundaicus can breed in wide range of

salinity hence it was reared in both fresh and brackish water. For establishing a

laboratory colony of An. sundaicus, wild caught females and immature were collected

from Ban Pak Nam, Muang Rayong district, Rayong Province. After morphological

identification of adult An. sundaicus, the mosquitoes were maintained by using the

standard rearing method in the insectary. Gravid females were held in a 30x30x30 cm

mosquito cage and kept in the insectary maintained at 28 ±1°C, relative humidity

between 70-80% and artificial light at 12 hours a day. Adults were offered 5%

multivitamin syrup and 5% sugar solution soaked in cotton pads in each bottle daily as

a source of energy (Figure 43). Eggs were collected from mosquitoes in an ovitrap by

placing a plastic bowl containing water and lined with filter paper. Eggs were held in

plastic bowls for hatching. Eggs hatched into larvae within 3-5 days, L1 larvae were

transferred to plastic tray (20x30x6 cm) for rearing (Figure 44). The larvae were fed

on fish meal powder and water at a ratio of 1:1. Water from the culture tray was

changed carefully every alternate day until pupation. The pupae were separated from

the larvae daily and placed in plastic bowls half filled with water. These plastic bowls

with pupae were placed in mosquito cage each day for emergence. Adults, after

emergence were offered 5% multivitamin syrup and 5% sugar solution soaked in

cotton pads in each bottle daily as a source of energy. The adults with 5-7 day old

were separated for artificial mating in each paper cup. 5% multivitamin syrup was

offered to male mosquitoes prior to artificial mating. After starveling the female

mosquitoes for 2-3 hours, they were offered hamster blood for egg development.

Then, artificial mating was conducted with male and female mosquitoes. After

artificial mating 2-3 days, female mosquitoes were transferred into the cage with

plastic bowl for egg oviposition. The developmental time was recorded.



Figure 43 Multivitamin syrup and sugar solution offered daily for mosquito adults

Figure44 Rearing larvae in plastic tray



Materials and procedure for artificial mating of Anopheles mosquito

Materials (Figure 45)

Pins mounted on small wooden sticks

Ethyl ether

Mosquito tubes

Glass container with lid

Cotton pads

Aspirator

Procedure

The adults with 5-7 day old were used for artificial mating.

1. After hamster blood fed female mosquitoes, gently aspirated approximately 5-10

blood fed females into an anesthetizing container (Figure 46).

2. Male mosquitoes were gently pierced the mid of the thorax with the pin (Figures 47)

3. Place females into the anesthetizing tube and leave it for 6-10 seconds.

4. Remove them from the tube.

5. Gently disperse the females onto a piece of paper and position ventral side up. Take

a mounted male, and then gently stroke the abdomen of the male over the female’s

abdomen to stimulate the claspers to open until the male clasps the female. If mated

successfully, they will remain attached for several seconds (Figure 48).

6. Place female together into a new cage to allow female to recover from the

anesthesia.

7. Ensure that the females are recovering from the anesthesia by gently blowing into

the recovery cage.



Figure 45 Materials for artificial mating of Anopheles mosquitoes

Figure 46 Blood fed female mosquitoes for artificial mating



Figure 47 Male prepared for artificial mating

Figure 48 Successful mating male was attached to the blood fed anesthetized female



4.6 Polymerase chain reaction for An. sundaicus species complex identification

After morphological identification of 30 An. sundaicus adult female from field

collection, some of the field collection and laboratory rearing were used to PCR

amplification for molecular identification. Adult female mosquitoes were extracted

genomic DNA and then amplified 3 genes, cytochrome oxidase I (COI), internal

transcribed spacer 2 (ITS2), and domain-3 (D3) of 28S rRNA, by PCR for

identification. The PCR products were done by 1.5% gel electrophoresis.

DNA extraction for An. sundaicus species complex identification

Genomic DNA was extracted from individual An. sundaicus using QIAamp

DNA kit and following manufacturer’s instructions (QIAGEN, GmbH, Germany).

180 µl tissue lysis buffer (ATL) was added to each 1.5 microcentrifuge tubes

containing An. sundaicus samples, after homogenized, 20 µl Proteinase K were added

and mixed by vortex. The samples were incubated at 56 °C overnight. Ten µl of

Rnase A was added and incubated at 40 °C for 30 minutes. Two hundred µl of lysis

buffer (AL) was added and briefly mix by vortex. The samples were incubated at 70

°C for 10 minutes. After briefly centrifuge, 200 µl of absolute ethanol was added into

samples then vortex and centrifuge. The only solution was carefully pipetted into

QIAamp spin column by pipet. The cap was closed and centrifuged for 1 min at 8,000

rpm. The column was placed into a new 2 ml collection tube, and discarded the tube

containing the filtrate. Five hundreds µl of washing buffer (AW1) was added to the

column tube and centrifuge for 1 min at 8,000 rpm (wash twice). Consequently,

centrifuge 14000 rpm for 3 minutes and discard washing buffer. The column tube was

transferred and labeled into a new 1.5 ml microcentrifuge tube and pipetted 150 µl of

Elution Buffer (AE) directly on to the membrane. The samples were incubation at

room temperature for 3 minutes and centrifuged 8000 rpm for 1 minute to elute the

DNA.



PCR amplification for identify An. sundaicus species complex

PCR amplification was carried out with DNA extract of An. sundaicus for

molecular identification using three genes oligonucleotide primers. These primers

were shown in Table 15 (Alam, Das, Ansari, and Sharma, 2006; Dusfour et al., 2004).

All PCR reactions were performed by the TGradient Thermocycler (Biometra,

Germany). The final volume of each PCR reaction was 50 µl (45µl master mix and 5

µl DNA template). The mixture set up for PCR reaction was displayed in the Table

16.

Table 16 Oligonucleotide primers of 3 nucleotide sequences to PCR amplification of

An. sundaicus

Primer names Primer sequences

COI-J-1718 (Forward primer)

COI-N-2191 (Reverse primer)

GGA GGA TTT GGA AAT TGA TTA GTT CC

CCC GGT AAA ATT AAA ATA TAA ACT TC

ITS2For (Forward primer)

ITS2Rev (Reverse primer)

ATC ACT CGG CTC GTG GAT CGA TG

GCT TAA ATT TAG GGG TAG TCA C

D3aF (Forward primer)

D3bR (Reverse primer)

GAC CCG TCT TGA AAC ACG GA

TCG GAA GGA ACC AGC TAC TA

Table 17 Mixture set up for PCR reaction

PCR Reagents Stock

concentration Final concentration

Quantify for 50 µl of reaction

mixture variable

Distilled water - - 33.5 µl

PCR buffer+KCl 10X 1X 5 µl

dNTPmix 10 mM 0.2 mM (125 µM ) 1 µl

Forward primer 1 µg/µl 100 ng 1 µl

Reverse primer 1 µg/µl 100 ng 1 µl

MgCl2 25 mM 1.5 mM 3 µl

Taq DNA

Polymerase 5 U/µl 2.5 U/µl 0.5 µl

Total volume - - 45 µl



The cycle of COI amplification reactions were as follow;


Step 2: 95 ˚C for 30 seconds.



Step 5: Repeat steps 2–4 for a total of 34 cycles



The cycle of ITS2 amplification reactions were as follow;



Step 3: 55.5 ˚C for 30 seconds.





The cycle of D3 amplification reactions were as follow;








The PCR products were examined by 1.5% gel electrophoresis. One point one

hundred and twenty five grams of SeaKem® LE agarose (USA) gel was prepared in 75

ml of 1XTBE buffer. The melted agarose mixture should be allowed to cool before

pouring into the gel cast, and then allowed to set for at least 30 minutes before

removing the combs. Five µl of each PCR product was loaded into the each well



simultaneously with 1 µl of 6X DNA loading dye (Fermentas), and a marker. Then

electrophoresis was carried out for one hour at 100 Volt, following staining by

ethidium bromide for 20 minutes and de-staining by tap water for 1 hour. DNA was

visualized and photographed by Gel Doc (G: Box HR) using GeneSnap program.

