Studies on the Mosquito Fauna in an Urban And

STUDIES ON THE MOSQUITO FAUNA IN AN URBAN AND SUBURBAN AREA IN PENANG AND THE LABORATORY EFFICACY OF MOSQUITO COILS CONTAINING DIFFERENT ACTIVE INGREDIENTS AGAINST SELECTED VECTOR MOSQUITOES

ROZILAWATI BINTI HARUN

UNIVERSITI SAINS MALAYSIA

2007

STUDIES ON THE MOSQUITO FAUNA IN AN URBAN AND SUBURBAN AREA IN PENANG AND THE LABORATORY EFFICACY OF MOSQUITO COILS CONTAINING DIFFERENT ACTIVE INGREDIENTS AGAINST SELECTED VECTOR MOSQUITOES

ROZILAWATI BINTI HARUN

Thesis submitted in fulfilment of the requirements for the Degree of Master of Science

July 2007

ACKNOWLEDGEMENT

I would like to express my profound gratitude to my supervisor Prof

Madya Dr. Zairi Jaal for his guidance, assistance and advices throughout this project period. Without his help, this work would not have been successful.

Special thanks to Mr Adanan the VCRU Research Officer, for giving me so much support during my study in USM, and my sincere thanks to the staff of

Vector Control Research Unit, USM for their help in the realization of this project.

To my lab mates and best friends: please find here the expression of my friendship. Finally I would like to extend my thanks to my colleagues and also my beloved family and fiancé for being understanding and patient, this is the special present for you all.

THANK YOU ALL.

ii TABLE OF CONTENTS

ACKNOWLEDGEMENTS ii TABLE OF CONTENTS iii LIST OF TABLES vii LIST OF FIGURES viii LIST OF PLATES ix LIST OF ABBREVIATION x LIST OF APPENDICES xi LIST OF PUBLICATIONS & SEMINARS xii ABSTRAK xiii ABSTRACT xiv

CHAPTER ONE: GENERAL INTRODUCTION 1

CHAPTER TWO: LITERITURE REVIEW 2.0 Introduction 1 2.1 Mosquitoes 4 2.2 Medical importance of mosquitoes 4 2.3 Dengue 5 2.4 Aedes as vectors 7 2.4.1 Biology of Aedes albopictus 11

2.4.1.1 Eggs 11

2.4.1.2 Larvae 12

2.4.1.3 Pupae 13

2.4.1.4 Adult 13

2.5 Other mosquito of medical importance 14

2.5.1 Culex quinquefasciatus 14

2.5.2 Culex gelidus 15

2.5.3 Mansonia uniformis 16

2.5.4 Subfamily Anophelinae 17

2.5.4.1 Anopheles peditaeniatus 19 2.5.4.2 Anopheles sinensis 20 2.5.4.3 Anopheles campestris 21 2.5.4.4 Anopheles vagus 21