The PCR products were sequenced by using service at MWG AG Biotech,

Germany and Sequencing unit of Research Centre Faculty of Medicine, Ramathibodi

Hospital. The number of samples for sequencing the COI partial mtDNA, ITS2, and

D3 genes was 10, 6, and 6 samples, respectively. These data were used to assess

evolutionary relationship. Consequently, the sequenced nucleotides were analyzed

and aligned with other An. sundaicus nucleotide sequences from other areas with

CLUSTAL X (Thompson et al., 1997). Phylogenic relationship, genetic distances and

trees were computed by MEGA4 (Tamura, Dudley, Nei, and Kumar, 2007). Average

number of substitutions per site was calculated using DnaSP 4.20.2 (Rozas et al.,

2003).

4.7 Insecticide susceptibility test

Adult bioassay test

This study was carried out to assess the susceptibility of An. sundaicus to the

diagnostic dose of 0.75% permethrin, 5% malathion and 0.05% deltamethrin.

Bioassays should be carried out preferably with 3-day old sugar fed laboratory strain

or 3-day old sugar fed F1 female progeny of field-collected vectors from the study

area. Exposures were made to the candidate insecticide using the WHO test kit

(WHO, 1981) as Figure 49. One hundred adult female An. sundaicus (20-25

adults/replicate) were used for test and control. The diagnostic dose of candidate

insecticides was filled in the test kit. Consequently, release the 20-25 adult An.

sundaicus females in to the test kit. Afterward, expose them to the insecticide in the

kit for one hour in the same time check the number of knockdown mosquitoes every

five minutes. Transfer the mosquitoes into the clean holding tube after one hour

exposure. Percent mortality was determined after 24 hours of holding period from the

total number of alive and dead mosquitoes in the replicates. When control mortality

was between 5% and 20%, the average observed mortality was corrected by Abbott’s

formula:



% test mortality - % control mortality x 100 100 - % control mortality

Susceptibility status of the insect species was categorized as per the WHO criteria: 98

to 100% mortality indicates susceptible, 81 to 97% mortality suggests the possibility

of resistance that needs to be confirmed, less than 80% mortality suggests resistant.

The results of this test indicated the susceptibility status of the An. sundaicus species

to the candidate insecticides. Probit analysis (Finney, 1971) was used to analyze the

50% lethal time (LT50) and the 95% lethal time (LT95) of 0.75% permethrin and 5%

malathion and 0.05% deltamethrin susceptibility to An. sundaicus mosquito.

Figure 49 WHO test kit



Larval bioassay test

The bioassay test was used to evaluate the susceptibility of An. sundaicus

larvae to temephos. The WHO standard method was used for bioassay test (WHO,

1996). One milliliter of the serial dilution concentration of temephos was added in test

cup and added water to the total volume of 250 ml. Twenty five of the late 3rd to early

4th instar larvae were used per cup and control solution, with 4-cup replications for

each concentration and one cup for control. The mortality rates were recorded after 24

hours exposure. Three subsequent sets of the experiment were carried out for each

test. Probit analysis (Finney, 1971) was used to analyze the 50% lethal concentration

(LC50) and the 95% lethal concentration (LC95) of temephos susceptibility against An.

sundaicus larvae.


Suchada Sumruayphol Results / 84

CHAPTER V

RESULTS

5.1 Bionomics of An. sundaicus s.l. in Pak-Nam, Rayong

A longitudinal entomological study was carried out in Ban Pak-Nam, Rayong

Province from May 2007 to April 2008 for investigating the bionomics of An.

sundaicus s.l.. A total 24 nights throughout the year (2 consecutive nights monthly)

from 18:00 to 24:00 for mosquito capture were conducted based on human landing

catch method (HLC). All 3 collection sites were selected at outside the patient

houses which were located at 12° 39' 39.7" N;101° 15' 44.4"E, 12° 39' 26.7" N;101°

16' 10.7"E and 12° 39' 17.2" N;101° 16' 56.7"E. After morphological and molecular

identification, in study area presented only one anopheles mosquito species which was

An. epiroticus or An sundaicus species A according to “Illustrated keys to the

mosquitoes of Thailand. IV. Anopheles” (Rattanarithikul et al., 2006). It was

predominant mosquito species without the primary malaria vectors observed in the

area from human landing catch method.

5.1.1 Mosquito density in study area

5.1.1.1 Mosquito composition in Pak-Nam, Rayong Province between 2007 and

2008

A total of 3,048 mosquitoes were captured within 12 months in study area.

There were 5 species found which were An. epiroticus, Cx. quinquefasciatus, Cx.

sitiens, Ae. aegypti and Ae. albopictus. The most abundant was An. epiroticus, Cx.

quinquefasciatus, Cx. sitiens, Ae. aegypti and Ae. albopictus which 43.8%, 42.6%,

8.9%, 4.63 and 0.07% respectively as shown in Table 18 and Figure 50. There was

only one species of malaria vector in this area that was An. epiroticus and presented at

highest number the collected mosquitoes.



Table 18 Mosquito composition in Ban Pak Nam since May 2007 to April 2008

Date

Mosquito species

An. epiroticus

(No.)

Cx. quinquefasciatus

(No.)

Cx. sitiens

(No.)

Ae. aegypti

(No.)

Ae. albopictus

(No.)

May 07 131 177 15 22 0

June 07 78 74 50 7 0

July 07 195 102 35 20 2

Aug 07 63 39 6 1 0

Sep 07 225 146 12 17 0

Oct 07 66 141 33 10 0

Nov 07 146 97 29 10 0

Dec 07 163 187 52 13 0

Jan 08 65 125 5 4 0

Feb 08 87 142 23 24 0

Mar 08 58 27 1 13 0

Apr 08 58 42 10 0 0

Total 1,335 1,299 271 141 2



Figure 50 Mosquito fauna in Ban Pak Nam from May 2007- April 2008

Ae. albopictus 0.07%

Ae. aegypti 4.63%

Cx. sitiens 8.9%

Cx.quinquefasciatus 42.6%

An. epiroticus 43.8%



5.1.1.2 Biting cycle of An. epiroticus

Mean biting rate of An. epiroticus from 18:00-24:00 since May 2007 until

April 2008 was examined. The mean number of An. epiroticus collected per person

per night varied from 10.2-37.6. On September, An. epiroticus were highest caught, it

was estimated that the mean biting rate was 37.6 bites/person/6 hours. The lowest

number was in January which was 10.2 bites/person/6 hours (Table 19).

An. epiroticus biting behavior revealed that it was gradually increased from the

evening (18:00-20:00) afterward it rapidly increased from 21:00-24:00. The pattern of

An. sundaicus biting cycle was unimodal. The peak of An. epiroticus number was at

midnight (23:00-24:00) with 6.58±0.82 An. epiroticus/person/hour (Figure 51).

Table 19 Biting cycle of An. epiroticus collected from May 2007 until April 2008

Date Mean number of An. epiroticus (bites/person/hour)

1800-1900 1900-2000 2000-2100 2100-2200 2200-2300 2300-2400

May 07 0 0.2 1.2 3.3 7.5 9.7

June 07 0 0.7 1.2 3 3.3 4.8

July 07 0 3.5 3.8 8.7 9.8 7

Aug 07 0 0.5 1.7 1.8 3.8 2.7

Sep 07 0.2 2.2 5.2 8.7 11 10.3

Oct 07 0 0.7 1 4.7 7.3 8.3

Nov 07 2.2 2.3 3.2 5.3 4 7.3

Dec 07 0.7 3.8 3.3 5.2 6.5 7.7

Jan 08 0.3 0.5 1.7 3 3.2 1.5

Feb 08 1 0.7 2 4.7 12 8.7

Mar 08 0.3 0.7 0.7 3 7 7.7

Apr 08 0.3 4 3 3 1 3.3

Total

mean 0.42 1.65 2.33 4.53 6.37 6.58



0

1

2

3

4

5

6

7

Time

Mea

n N

o. o

f An.

epi

rotic

us/p

erso

n/ho

ur

18:00 19:00 20:00 21:00 22:00 23:00

Figure 51 Biting cycle of An. epiroticus from 18:00-24:00 since May 2007 to April

2008



0

5

10

15

20

25

30

35

40

Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec

Months

Mea

n N

o. 0

f An.

epi

rotic

us p

er

pers

on p

er n

ight

0

50

100

150

200

250

300

350

400

450

Rai

nfal

l (m

mAn. epiroticus Malaria cases Rainfall (mm)

5.1.1.3 Seasonal abundance of An. epiroticus

The seasonal abundance of An. epiroticus was carried out by human landing

catch. The result showed fluctuate pattern distribution of An. epiroticus throughout

the year (Figure 52). The mean number of An. epiroticus was highest in September

and the lowest in January with 37.6 and 10.2 bites/person/6 hours, respectively.