iii

2.5.4.4 Anopheles subpictus 22

2.6 Physical factors 22 2.6.1 Rainfall 23

2.6.2 Temperature 24

2.6.3 Relative humidity 24

2.7 Surveillance of mosquitoes 25

2.7.1 The oviposition trap (ovitrap) 25

2.8 Control of vector mosquitoes 28 2.8.1 Control approaches 28 2.8.2 Household insecticides-mosquito coil 30

2.8.3 Pyrethroid insecticides in mosquito coil 31

2.8.4 Factors influencing laboratory efficacy 32

CHAPTER THREE: SURVEILLANCE OF MOSQUITO FAUNA IN AN URBAN AND SUBURBAN AREA IN PENANG

3.0 Introduction 34 3.1 Materials and methods 36

3.1.1 Description of study sites 36 3.1.1.1 Kampung Pasir Gebu 36 3.1.1.2 Taman Permai Indah (TPI) 39

3.1.2 Sampling techniques 42

3.1.2.1 Adult collection 42

3.1.2.1 (a) Bare-leg Catch 42

3.1.2.1 (b) Cow-baited trap 44

3.1.2.2 Immature collection 46

3.1.2.2 (a) Ovitrap surveillance 46

3.1.3 Mosquito identification 49 3.1.3.1 Larval identification 49 3.1.3.2 Adult identification 52

3.1.4 Culture methods 55

3.1.4.1 Egg hatching 55 3.1.4.2 Larval rearing 55

3.1.4.3 Pupal rearing 55 3.1.4.4 Adult Rearing 56

3.1.4.5 Egg Collections 56

3.1.5 Meteorological data 57 3.1.6 Data analysis 57

3.2 Results 58

3.2.1 Meteorological condition 58

3.2.2 Mosquito species 62

3.2.2.1 Bare leg catch (BLC) 66

3.2.2.2 Cow baited trap (CBT) 66

3.2.3 Ovitrap surveillance 68

3.2.3.1 Ovitrap index (OI%) 68 3.2.3.2 Egg abundance 71

3.2.3.2 (a) Taman Permai Indah (TPI) 71

3.2.3.2 (b) Kampung Pasir Gebu, Penaga 71

3.2.3.3 Correlations 73

3.2.3.3 (a) Taman Permai Indah 73

3.2.3.3 (b) Kampung Pasir Gebu, Penaga 78

3.2.3.4 Total larvae hatched 82

3.3 Discussions 84

v CHAPTER FOUR: LABORATORY EFFICACY OF HOUSEHOLD INSECTICIDES (MOSQUITO COIL) WITH DIFFERENT ACTIVE INGREDIENTS AGAINST SELECTED VECTOR MOSQUITOES

4.1 Introduction 94

4.2 Materials and methods 95

4.2.1 Mosquito strains 95

4.2.2 Test chamber 96 4.2.3 Active ingredients 96

4.2.4 Efficacy of mosquito coil test 97 4.2.5 Statistical analysis 97

4.2 Results 99

4.2.1 Bioassay 99

4.2.1.1 Bioefficacy of 0.04% prallethrin 99

4.2.1.2 Bioefficacy of Product B (0.15% d-transallethrin) 102

4.2.1.3 Bioefficacy of Product C (0.30% d-allethrin) 104

4.2.1.4 Boefficacy of Product D (0.30% d-allethrin) 106

4.2.1.5 Bioefficacy of Product E (0.20% d-allethrin) 108

4.3 Discussions 110

CHAPTER FIVE: SUMMARY AND CONCLUSION 113

BIBLIOGRAPHY 115

APPENDICES 133

vi LIST OF TABLES

Page

3.1 Correlation between several parameters in Taman Permai 75 Indah

3.2 Correlation between several parameters in Kampung Pasir 79 Gebu

3.3 Total number of eggs collected and total number of Aedes 83 albopictus larvae produced including the sex ratio of adults Aedes albopictus produced from the eggs collected in both study sites. 4.1 Knockdown time (minutes) and mortality percentage of product 100 A (0.04% prallethrin) against Anopheles sinensis, Aedes aegypti, Aedes albopictus, and Culex quinquefasciatus using the glass chamber method

4.2 Knockdown time (minutes) and mortality percentage of product 103 B (0.15% d-trans allethrin) against Anopheles sinensis, Aedes aegypti, Aedes albopictus and Culex quinquefasciatus using the glass chamber method

4.3 Knockdown time (minutes) and mortality percentage of 105 product C (0.30% d- allethrin) against Anopheles sinensis, Aedes aegypti, Aedes albopictus, and Culex quinquefasciatus using the glass chamber method.

4.4 Knockdown time (minutes) and mortality percentage of 107 product D (0.30% d-allethrin) against Anopheles sinensis, Aedes aegypti, Aedes albopictus, and Culex quinquefasciatus using the glass chamber method

4.5 Knockdown time (minutes) and mortality percentage of 109 product E (0.20% d-allethrin) against Anopheles sinensis, Aedes aegypti, Aedes albopictus, and Culex quinquefasciatus using the glass chamber method

vii

LIST OF FIGURES

Page

3.1 Meteorological conditions obtained from the Malaysian 60 Meteorological Services Department in Bayan Lepas, reflective of Taman Permai Indah, Sg Dua, Penang