Figure 52 The seasonality of An. epiroticus within 12 observed months compared

with malaria cases and rainfall



Table 20 Observed An. epiroticus, malaria cases, and meteorological data in Pak Nam,

Rayong during study period

Month

Mean No. of

An. epiroticus

(bites/person/6 hours)

Malaria

cases

Rainfall

(mm)

Average of

RH (%)

Average temperature

(°C) (range)

May 2007 21.9 1 416.3 81 28.9 (23.9-35.3)

Jun 2007 13 3 227.8 80 29.4 (24-35.5)

Jul 2007 32.8 1 192.7 79 28.8 (24.3-34.5)

Aug 2007 10.5 0 128 77 29 (24-34.7)

Sep 2007 37.6 1 212.2 80 28.4 (22.3-32.8)

Oct 2007 22 2 76.9 76 27.8 (23-33.8)

Nov 2007 24.3 2 15.6 63 26.5 (17.2-33.5)

Dec 2007 27.2 1 0 73 26.4 (17.2-33.5)

Jan 2008 10.2 0 3.7 70 26.4 (19.8-33.2)

Feb 2008 29.1 1 58.5 74 26.9 (19.8-32.8)

Mar 2008 19.4 0 44 75 28 (20.2-32.6)

Apr 2008 14.6 0 104 76 29.2 (24.2-34.7)

The correlation study between rainfall, relative humidity (RH), and mean

number of An. epiroticus was calculated with t-test by SPSS11.5. The result showed

that the correlation between rainfall, relative humidity, and mean number of An.

epiroticus was not significant. The p-value between relative humidity and mean

number of An. epiroticus was 0.69 which more than 0.05 confident interval (not

significance). In the same way, the correlation between relative humidity and mean

number of An. epiroticus was not significant (significant value was 0.707 > 0.05

confident interval). There were no correlation between rainfall, relative humidity, and

mean number of An. epiroticus in this study.



5.1.1.4 Larval prevalence

Larval survey was carried out by dipping method for studying breeding

habitats of An. epiroticus from May 2007 through April 2008. A total of 38 cement

ponds within 4 houses were selected for larval collection (Figure 53). All 4 sites were

selected nearby the patient houses. Water quality was estimated as well by HACH

portable equipment. The physical and chemical characters of An. epiroticus breeding

places were examined. Total dissolved solid (TDS), and temperature were measured

for physical character whereas, pH, conductivity, salinity, and dissolved oxygen (DO)

were checked for chemical character.

In the area, An. epiroticus larvae were presented together with other mosquito larvae

which were Aedes, and Culex. The highest proportion between An. epiroticus and

other larvae was 23.01:69.7 in May. An. epiroticus breeding places were varied from

fresh (salinity < 0.5 ppt), brackish (salinity = 0.5-30 ppt), and salt (salinity > 30 ppt)

water with sunlit open cement ponds and algae (Table 21). The mean salinity was

9.11-52.42 ppt (range from 0.5 to 119.4 ppt) within 12 months. The mean number of

An. epiroticus larvae ranged from 0.97 through 23.01 per dip in December and May,

respectively. The mean pH of An. epiroticus breeding places was not much different

which ranged from 8.15 to 8.73. The mean of conductivity was varied from 15.47

through 75.2 microseimen / cm (ms/cm) on May and March, respectively this value

was two time greater than the average total dissolved solid (TDS) which TDS was

7.73-37.6 g/l in the same months. The dissolved oxygen was highest in November

with 6.27 mg/l and the lowest in March with 3.46 mg/l. The mean temperature was

24.6-32.76 ˚C. However, the mean value ±SE (range) of larvae and measured water

quality in breeding place of An. epiroticus in Pak Nam sub-district, Rayong Province

were shown in Table 21.



NF

= N

on c

olle

ctin

g da

ta

Tab

le 2

1 M

ean

valu

e ±S

E (r

ange

) of l

arva

e an

d m

easu

red

wat

er q

ualit

y in

bre

edin

g pl

ace

of A

n. e

piro

ticus

in P

ak N

am su

b-

dis

trict

, Ray

ong

Prov

ince



A B

C D

Figure 53 A-H showed breeding places of An. epiroticus in Ban Pak Nam, Rayong

Province



E F

G H

Figure 53 (continued) A-H showed breeding places of An. epiroticus in Ban Pak Nam,

Rayong Province



5.1.2 Parity rate of An. epiroticus in study area

The parity rate is the indirect method to estimate longevity of the mosquito

vectors. After morphological identification of collected mosquitoes An. epiroticus

were dissected for examining the ovary status. In the study area, a total of 1,215 An.

epiroticus females were studied parity rate. The result of parity rate was shown in

Table 22.

The parity rate of An. epiroticus was very high throughout the year from 61%

to 90%. The lowest parity rate was in November with 61%. The parity rate reached

the highest value on January with 90%. This result showed that the proportion old An.

epiroticus was much greater than the young one.

We estimated the expectation of life of An. epiroticus in study areas using the

equation for expectation of life.

The expectation of life = 1/(-lnp) day

p = n√P/(P+N)

p = daily probability of survival

n = gonotrophic cycle (day)

P = number of parous

N = number of nulliparous

According to the result total number of An. epiroticus was 1,251 individuals

with 902 and 313 of parous and nulliparous respectively.

p = 3√902/(902+313)

= 3√0.74

= 0.905 = 90.5%

Expectation of life = 1/(-lnp)

= 1/(-ln 0.905)

= 1/0.0998

= 10.02 days

According to rearing An. epiroticus in laboratory, the gonotrophic cycle was 3

days. Therefore, the very high daily probability of survival was estimated with 0.905

or 90.5%.



Table 22 The parity rate of An. epiroticus in the area from May 2007 to April 2008

Month No. of dissected

An. epiroticus No. of parous No. of nulliparous Parity rate (%)

May 2007 113 70 43 62

Jun 2007 70 49 21 70

Jul 2007 180 141 39 78

Aug 2007 60 40 20 67

Sep 2007 200 160 40 80

Oct 2007 60 50 10 83

Nov 2007 140 85 55 61

Dec 2007 150 115 35 77

Jan 2008 60 54 6 90

Feb 2008 80 60 20 75

Mar 2008 50 40 10 80

Apr 2008 52 38 14 73

Total 1,215 902 313 74

A B

Figure 54 Parous (A) and nulliparous (B) of dissected An. epiroticus ovary



5.1.3 Infection rate of An. epiroticus to plasmodium parasite

Nested PCR (Snounou, and Singh, 2002) and Real-time PCR (Swan et al.,

2005) were used to detect plasmodium species in adult female of An. epiroticus. Only

the parous An. epiroticus were divided into 2 parts which were head-thorax and

abdomen portion for DNA extraction. After nested PCR with genus plasmodium

positive, they were subjected in Real-time PCR to identify species of plasmodium in

An. epiroticus. A total of 926 An. epiroticus were detected to plasmodium infection.

926 of head-thorax and abdomen portions were tested by nested PCR. We found 9

(0.97%) of head-thorax portions with genus plasmodium positive. After real-time

PCR detection of 9 positive samples, the result showed that 6 were P. falciparum

(66.7%), and 3 were P. vivax (33.3%). The abdomen portions were not found positive

with genus plasmodium. Results from the Nested and Real-time PCR were shown in

the Figure 55 and Figure 56. However, the summary of positive malaria infection in

An. epiroticus was demonstrated in Table 23.

The infective mosquitoes were detected in four months which were January,

February, May, and July with 3, 2, 1, and 3 infective samples, respectively. All

positive samples were found in dry cool season (January and February) and rainy

season (May and July). The highest positive amounts were in January, and July with

each three samples. In February and May, there were less infection 2, and 1 sample.

Six out of nine were P. falciparum whereas, three were P. vivax infection.

The entomological inoculation rate (EIR), calculated as the number of positive

sporozoite An. epiroticus bites received by one person in one year for each

plasmodium species. The An. epiroticus EIA in Ban Pak-Nam was estimated by

equation.

h = ma*s

h = mean biting rate of infective mosquito per person per night

ma = mean biting rate of mosquito per person per night (22 for An. epiroticus

according to mean number of An. epiroticus (bites/person/6 hours) within 12 months

was 21.88)

s = sporozoite rate (0.97%)

Therefore, h = 22*0.97/100 = 0.21



The h of An. epiroticus in Ban Pak-Nam was 0.21. It was mean biting rate infective

An. epiroticus per person per night. The total estimated EIR was 76.65 (0.21x365)

positive bites per person per year. This revealed that approximately every five days a

person may received an infective bite.

Figure 55 2% agarose gel electrophoresis of An. epiroticus positive samples with

genus Plasmodium Lane1: 0.5 µg of 100 bp DNA ladder, lane 2-10: genus positive

Plasmodium samples, lane 11-14: malaria infected human blood for P. falciparum, P.

vivax, P. ovale and P. malariae as positive control, lane 15: negative control

500 bp

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15



Figure 56 Result of Real time PCR shows melting curve of species-specific

plasmodium in An. epiroticus. The annealing temperature of P. vivax and

P. falciparum were 51.8 - 55.5 and 60.0 ± 2.0 °C, respectively.



Table 23 Result of plasmodium detection in An. epiroticus head-thorax portions

Month No. of head-thorax

portion tested

No. of positive

An. epiroticus

No. of

P.falciparum

No. of

P. vivax

May 2007 70 1 0 1

Jun 2007 49 0 0 0

Jul 2007 141 3 3 0

Aug 2007 40 0 0 0

Sep 2007 160 0 0 0

Oct 2007 50 0 0 0

Nov 2007 85 0 0 0

Dec 2007 115 0 0 0

Jan 2008 54 3 2 1

Feb 2008 60 2 1 1

Mar 2008 50 0 0 0

Apr 2008 52 0 0 0

Total 926 9 (0.97%) 6 (66.7%) 3 (33.3%)




According to biological study of An. epiroticus, we established laboratory

colony from approximately 100 collected gravid females from study sites. After

morphological identification of adult An. epiroticus were reared in insectary. The

biology of An. epiroticus was set under laboratory condition.

The average number of eggs per female was 83.2 ranging from 68 to 123. The

mean hatching rate was 82.7%. The average of larval development from first instar

larvae to the fourth instar larvae was 20.4 days. The pupae duration was 2-3 days.

The average male and female ratio in adult stage estimated was 1:1. The mean

emergence rate was 72%. The longevity of An. epiroticus was 29-54 days (Table 24).

Table 24 The biology of An. epiroticus under laboratory condition

Criteria Number

Number of eggs per female 68-123 eggs

Hatching rate 82.7%

Larval development (L1-L4) 13-35 days

Pupae duration 2-3 days

Emergence rate 72%

Sex ratio (male: female) in adult stage 1:1

Adult longevity (unfed female) 29-54 days



5.2 Molecular identification of An. sundaicus s.l. from Pak-Nam, Rayong

After morphological identification of all An. epiroticus adult female from field

collection, PCR was used for molecular identification. Some of the An. epiroticus

adult female were extracted genomic DNA and then detected with 3 genes were

cytochrome oxidase I (COI), internal transcribed spacer 2 (ITS2), and domain-3 (D3)

of 28S rRNA by PCR. Gel electrophoresis was conducted for detection the PCR

product in each sample (Figure 57). The PCR products were sent to DNA sequencing

at Bioservice Unit (BSU). The number of samples for sequencing the COI partial

mtDNA, ITS2, and D3 genes was 10, 6, and 6 samples, respectively. These data were

used to assess evolutionary relationship. Consequently, the sequenced nucleotides

were analyzed and aligned with other areas of An. sundaicus nucleotide sequences

with CLUSTAL X (Thompson et al., 1997). Phylogenic relationship, genetic

distances and trees were computed by MEGA4 (Tamura et al., 2007) between the taxa.

Average number of substitutions per site was calculated using DnaSP 4.20.2 (Rozas et

al., 2003).



500 bp

1,000 bp

Figure 57 1.5% agarose gel electrophoresis of COI, ITS2, and D3 of An. epiroticus,

lane1; 100 basepair (bp) DNA ladder, lane2-3; 650bp of ITS2, lane 4-5; 400 bp of D3,

lane 6-7; 750 bp of COI

1 2 3 4 5 6 7



5.2.1 COI sequence results

COI sequences of An. sundaicus s.l. from many resources (Table 25) were

studied. The An. sundaicus COI sequences were aligned by CLUSTAL X (Thompson

et al., 1997). Multiple alignment of the COI gene for An. sundaicus s.l. sequences

was shown in Figure 58. 17 COI sequences for pairwise analysis were shown in Table

26. Pairwise distance (p-distance) of 17 COI sequences was shown in Table 27. The

p-distance is the number of base differences per site from analysis between sequences.

The p-distance was calculated using the p-distance algorithm and plylogenetic tree

was constructed by MEGA4 (Tamura et al., 2007), using Neighbor-Joining method.

The evolutionary relationship of COI gene of all taxa represents the evolutionary

history of the taxa analyzed. Branches represent partitions reproduced in less than

50% bootstrap replicates are collapsed. The percentage of replicate trees in which the

associated taxa clustered together in the bootstrap test (1000 replicates) is shown next

to the branches. The tree is drawn to scale, with branch lengths in the same units as

those of the evolutionary distances used to infer the phylogenetic tree. The

phylogenetic tree of COI gene was shown in Figure 59.

According to multiple alignments between An. epiroticus from Ban Pak Nam

and An. epiroticus from laboratory rearing both fresh and brackish water habitats, the

COI sequences of all sequence samples were identical.



Table 25 COI gene sequences of An. sundaicus s.l. and An. minimus from many

resources for sequence analysis

No. of

sequence Code Description

1 RY_Field An. epiroticus from Ban Pak Nam

1 RY_Lab An. epiroticus from laboratory rearing

7 CB_1-CB_7 An. epiroticus from Chantaburi Province 1-7 samples

8 PN_1-PN_8 An. epiroticus from Phang Nga Province 1-8 samples

1 Pattani An. epiroticus from Genbank (AY672357) of Pattani Province

1 Phangnga An. epiroticus from Genbank (AY672355) of Phang Nga Province

1 Vietnam An. epiroticus from Genbank (AF222324) of Vietnam

1 Indonesia An. sundaicus from Genbank (AY672404) of Indonesia

1 Malaysia An. sundaicus from Genbank (AY672379) of Malaysia

1 An. minimus An. minimus from Genbank (EU143299) from India

Table 26 The explanation of 17 COI nucleotide sequences for p-distance analysis

No. of sequence Code Description

1 CB_6 An. epiroticus from Chantaburi Province sample 6

2 PN_3 An. epiroticus from Phan Nga Province sample 3









11 RY_Lab An. epiroticus from Rayong Province from laboratory





16 An. epiroticus An. epiroticus from Genbank (AF222324) of Vietnam

17 An. minimus An. minimus from Genbank (EU143299) from India



Table 27 The pairwise distance (p-distance) of all 17 COI nucleotide sequences

No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

1

2 0

3 0 0

4 0.01 0.01 0.01

5 0.01 0.01 0.01 0

6 0.01 0.01 0.01 0 0

7 0.01 0.01 0.01 0.01 0.01 0.01

8 0.01 0.01 0.01 0.01 0.01 0.01 0

9 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01

10 0.01 0.01 0.01 0.02 0.02 0.02 0.01 0.01 0.01

11 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0

12 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01

13 0.03 0.03 0.03 0.02 0.02 0.02 0.03 0.03 0.02 0.03 0.03 0.02

14 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.03 0.02 0.02 0.02

15 0.03 0.03 0.03 0.02 0.02 0.02 0.03 0.03 0.02 0.03 0.03 0.02 0 0.02

16 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.02 0.01 0.01 0.02 0.01 0.02

17 0.13 0.13 0.13 0.13 0.13 0.13 0.13 0.13 0.12 0.13 0.12 0.12 0.12 0.12 0.13 0.12

The result of comparison p-distance value of COI sequences of An. epiroticus

from 3 populations, Rayong, Phang Nga and Chanta Buri Province and An. sundaicus

s.l. (AY672357, AY672355, AF222324, AY672404, and AY672379) showed that

there were 0-3% nucleotide variation (0.00-0.03 p-distance value). In contrast,

comparison between An. minimus as outgroup taxon the nucleotide variation was 12-

13% (0.12-0.13 p-distance value). The number of polymorphic sites between An.

epiroticus from this study and sequences from Phang Nga and Chanta Buri Province

(Ruangsittichai et al., 2008) was estimated by DnaSP 4.20.2 (Rozas et al., 2003) the

data were shown in table 28.