3.2 Meteorological conditions obtained from the Malaysian 61 Meteorological Services Department in Butterworth, reflective of Kampung Pasir Gebu

3.3 Mosquito composition in Kg. Pasir Gebu, Penaga 63

3.4 Total percentage of mosquito species caught 64

3.5 (A) Percentage of mosquitoes collected using BLC in KPG 67 3.5 (B) Percentage of mosquitoes collected using CBT in KPG 67 3.6 Ovitrap index in Taman Permai Indah (TPI) and Kg Pasir Gebu 69 (KPG) for 14 months of sampling

3.7 (A) The distribution of Aedes albopictus and other mosquitoes 70 present in the ovitraps in Taman Permai Indah

3.7 (B) The distribution of Aedes albopictus and other mosquitoes 70 present in the ovitraps in Kampung Pasir Gebu

3.8 Mean number of mosquito eggs per ovitrap collected in both 72 study areas from 14 months of sampling

3.9 Correlation between ovitrap index and mean number of eggs 74 collected in Taman Permai Indah

3.10 Correlation between ovitrap index and rainfall in Taman Permai 76 Indah

3.11 Correlation between mean number of eggs and rainfall in 77 Taman Permai Indah (TPI)

3.12 Correlation between ovitrap index and rainfall in Kampung 80 Pasir Gebu (KPG)

3.13 Correlation between mean number of eggs and rainfall in 81 Kampung Pasir Gebu (KPG)

viii

LIST OF PLATES

Page

2.1 The characteristics used for identifying mosquitoes of general 18 importance

3.1 Paddy is the main crop grown in Kampung Pasir Gebu 37

3.2 Houses in Kampung Pasir Gebu 37

3.3 Natural aquatic plants in Kampung Pasir Gebu 38 3.4 Chicken coop built close to the residence houses in 38 Kampung Pasir Gebu

3.5 Taman Permai Indah, Sg Dua 40 3.6 Flat buildings in Taman Permai Indah 40 3.7 A small river which flows through Taman Permai Indah 41

3.8 Concrete drainage in Taman Permai Indah 41 3.9 The bare leg catch (BLC) technique 43 3.10 The cow baited trap (CBT) 45

3.11 An ovitrap with a paddle 47 3.12 Comb teeth and thoracic spine of Aedes albopictus 50 3.13 Comb teeth and thoracic spine of Aedes aegypti 51 3.14 Aedes albopictus- single silver broad line on the thorax 53 3.15 Aedes aegypti - silvery straight and curved lines (lyre-shaped) 54 on the thorax

4.1 Glass chamber (70x70x70 cm) used for the test 98 4.2 A piece of 0.5g tested coil ignited at both ends in the 98 glass chamber

LIST OF ABBREVIATION

Ae. Aedes An. Anopheles Ma. Mansonia Cx. Culex KT Knockdown time min Minute TPI Taman Permai Indah KPG Kampung Pasir Gebu OI ovitrap index RH relative humidity 0C Celsius % Percentage + plus minus SE Standard error Temp Temperature P Significant R Correlation

LIST OF APPENDICES

Page

A Test of Normality (TPI) 133

B Test of Normality (KPG) 134

C Independent sample t test for the total ovitrap index and the 135 mean number of eggs collected between TPI and KPG

D Independent sample t test, for ovitrap index and means of 136 eggs collected during the high and low rainfall seasons in Taman Permai Indah

E Independent sample t test, for ovitrap index and means of eggs 136 collected during the high and low rainfall seasons in Kampung Pasir Gebu.

LIST OF PUBLICATIONS & SEMINARS

Page

1.1 Laboratory efficacy of prallethrin coil against Aedes 137 albopictus, Aedes aegypti and Culex quiquefasciatus. Rozilawati H & Zairi J in Malaysian Society of Parasitology and Tropical Medicine 1st Asean Congress of Parasitology and Tropical Medicine & 40th Annual Scientific Seminar – Tropical Diseases and Vectors: Management and Control. 23-24 March 2004,. Grand Seasons Hotel, Kuala Lumpur.