Table 28 The number of polymorphic sites between An. epiroticus from this study*

Sites Number Positions

Invariable (monomorphic) sites 593

Variable sites (polymorphic) sites 30

Singleton variable sites 6 229, 238, 364, 478, 589, 622

Parsimony informative sites 24

- Parsimony informative sites

(two variants)

21

22, 34, 37, 46, 178, 196, 223, 224,

247, 296, 298, 328, 359, 361, 379,

427, 445, 508, 530, 560, 577

- Parsimony informative sites

(three variants)

3 283, 346, 412

*These data were calculated by using CB, RY and PN sequences



Figure 58 Multiple alignment of COI sequences of An. sundaicus s.l.

. represented the similar nucleic acid sequences



Figure 58 (continued) Multiple alignment of COI sequences of An. sundaicus s.l.




Figure 59 Phylogenetic analysis of COI sequences using Neighbor-Joining method of

An. sundaicus sequences from various sites whereas An. minimus was outgroup taxon

CB 6

CB 1

PN 3

CB 4

RY Lab

PN 6

PN 2

PN 4

PN 8

PN 7

PN 1

CB 3

CB 2

Pattani AY672357

Vietnam AF222324

Phangnga AY672355

PN 5

CB 7

Indonesia

Malaysia

An. minimus

95

92

7592

90

8558

38

35

31

49

40

4525

26

40

0.001



The phylogenetic tree of COI gene showed strong distinguish discrimination of

An. epiroticus in this study from An. sundaicus s.l. from Malaysia, Indonesia. All of

the An. epiroticus sequences from this study were closely related to An. epiroticus

(AF222324) from Vietnam. Even though, there was a lot of variation within An.

epiroticus COI sequences in this study nevertheless they are together in the same

cluster.

5.2.2 ITS2 sequence results

Six ITS2 nucleotide sequences of An. sundaicus s.l. from many resources were

studied and aligned by CLUSTAL X (Thompson et al., 1997).

The description of 13 ITS2 sequences for p-distance analysis was explained in

the Table 29. Pairwise distance (p-distance) of 13 ITS2 sequences was shown in

Table 30. The results were based on the pairwise analysis of 13 sequences that were

conducted in MEGA4 using the p-distance algorithm and using Neighbor-Joining

method (Tamura et al., 2007) between ITS2 sequences of An. epiroticus from this

study (RY), An. sundaicus s.l. from India and Malaysia, and An. epiroticus from

Vietnam (An. epiroticus).

There were no polymorphic sites observed by DnaSP 4.20.2 (Rozas et al., 2003)

between sequences of An. sundaicus s.l. and An. epiroticus (AY789168).



Table 29 The description of 13 ITS2 nucleotide sequences for p-distance analysis

No. of

sequence Code Description

1 RY_1 An. epiroticus from Rayong Province sample 1











12 An. epiroticus An. epiroticus from Genbank of Vietnam (AY789168)

13 An. minimus An. minimus from Genbank (AY841147)

Table 30 P-distance value of An. epiroticus, An. sundaicus s.l. and An. minimus

No. 1 2 3 4 5 6 7 8 9 10 11 12 13

1

2 0

3 0 0

4 0 0 0

5 0 0 0 0

6 0 0 0 0 0

7 0 0 0 0 0 0

8 0 0 0 0 0 0 0

9 0 0 0 0 0 0 0 0

10 0 0 0 0 0 0 0 0 0

11 0 0 0 0 0 0 0 0 0 0

12 0 0 0 0 0 0 0 0 0 0 0

13 0.52 0.52 0.52 0.52 0.52 0.52 0.52 0.52 0.52 0.52 0.52 0.52



The result of comparison p-distance value of ITS2 sequences of An. epiroticus

from 3 populations, Rayong, Phang Nga and Chanta Buri Province and An. sundaicus

s.l. (AY789168) showed that there were no nucleotide variation (0 p-distance value)

within 3 population (Rayong, Phang Nga and Chantaburi Province) and An. epiroticus

sequence from Genbank. In An. minimus comparision, the nucleotide variation was

52% (0.52 p-distance value). The number of polymorphic sites between An. epiroticus

from this study and sequences from Phang Nga and Chantaburi Province

(Ruangsittichai et al., 2008) was estimated by DnaSP 4.20.2.

The ITS2 sequences for multiple alignment using 534 bp of An. epiroticus

from this study sequence were compared with Genbank sequences of 572 bp (Figure

60). An. epiroticus which accession number AY789168 from Viet Nam, 572 bp An.

sundaicus which accession number AY691517 from India: Car Nicobar Island and

571 bp An. sundaicus accession number AF369550 from Malaysia: Lundu. From the

results the sequences of An. epiroticus from this study were identical to An. epiroticus

ITS2 sequences. Moreover, the ITS2 sequences of all samples were identical between

An. epiroticus from Ban Pak Nam and An. epiroticus from laboratory rearing both

fresh and brackish water habitats.

We found 3 polymorphic sites between An. epiroticus from Rayong Province

and An. sundaicus from India and Malaysia at position 431 (C→T), 490 (T→G) and

555 (insertion to be C).



Figure 60 Alignment of the ITS2 sequences for An. sundaicus s.l.

.; the identical nucleic acid sequence, Epiroticus; AY789168, India; AY691517,

and Malaysia; AF369550



Figure 61 Phylogenetic tree of ITS2 sequences using NJ and 1,000 bootstraps within

MEGA4 for An. epiroticus from this study (RY) with An. sundaicus from India and

Malaysia and An. epiroticus from Vietnam whereas An. minimus was outgroup taxon.

The phylogenetic analysis of ITS2 sequences was done and showed that there

were well separated into two clades (Figure 61). They are the first clade of An.

epiroticus from Rayong and An. epiroticus (AY789168) from Vietnam and the second

clade of An. sundaicus s.l. from Malaysia and India. The evolutionary relationship

between An. sundaicus s.l. from Malaysia and India was closer than An. epiroticus.

All of the An. epiroticus sequences from Rayong were closely related to An. epiroticus

from Vietnam.

5.2.3 D3 sequence results

D3 sequences of An. epiroticus from Rayong, An. sundaicus from India were

aligned by CLUSTAL X (Thompson et al., 1997) (Figure 62). Pairwise distance (p-

distance) of all 11 nucleotide sequences was shown in Table 31. We used the D3 An.

epiroticus sequences within 3 Provinces which were Rayong (RY_1-RY_3),

Chantaburi (CB_1-CB_3), Phangnga (PN_1-PN_3), and An. sundaicus species D from

India (AY691516). There were no different between D3 sequences of An. epiroticus

sequences from Ban Pak Nam and An. epiroticus from laboratory rearing both fresh

and brackish water habitats.

RY

An. epiroticus

India

Malaysia

An. minimus

60

58

0.05



We compared D3 sequences of An. epirotius from Rayong, An. sundaicus from

only India: Nancowry Island, Andaman-Nicobar (AY691516) and An. minimus

(AM039906). The phylogenetic tree was shown in Figure 63.

After analysis by DnaSP 4.20.2 (Rozas et al., 2003), there were no

polymorphic sites between sequences of An. sundaicus from Rayong and An.

sundaicus from India (AY691516).

Table 31 P-distance of An. epiroticus, An. sundaicus D, and An. minimus

Code RY_1 RY_2 RY_3 CB_1 CB_2 CB_3 PN_1 PN_2 PN_3 India An. minimus

RY_1

RY_2 0

RY_3 0 0

CB_1 0 0 0

CB_2 0 0 0 0

CB_3 0 0 0 0 0

PN_1 0 0 0 0 0 0

PN_2 0 0 0 0 0 0 0

PN_3 0 0 0 0 0 0 0 0

India 0 0 0 0 0 0 0 0 0

An. minimus 0.13 0.13 0.13 0.13 0.13 0.13 0.13 0.13 0.13 0.13

According to p-distance results, D3 sequences of An. epiroticus from Thailand

and India were identical with p-value was 0. While comparison with D3 sequences of

An. minimus, the nucleotide variation was 13% (p-value was 0.13).