1.2 Species composition of adult mosquitoes and ovitrap 138 method in a rural area of Penang. Rozilawati H & Zairi J in 2nd Life Sciences Postgraduate Conference-Life Sciences: The Power to Explore, Discover & Simulate. 1-3 April 2004. Universiti Sains Malaysia., Pulau Pinang.

1.3 Laboratory efficacy of 0.12% d-transallethrin coil against 139 Anopheles sinensis, Aedes albopictus, Aedes aegypti and Culex quinquefasciatus- Rozilawati H & Zairi J in Malaysian Society of Parasitology and Tropical Medicine 41st Annual Scientific Seminar –Natural Product in the Management of Tropical Diseases. 10-11 March 2005. Grand Seasons Hotel, Kuala Lumpur.

1.4 Seasonal abundance of Aedes albopictus in selected urban 140 and suburban area in Penang, Malaysia. Rozilawati H, Zairi J & Adanan CR in 43rd Annual Scientific Seminar Of MSPTM & Centenary Celebration of the Royal Society of Tropical Medicine and -Advances of Biotechnology in Tropical Medicine"Diseases. 20-22 March 2007. Grand Seasons Hotel, Kuala Lumpur.

1.5 Rozilawati H, Zairi J & Adanan CR. 2007.Seasonal 141 abundance of Aedes albopictus in selected urban and suburban area in Penang, Malaysia. Tropical Biomedicine 24(1): 83-94.

xii KAJIAN TERHADAP KEPELBAGAIAN FAUNA NYAMUK DI KAWASAN BANDAR DAN PINGGIR BANDAR DI PULAU PINANG DAN KEBERKESANAN LINGKARAN UBAT NYAMUK YANG BERLAINAN KANDUNGAN BAHAN AKTIF TERHADAP NYAMUK VEKTOR TERPILIH

ABSTRAK

Satu kajian terhadap kepelbagaian fauna nyamuk di kawasan bandar dan pinggir bandar di Pulau Pinang telah dijalankan selama 14 bulan iaitu di Taman Permai Indah (kawasan pulau) dan Kampung Pasir Gebu (semenanjung). Dengan mengunakan teknik perangkap umpan lembu dan tangkapan menggunakan umpan kaki, telah didapati bahawa sebanyak 675 (22.80%) Culex gelidus yang merupakan spesis terbanyak di Kg Pasir Gebu diikuti oleh 514 (17.37%) Anopheles peditaeniatus, 383 (12.94%) Anopheles sinensis, 254 (8.58%) Mansonia uniformis, 252 (8.52%) Anopheles subpictus, 192 (6.49%) Anopheles campestris dan 135 (4.57%) Anopheles vagus. Nyamuk-nyamuk tersebut telah didapati lebih tertarik kepada umpan lembu berbanding umpan manusia. Penyampelan populasi telur di luar kediaman juga telah dijalankan dengan menggunakan perangkap telur atau ovitrap. Aedes albopictus merupakan spesis Aedes yang paling banyak di kawasan ini. Aedes egypti dan Culex quinquefasciatus juga telah didapati berada di dalam ovitrap yang sama tetapi hanya pada peratusan yang rendah. Keputusan ini menunjukkan bahawa Aedes albopictus adalah vektor denggi utama di kawasan kajian. Satu korelasi yang kuat telah didapati antara jumlah hujan dan populasi telur di kedua-dua kawasan kajian (r=0.982 dan r=0.918). Jumlah telur yang dikumpul pada musim hujan yang rendah adalah lebih banyak berbanding musim hujan yang tinggi. Nisbah antara nyamuk jantan dan betina yang terhasil juga menghampiri satu (TPI= 0.93+ 0.33 dan KPG= 0.97+0.42). Keberkesanan formulasi lingkaran ubat nyamuk terpilih telah dijalankan di makmal terhadap empat vektor nyamuk terpilih (Aedes aegypti, Aedes albopictus, Culex quinquefasciatus dan Anopheles sinensis). Anopheles sinensis merupakan spesis yang paling rentan terhadap semua formulasi, manakala Culex quinquefasciatus merupakan spesis yang paling rintang berdasarkan kepada keputusan nilai KT50, KT95 dan kadar kematian. Strain nyamuk di makmal juga telah didapati lebih rentan berbanding strain nyamuk dari lapangan.