Figure 62 Alignment of the D3 sequences of An. epiroticus in Rayong (Thailand), An.

sundaicus from India, and An. minimus

* represented consensus sequence



Figure 63 Phylogenetic analysis of D3 sequences of An. sundaicus s.l. from Thailand,

(Rayong, Chantaburi, and Phangnga), An. sundaicus (AY691516) from India and An.

minimus as outgroup taxon

The phylogenetic analysis represented D3 sequences of An. sundaicus s.l and.

An. sundaicus D (AY691516) from India were in the same clade.

RY 1

CB 3

RY 3

PN 3

India

PN 1

CB 1

RY 2

PN 2

CB 2

0.01

100

An minimus



5.3 Insecticide susceptibility test of An. epiroticus

An. epiroticus larvae and adults were tested with larvicide and adulticides.

Temephos was used for larvicide testing. 5% malathion, 0.75% permethrin and 0.05%

deltamethrin were evaluated for adulticide susceptibility. 50% and 95% lethal

concentration (LC50, LC95) were calculated for temephos. 50% and 95% lethal time

LT50, LT95) of malathion and knockdown time (KT50, KT95) of pyrethroid group

(0.75% permethrin and 0.05% deltamethrin). Probit analysis was used to estimate all

insecticides against An. epiroticus (Finney, 1971). The results were shown in Table

32-35.

Table 32 Bioassay result of temephos against An. epiroticus larvae

Dose (ppm) Larval No. Killed Observed mortality (%) Corrected mortality (%)

0.0025 240 52 21.7 21.7

0.0050 240 140 58.3 58.3

0.0100 240 228 95 95

0.0200 240 240 100 100

% control mortality: 0 (0/60)

Table 33 Lethal concentration (LC) and range of An. epiroticus to temephos

LC (%) Temephos concentration (ppm) Range

2 0.001 0.00106-0.00146

50 0.004 0.00386-0.00439

90 0.009 0.00789-0.00958

95 0.010 0.00957-0.01207

98 0.013 0.01185-0.01569

Level of conference at 95%

The LC50 of An. epiroticus to larvicide (temephos) was 0.004 ppm ranging

from 0.00386-0.00439. The LC95 was 0.01 ppm ranging from 0.00957-0.01207.



Table 34 Susceptibility of An. epiroticus against 3 adulticides by using diagnostic

doses

Insecticides KT 50 (mins) KT 95 (mins) % Mortality

5% Malathion 25.79 44.89 100

0.75% permethrin 15.30 29.74 100

0.05% deltamethrin 15.11 26.11 100

Adult bioassay test results of An. epiroticus against 3 adulticides showed that it

was susceptible to all insecticides since 100% mortality at 24 hours after insecticide

exposure. The KT50 against 5% Malathion, 0.75% permethrin and 0.05%

deltamethrin was 25.79 minutes, 15.30 minutes and 15.11 minutes, respectively

(Table 34).

Table 35 Summary the insecticide susceptibility of An. epiroticus in Ban Pak-Nam,

Rayong

Temephos (ppm) 5% malathion 0.75% permethrin 0.05% deltamethrin

LC50 LC95 KT50 KT95 KT50 KT95 KT50 KT95

0.004 0.010 25.79 44.89 15.30 29.74 15.11 26.11


Suchada Sumruayphol Discussion / 124

CHAPER VI

DISCUSSION

Malaria in study area

From 2002-2008, malaria cases in Ban Pak Nam occurred every year both Thai

and non Thai. The most prevalent parasite was P. vivax with 96% and 4% with P.

falciparum. All P. falciparum cases were non Thai patients. All Thai patients were P.

vivax cases. Non Thai patients were occurred both P. falciparum and P. vivax.

6.1 Bionomics of adult and larval of An. epiroticus

Since An. sundaicus s.l. have high level of variation both ecology and behavior

therefore, it is necessary to study bionomics of them for understanding the biology and

led to an effective vector control.

6.1.1 Mosquito density

An. epiroticus was the predominant mosquito in this area with 43.8% (1,335).

The highest mean number of An. epiroticus was in September (37.6 bites/person/half

night), the month with high rainfall at 212.2 mm.

Its biting activity occurred throughout the night with highest peak at 2300-

2400. A mean of man-biting rate was varied from 10.2-37.6 bites/person/6 hr or half

night. The highest rate was in September with mean number 37.6 bites/man/half night.

Contrasting observation the observation the lowest rate was in January with mean

number 10.2 bites/man/6 hours. Higher density was also observed in Vietnam with

190 bites/man/night (Trung et al., 2004). There was not a significant relationship

between mean numbers of An. epiroticus and rainfall or between incidence and rainfall.

The biting pattern of An. epiroticus was highest peak during 23:00-24:00 with

6.58±0.82 bites/person/half night.

The result of seasonal abundance showed fluctuate pattern distribution of An.

epiroticus throughout the year in Pak-Nam, Rayong. The mean number was highest in



September (the month with high rainfall) with 37.6 bites/man/6 hours and rainfall at

212.2 mm. The lowest mean number was occurred in January (dry-cool season) at

10.2 bites/man/6 hours and 3.7 mm of rainfall. The highest and the lowest mean

number of An. epiroticus were shown in the rainy season and dry-cool season. There

were no correlation between rainfall, relative humidity (RH), and mean number of An.

epiroticus from this study.

Larvae can breed in fresh water (<0.5 g salt/liter or ppt), brackish water (> 0.5

and < 30 g salt/liter or ppt), and salt water (> 30 g salt/liter or ppt) habitats which

salinity ranging from 0.4 ppt through 178 ppt. This is agree with the study of Linton

et al (2001) and Nanda et al (2004) that An. sundaicus s.s. and An. sundaicus D from

Malaysia and India can breed in both brackish and fresh water habitats. However, it

has been found in only brackish water habitats (Dusfour et al., 2007) while in this

study it was found in all water habitats including fresh water, brackish water, and salt

water. The larvae were found with filamentous algae habitat with the pH was wider

ranged from 5.64 to 10.33 whereas the studies from India, Vietnam, and Indonesia

with pH ranged 7-8.5 (Dusfour et al., 2004a). Thus, larval differences do not support

An. sundaicus complex since they are the same species.

Not only An. sundaicus s.l. larvae can tolerant in wide range salinity but also

An. farauti s.s., An. bwambae, An. gambiae, An. merus, and An. melas can breed in

brackish water and fresh water (Dusfour et al., 2004a).

6.1.2 Parity rate of An. epiroticus in Pak-Nam, Rayong

A total of 1,215 An. epiroticus was dissected for parous examination. The

parity was very high throughout the year ranged from 61% through 90% in January

and sporozoite was detected in this month in three mosquitoes. This revealed that the

old mosquitoes are important to transmit malaria parasite.

In this study 74% out of 1,215 An. epiroticus was parous indicating that the

survival rate was high when compared with Trung et al (2004) who found that 47%

out of 11,002 was parous.

The parity rate from this study (74%) related to An. sundaicus D from

India which presented high parity rate with 73.38% (Kumari and Sharma, 1994).

Moreover, the parity rate of An. epiroticus was high throughout the year and the



expectation of life of An. epiroticus was 10.02 days from parity rate. Therefore, it has

higher chance of being infected with plasmodium parasite. Since parous (older)

anopheline are more important for epidemiology than nulliparous mosquitoes.

6.1.3 Malaria infection in An. epiroticus

There are no certain confirmation data about malaria infection of An. epiroticus

in Thailand. It was only reported that An. sundaicus species A is involved in malaria

transmission in southern Thailand by available distributional and epidemiological data

(Prasittisuk, 1985). This was the first evidence of natural sporozoite infection of An.

epiroticus.

All 926 of head-thorax and 926 abdomen portions were tested by nested PCR.

9 out of 926 of head-thorax portions were genus plasmodium positive. This study

revealed 0.97% sporozoite rate of An. epiroticus which was much higher than the

infection rate of An. sundaicus from Indonesia wirh 0.07% sporozoite rate by salivary

gland dissection (Collins et al., 1979). In accordance with the An. sundaicus in

Malaya of Malaysia, the sporozoite rate was very low at 0.04% from 11,000 samples

by salivary gland dissection (Reid, 1968). This may be due to the low sensitivity of

dissecting method. The abdomen portions were not positive with genus plasmodium.

The 9 positive head-thorax portions were further subjected to Real-time PCR for

identification of plasmodium species. The result showed that 6 were P. falciparum

and 3 were P. vivax. The highest positive samples (3) were found in January with the

highest parity rate (90%).