xiii STUDIES ON THE MOSQUITO FAUNA IN AN URBAN AND SUBURBAN AREA IN PENANG AND THE LABORATORY EFFICACY OF MOSQUITO COILS CONTAINING DIFFERENT ACTIVE INGREDIENTS AGAINST SELECTED VECTOR MOSQUITOES

ABSTRACT

A study of the mosquito fauna in an urban and suburban area in Penang Island was carried out for 14 months namely in; Taman Permai Indah (on the island) and Kg Pasir Gebu (mainland). Using the cow baited trap and bare leg catch techniques, it was found that Culex gelidus totalling 675 (22.80%) was the most abundant in Kg Pasir Gebu followed by 514 (17.37%) Anopheles peditaeniatus , 383 (12.94%) Anopheles sinensis, 254 (8.58%) Mansonia uniformis, 252 (8.52%) Anopheles subpictus, 192 (6.49%) Anopheles campestris and 135 (4.57%) Anopheles vagus. The mosquitoes were more attracted to cow than human. Outdoor ovitrap surveys were also carried out in the urban and suburban sites and it was found that Aedes albopictus was the most abundant Aedes species in this area, even though a small percentage of Aedes aegypti and Culex quinquefasciatus was found to breed simultaneously in the same ovitrap. This indicated that the main dengue vector is Aedes albopictus. A strong correlation was found between rainfall and number of eggs in both of the study sites(r=0.982 and r=0.918). The eggs collected were more abundant during low rainfall (dry season) than during higher rainfall (wet season). The ratio between males and females that emerged from the eggs collected was also close to one (TPI=0.93+ 0.33 and KPG=0.97+0.42). The effectiveness of five selected mosquito coil formulations was also studied against four selected vector mosquitoes (Aedes aegypti, Aedes albopictus, Culex quinquefasciatus and Anopheles sinensis). Anopheles sinensis was the most susceptible against all the formulation, whereas Culex quinquefasciatus was found to be the most tolerant species against all the formulation based on the KT50, KT95 and mortality values. The laboratory strain mosquitoes were also more susceptible than the field strain.

xiv

CHAPTER ONE

GENERAL INTRODUCTION

Mosquitoes are small insects belonging to the family Culicidae of the order

Diptera. Mosquitoes are unquestionably the most important vectors of diseases

(Brenda et al., 2000).They are important because of the effects on human welfare by direct annoyance as nuisance biters and most of all due to the role they play in the transmission of diseases (Service, 1993). Mosquitoes are still a persistent problem in Malaysia. According to Abu & Salmah (1990), in

Malaysia including Sabah and Sarawak, there are 431 species representing 20 genera of mosquitoes.

Studies on the distribution and relative abundance of mosquitoes which frequent houses in urban/suburban areas indicated that Culex quinquefasciatus

(Say), Aedes albopictus (Skuse) and Aedes aegypti (Linnaeus) are the most abundant (Yap, 1975; Yap et al., 1978; Yap & Thiruvengadam, 1979; Yap et al.

1990a,b). Mosquito surveillance therefore plays an important role in formulating a good control programme (Service, 1993).

Mosquito-borne diseases such as dengue fever (DF) and dengue hemorrhagic fever (DHF) are the most important arthropod-borne viral diseases of public health significance. Their geographical spread is increasing: only five countries documented dengue in the 1950’s but to date there are more than 100 countries around the world reporting the incidence of DF and DHF (Guha-Sapir

& Schimmer, 2005). Several important factors also have influenced the epidemiology of dengue.

1 Aedes (Stegomyia) albopictus (Skuse), 1894 known as the Asian tiger mosquito and Aedes (Stegomyia) aegypti (Linnaeus), 1762 (Diptera: Culicidae) are the principal dengue vectors and to date have become the main vectors in the transmission of dengue and dengue haemorrhagic fever in the tropical and subtropical regions (Smith, 1956; Rudnick et al., 1965; Hammond, 1966;

Knudsen, 1995). The distribution of Ae.aegypti and Ae.albopictus in Peninsular

Malaysia has been well established (Lee, 1990).

In a study in an endemic dengue area in Selangor by Chen et al. (2005), it was found that mixed breeding of Ae. aegypti and Ae. albopictus occurred in the same container outdoors and indoors. Therefore, both mosquito species play an important role of in the transmission of the dengue virus. Furthermore it was reported by Lee & Inder (1993) that Ae. aegypti and Ae. albopictus are incriminated as dengue vectors in Malaysia.

Aedes albopictus is indigenous in tropical Asia but presently the distribution is world wide. The high incidence of dengue is closely associated with the abundance of the vectors. It was also reported that the abundance of the vectors is associated with environmental factors such as rainfall, temperature and relative humidity (Okogun et al., 2003), while the wet seasons are associated with the higher prevalence of mosquito borne diseases.

Several control measure are available in combating mosquitoes. One of the most widely used mosquito control approaches is personal protection. The usage of household insecticides is the most favoured personal protection method used by consumers. Among them, mosquito coil is still widely used in

2 Southeast Asia. It is important to test the effectiveness of coils being used to avoid resistance development in mosquitoes and other side effects.

Therefore, this study was conducted to look at the mosquito fauna in selected urban and suburban areas in Penang Island and to determine the efficacy of several mosquito coil formulations against selected vector species. The general objectives of this study are:

 To determine the composition and seasonal abundance of mosquitoes in

a selected urban and suburban areas in Penang Island.

 To determine the density, distribution and other physical parameters

relating to the fluctuations of Aedes albopictus.

 To determine the laboratory efficacy of several formulations of mosquito

coils against laboratory and field strains of Aedes aegypti, Aedes

albopictus, Culex quinquefasciatus and Anopheles sinensis.

3 CHAPTER TWO

LITERITURE REVIEW

2.0 Introduction

2.1 Mosquitoes

Mosquitoes are placed in the family Culicidae, suborder Nematocera of the order Diptera, the true flies (Barry & William, 1996). Culicidae contains 3500 species which are divided into three subfamilies: Toxorhynchitinae, Anophelinae and Culicinae (Knight & Stone, 1977). Anopheles, Culex, Aedes, Mansonia,

Haemagogus, Sabethes and Psorophora are genera of mosquitoes that are of medical importance because of their habit of biting humans for blood (Service,

1995a; Abu Hassan &Yap, 1999).

2.2 Medical importance of mosquitoes

Mosquitoes are very successful vectors. Some species are capable of transmitting diseases such as dengue, yellow fever, chingkungunya and

Japanese encephalitis (viruses), malaria (protozoa) and filariasis (nematode).

Aedes are of major concern in Malaysia because they transmit the dengue virus

(Lee, 2000). There are other species that are also of major concern as vectors such as Anopheles sp., mosquitoes that transmit malaria and filariasis

(Sulaiman, 2000), Culex sp. which transmit the Japanese encephalitis and urban filariasis (Adanan et al., 2000), and Mansonia sp. which are known vectors of filariasis (Chang, 2000). Some other arboviruses that can be transmitted by mosquitoes are Eastern Equine Enchephalitis (Coquilletidia perturbans), Ross River, Murray Valley Encephalitis (Culex annulirostris),

4 Sindbis, West Nile Virus (Cx. univittatus), Venuzuelan Equine encephalitis, St.

Louis Encephalitis, Rift Valley Fever (Cx. pipiens), Western Equine Encephalitis

(Cx. tarsalis), Japanese Encephalitis (Cx. tritaneiorhynchus), yellow fever (Ae. aegypti, Ae. africanus, Ae. simpsoni, Haemagogus sp.) and La Crosse

Encephalitis (Aedes triseriatus)(Monath, 1988).

2.3 Dengue

In tropical countries around the world, dengue is one of the most common viral diseases spread to humans by mosquitoes. Tens of millions of cases of dengue fever and up to hundreds of thousands of cases of dengue hemorrhagic fever occur each year. Globally an estimated 2 billion people are at risk of dengue while over 100 million people a year are infected with about 100,000 deaths (Gubler, 1997; CDC, 2005). Dengue remains of great public health importance in many tropical countries, causing considerable morbidity and significant mortality.