Despite P. vivax was the predominant species of malarial patients in the area,

accounting for 96% of all infection, P. falciparum infection in An. epiroticus was

66.7% and P. vivax was 33.3% by nested PCR and real time PCR technique. This is

interesting to point out that although P. vivax is the most frequent parasite in this area

its infection was lower than P. falciparum. This is not beyond all reasonable doubt as

there are a lot of illegal foreign workers containing P. falciparum in their blood but

they do not receive curing from hospital. Moreover, they almost work (fishery)

outside the houses at night rendering more chance for this mosquito bite resulted in

more P. falciparum was detected in An. epiroticus than P. vivax. By considering

various possible solutions, firstly, these positive samples were all sporozoites in the



head-thorax portions. There were no oocysts positive in all samples of abdomen

portions. Secondly, very high parity rate (up to 90% in January) confirmed this reason.

When compared the sporozoite rate to Malaysian An. sundaicus, low sporozoite rate

(0.04%) was detected in natural population (Sandosham and Thomas, 1983). It is

sensible because it is a prolific breeder, huge number of female may resulting in low

infection rate (Poolsuwan, 1995). Despite the high density (190 bites/man/night) of all

11,002 An. epiroticus occurred in Vietnam but there were no malaria infection

detected by ELISA method (Trung et al., 2004). This situation was simultaneously

occurred with the low parity rate (47%) in this study site. This may reflect low vector

status of An. epiroticus in study area (Trung et al., 2004). Dousfour et al (2004) also

stated that the large increase in mosquito density lead to reducing in malaria

transmission. Decreasing of mosquito longevity also reflected the incomplete

development of plasmodium into sporozoite (Coosemans et al., 1992). Although, An.

sundaicus is the important malaria vector in Car Nicobar island of India but there were

no sporozoite or oocyst positive detection (Kumari and Sharma, 1994).

All knowledge assumed that An. epiroticus is an effective vector in coastal

areas of Thailand and other countries but there were no reliable evidence for

sporozoite positive. This is the first report for sporozoite positive in An. epiroticus.

The present study reported EIR of An. epiroticus in study area which was 76.65

infective bites/person/year or every five days a person may received an infective bite.

In general, there are no EIR study of An. epiroticus was estimated elsewhere.

Reid stated that in nature An. sundaicus has low sporozoite rate and low

attraction to human blood yielding no important in malaria epidemic (Reid, 1961).

But in study area, high density of An. epiroticus (up to 37.6 bites/person/half night in

September) was observed. Moreover, there were less animal and abundant people

presented in this area. Therefore, its role in malaria transmission is very important. It

may be concluded that malaria transmission by An. epiroticus come from high

population density and Colless (1952) notified that it required high density to cause

epidemics of malaria.




The average number of eggs per female was 83.2 ranging from 68 to 123. This

was correlated with Das et al (2004) who showed that the average number of eggs laid

by female was 74.5 (Das et al., 2004).

The mean hatching rate of An. epiroticus was 82.7%, it was greater than An.

sundaicus from India which was 77.8% (Das et al., 2004). The average of larval

development from first instar larvae to the fourth instar larvae was 20.4 days. The

pupae duration was 2-3 days. The average sex ratio was 1:1. The mean emergence

rate was 72%. The longevity of An. epiroticus was 29-54 days.

6.2 Molecular identification of An. epiroticus

The mean pairwise distance (p-distance) based on COI sequences of An.

epiroticus from Rayong was 1.4% (0-3%), it was correlated to Linton et al (p-distance

of An. epiroticus from Cambodia, peninsular Malaysia, Thailand, and Vietnam was

1.2%). Moreover, it was closely to 1.1% p-distance of An. epiroticus from Trat and

Phang Nga Province (Thailand) and 1.7%-1.9% p-distance was observed between

Thailand and Vietnam An. epiroticus (Dusfour et al., 2004).

COI sequences from Rayong obtained 30 polymorphic sites (24 was parsimony

informative sites, 6 was singleton variable sites). In comparison the study of Linton et

al (2001), found that there were five polymorphic sites (3 was parsimony informative,

other 2 was singleton variable sites) within Malaysian COI sequences. It implied that

Thailand COI sequences have more intraspecific variation than Malaysian COI

sequences. Its reason is the small population size at the collection time, perhaps as a

result of limited habitats for the immature stages (Linton et al., 2001). 23 variable

sites (polymorphism) from COI gene amplified for An. sundaicus s.l. from Vietnam,

Thailand, and Malaysian Borneo were observed (Dusfour et al., 2004). Thirty-eight

variable sites were detected in alignment of partial COI gene of An. epiroticus from

Cambodia, peninsular Malaysia, Thailand, and Vietnam and An. sundaicus s.s. from

Malaysian Borneo (Linton et al., 2005). There was high nucleotide variation within

COI sequences of An. epiroticus.



Based on COI gene sequence alignment, all three province sequences were

well separated with Indonesia and Malaysian sequences from Genbank. An. epiroticus

was located within the same clade in phylogenetic tree with three province sequences

from this study by NJ method.

There was none intraspecific differences among ITS2 sequences from this

study (0 p-distance value). In comparison the variability of ITS2 sequences of six An.

epiroticus samples with An. epiroticus from Genbank, An. sundaicus from India and

Malaysia, it revealed no variable site within specimens from all sequences of An.

epiroticus from Genbank.

Three polymorphic sites were found between these sequences with Indian and

Malaysian sequences. ITS2 sequences from this study have 533 bp whereas Linton et

al (2005) got 663 bp but three variable sites were identical with An. epiroticus from

Genbank. When compared the sequences of An. epiroticus from Thailand and

Vietnam with An. sundaicus s.s. from Sarawak (Linton et al., 2005). It indicated that

ITS2 amplicons of An. epiroticus (603 bp) obtained one base longer than An.

sundaicus (662 bp), at base 479, 538, and 603 showing transition T↔C, transversion

G↔T, and indel C↔gap, respectively. Similar to this study at base 431, 490, and 555

of An. epiroticus sequence occurred the same situation when compare with An.

sundaicus s.s (Malaysia) from Genbank. In comparison the sequences of An.

epiroticus from Rayong with An. sundaicus D from India, there was only one

transition occurred (T↔C) at base 431 whereas at base 490 and 555 were identical to

An. epiroticus sequences from this study. This result was similar to the study of An.

sundaicus D from India by Alam et al (2006) who found one transition when

compared with An. epiroticus at base 431 (C↔T) whereas An. sundaicus s.s. from

Malaysia displayed differences at two positions from An. sundaicus D. First variable

position was a transversion (G↔T) at position 490 and second, a deletion of ‘C” at

position 555. These three sites can be used to distinguish An. epiroticus, An.

sundaicus s.s., and An. sundaicus D.

In conclusion, the ITS2 comparison showed a single nucleotide substitution

which can distinguish An. sundaicus D (India) from An. epiroticus (Thailand and

Vietnam) while a substitution and addition of an extra base in An. sundaicus D can

differentiate it from An. sundaicus s.s from Malaysia (Linton et al., 2001).



Therefore, ITS2 sequences can be used for identify An. epiroticus and other An.

sundaicus complex group that agree with Linton et al (2005) suggested that only ITS2

sequences can be used for differentiate An. sundaicus species complex.

The high mean intraspecific sequence divergence of COI can easily distinguish

An. epiroticus from An. sundaicus whereas ITS2 sequences showed indel at base 603

which was easier for species separation (Linton et al., 2005).

The comparison was done only with Indian sequences since there were no

others studies of D3 sequence. There were no different between An. epiroticus from

this study and An. sundaicus from India based on D3 with 0% p-value and no

polymorphic sites observed. All sequences were in the same clade of phylogenetic

tree. Therefore, D3 gene sequences were inappropriate for distinguishing the An.

sundaicus species complex group. Using D2 region of 28s rDNA also poorly resolved

the An. maculipennis complex (Porter and Collins, 1996).

6.3 Insecticide susceptibility of An. epiroticus

According to high population density, the control should pinpoint to decrease

vector population and vector human contact by using insecticide spraying. Based on

the overall results, the An. epiroticus larvae and adults in Pak-Nam, Rayong were

susceptible to all insecticide tested. The larvae of An. epiroticus was susceptible to

temephos with LC50 was 0.004 ppm and LC95 was 0.01 ppm.

An. epiroticus adult was also susceptible to three tested insecticides since

100% mortality was obtained at 24 hours after insecticide exposure. The KT50 of An.

epiroticus against 5% malathion, 0.75% permethrin and 0.05% deltamethrin was 25.79

minutes, 15.30 minutes and 15.11 minutes, respectively.

Therefore, temephos can be used for larvicidal control of An. epiroticus.

Malathion, permethrin, and deltamethrin were suited for adulticide in the area. The

density and behavior of An. epiroticus was mainly high at midnight while people were

asleep. Therefore, the insecticide-treated net is additionally recommended for malaria

control in Pak-Nam, Rayong.

There was no difference among the organophosphate and pyrethroid adulticide

used in this study. However, deltamethrin seemed to be the best one.



CHAPTER VII

CONCLUSION

In the past, there was barely information for An. sundaicus s.l. mosquito i.e.

bionomics, species complex, and insecticide susceptibility. This study proved that An.

sundaicus s.l. from field collection was An. epiroticus or An. sundaicus species A by

molecular identification. This study was complete study for give the worthy

knowledge of this mosquito species. The knowledge from this project is very useful

for malaria vector control in the coastal areas of Thailand. In Pak-Nam, Rayong

Province, it was predominant with 43.8%. This is the urban area with presented the

urban mosquito (Cx. quinquefasciatus) at 42.6%. The mean number of An. epiroticus

varied from 10.2-37.6 per person per half night. The highest number was found on

September at 37.6 bites/person/half night. It was gradually increased from the evening

(18:00-20:00) and rapidly increased from 21:00-24:00. According to the survey of

mosquito breeding place, An. epiroticus larvae coexisted with Aedes, and Culex larvae.

Breeding places varied from fresh, brackish, and salt water, typically with full sunlight

and mats of green algae on the water surface. The mean number of An. epiroticus

larvae ranged from 0.97 through 23.01 per dip in December and May, respectively.

This study revealed that An. epiroticus was related to malaria transmission in Pak-

Nam, Rayong. It was the first report in Thailand that An. epiroticus contained

sporozoite in their salivary gland. The sporozoite rate was 0.97%. This was due to

sporozoite infection detected in head-thorax portions by nested PCR and real time

PCR. The P. falciparum was greater than P. vivax in An. epiroticus with 6 and 3

samples, respectively from a total of 926 samples. This species has long life due to the

fact that parity rate was up to 90%. The COI and ITS2 sequences can be used for

distinguish An. epiroticus and An. sundaicus s.l. in the field work. All tested

insecticides (temephos, malathion, deltamethrin, and permethrin) can be used for An.

epiroticus control in Pak-Nam, Rayong since it is susceptible.


Suchada Sumruayphol References / 132

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APPENDIX


Suchada Sumruayphol Appendix / 144

APPENDIX A

MORPHOLOGICAL KEYS FOR ADULT FEMALE OF

ANOPHELES EPIROTICUS (Rattanarithikul et al., 2006)

Adult morphology



Abbreviations of Adult



APPENDIX B

METEOROLOGICAL DATA IN STUDY AREA

Table 35 Temperature and relative humidity at Muang Rayong weather station

(weather station code: 478201) from May 2007 to April 2008

Month-Year

Maximum temperature (°C)

Minimum temperature (°C)

Mean temperature (°C)

Average Relative- Humidity (%)

May-07 35.3 23.9 28.9 81 Jun-07 35.5 24 29.4 80 Jul-07 34.5 24.3 28.8 79 Aug-07 34.7 24 29 77 Sep-07 32.8 22.3 28.4 80 Oct-07 33.8 23 27.8 76 Nov-07 33.5 17.2 26.5 63 Dec-07 33.5 17.2 26.4 73 Jan-08 33.2 19.8 26.4 70 Feb-08 32.8 19.8 26.9 74 Mar-08 32.6 20.2 28 75 Apr-08 34.7 24.2 29.2 76



Table 36 Rainfall (mm) at Muang Rayong from May 2007 to April 2008

Date Month-Year 1 2 3 4 5 6 7 8 9 10 11 12 13 14

May-07 21.3 50.4 15.1 22.1 68 128.4 4.4 0 T 30.1 T 2.4 13 18.6

Jun-07 1.8 41.5 4.9 2.8 7 0 0 0 T 2 11.3 0 0 0

Jul-07 1 0.3 29.3 1.5 65.7 47.5 1.3 0 0 0 1 T 1.7 0.2

Aug-07 6.5 0 T 0 10.9 T 0.9 T 0.2 15.7 0 0 0 0

Sep-07 0 3.4 0.6 0 0 0 0 0.4 0 1.3 5.6 T 26.5 7

Oct-07 0 0 0 0 0 13.7 T 0 0 5 0.4 0 5.4 18.5

Nov-07 0 0 0 0 0 0 0 0 0 0 0 0 15.6 0

Dec-07 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Jan-08 0 0 0 0 0 0 0 0 0 0 0 0 0 2.8

Feb-08 0.7 1.8 14.8 10.3 0 0 0 0 T T 21.1 9 0.5 0

Mar-08 0 0 0 0 0 0 43.7 T 0 0 T 0 0 0.2

Apr-08 0 2.6 6.6 0.2 48.8 17.1 T 0 0 0 0 0 0 0

Date Month-Year 15 16 17 18 19 20 21 22 23 24 25 26 27 28

May-07 17.1 T 12.8 0 0 0 0 0 0 0 0 0 0 2.3

Jun-07 10 0.2 23.9 0 24.7 18.2 0.2 0 51.6 T 2.6 0.3 13.5 6.8

Jul-07 0 2.5 9.9 0 0.5 0 0 T 0.8 0 0 T 6 0

Aug-07 0 0 0 0 0 0 0.5 0.1 4.3 0 0.2 0.1 25.6 61.2

Sep-07 5.1 17.3 18 1.2 4.8 47.7 2.3 0 39 0 0.7 0.9 25.6 2.1

Oct-07 0.5 0 11.2 0 0 0 0 0.5 1.5 3 0 0.6 T 12.1

Nov-07 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Dec-07 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Jan-08 0 0 0 0 0 0 0 0 0 T 0 0 0 0.2

Feb-08 0 0 0 0 0 0 0 0 0 0.3 0 0 0 0

Mar-08 0 0 0 0 0 0.1 0 0 0 0 0 0 0 0

Apr-08 0 0 T 0 0 0 0 4.5 0 0 1.7 0 0.5 17.7

Date Month-Year 29 30 31 Total

May-07 6.8 0 3.5 416.3

Jun-07 T 4.5 - 227.8

Jul-07 0.8 21.4 1.3 192.7

Aug-07 0.8 1 0 128

Sep-07 2.2 0.5 - 212.2

Oct-07 4.5 T 0 76.9

Nov-07 0 0 - 15.6

Dec-07 0 - - -

Jan-08 0 0 0.7 3.7

Feb-08 0 - - 58.5

Mar-08 0 0 0 44

Apr-08 4.3 0 - 104



APPENDIX C

FORMULA OF TBE BUFFER

Formula for preparing TBE running buffer for agarose gel electrophoresis

TBE running buffer (5x)

Tris base 52 g

Boric acid 27.5 g

Disodium EDTA·2H2O 4.65 g

Water to 1 liter (Adjust pH to 8.3)

Working solution: 0.5x



APPENDIX D

SEQUENCES OF AN. EPIROTICUS DNA

COI gene sequences of collected An. epiroticus from Ban Pak Nam Rayong

Province



COI gene sequences of An. epiroticus from laboratory rearing



ITS2 gene sequences of collected An. epiroticus from Ban Pak Nam Rayong

province



D3 gene sequences of collected An. epiroticus from Ban Pak Nam Rayong

province



BIOGRAPHY

NAME : Miss Suchada Sumruayphol

DATE OF BIRTH : March 30, 1979

PLACE OF BIRTH : Bangkok, Thailand

INSTITUTION ATTENDED : Kasetsart University, 1997-2000:

Bachelor of Science (Agriculture)

: Mahidol University, 2001-2004:

Master of Science (Appropriate Technology for

Resources and Environmental Development)

: Mahidol University, 2004-2009:

Doctor of Philosophy (Tropical Medicine)

HOME ADDRESS : 68 Moo 3 Tambon Thepmongkon, Bangsai

district, Pranakornsriayutthaya Province

MOBILE PHONE : 086-599-7174

E-MAIL ADDRESS : [email protected]


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