Dengue occurs in subtropical and tropical countries in the world (CDC,

2004). The spread of dengue is now considered a worldwide problem, since the global prevalence of dengue has grown dramatically in recent decades (WHO,

2002).

Dengue is a disease caused by a retrovirus belonging to the family of

Flaviviridae, genus Flavivirus (Urdaneta et al., 2005). It is transmitted by a mosquito vector of the genus Aedes. There are 4 serotypes of dengue virus

(DENV-1, DENV-2, DENV-3 and DENV-4) and all are co-circulating in Malaysia

(Gubler & Clark, 1996; Abubakar & Shafee, 2002). In 2005, dengue was the

5 most important mosquito-borne viral disease affecting humans; its global distribution is comparable to that of malaria, and an estimated 2.5 billion people live in areas at risk for epidemic transmission (CDC, 2005).

In Malaysia, the first reported DHF cases was in Penang in 1962

(Rudnick et al., 1965) while classical dengue was first reported in 1901-1902 in

Penang by Skae (1902). Major outbreaks were reported in 1974, 1978, 1982,

1990 and 1995 (Lam, 1993; Poovaneswari, 1993; Hairi et al., 2003). Since then, the disease has become endemic throughout the country (Singh, 2000). Up until

November 2005, there were 3098 cases reported in Penang, with 7 deaths

(MOH, 2005). In the last decade, cases of dengue have become more severe

(Hairi et al., 2003). The incidence rate of dengue has increased from 8.5 to

123.4 per 100, 000 respectively in 1988 and 1998 (Chua et al., 2005). The infection is predominant in urban areas where 61.8% of the total population lives and the rapid industrial and economic development created many man made opportunities for Aedes mosquito breeding (Teng & Singh, 2001).

Dengue vaccines have been touted as the most effective control measure for the disease (Lam, 1994). However no licensed vaccine is available to date. As there is no effective vaccine to prevent and no specific treatment for dengue, vector control remains the best strategy to prevent the disease. In

Malaysia, four strategies are applied: (1) Anti-larval measures; (2) Anti-adult measures; (3).Health education and (4) Enforcement of the Destruction of

Disease Bearing Insects Act (DDBIA) (Hairi et al., 2003). Vector control is the only option currently available to contain dengue outbreaks (Arunachalam,

1999).

2.4 Aedes as vectors

Aedes aegypti and Aedes albopictus are important vectors of dengue in

Malaysia (Vythilingam et al., 1999). According to Macdonald (1956), in

Peninsular Malaysia (known as Malaya before), Ae. albopictus is a very common species and its breeding preferences overlapped those of Ae. aegypti.

Reid (1954), Smith (1956), Rudnick et al. (1965) and Hammond (1973) have also reported that Ae. aegypti and Ae. albopictus are dengue vectors and to date have become the main vectors in the transmission of dengue and dengue haemorrhagic fever in tropical and subtropical regions worldwide (Yap et al.,

1994; Knudsen, 1995; CDC, 2004). The distribution of Ae.aegypti and

Ae.albopictus in Peninsular Malaysia is well established (Lee, 1990). A recent taxonomic review proposed to elevate the subgenus Stegomyia to the rank of genus. The new nomenclature proposed for these two species are, Stegomyia albopicta (Skuse), 1894 and Stegomyia aegypti (Linnaeus), 1762

(Inform'ACTION, 2005). In the present work, and to avoid confusion, we will continue to use the first nomenclature i.e. Aedes albopictus and Aedes aegypti.

7 The classification of Aedes aegypti and Aedes albopictus is shown below

(Knight & Stone, 1977):

Aedes aegypti (Linnaeus, 1762) Aedes albopictus (Skuse,

1894)

Kingdom: Animalia Kingdom: Animalia

Phylum: Artropoda Phylum: Artropoda

Class: Insecta Class: Insecta

Order: Diptera Order: Diptera

Family: Culicidae Family: Culicidae

Subfamily: Culicinae Subfamily: Culicinae

Genus : Aedes Genus : Aedes

Species: aegypti Species: albopictus

8 Whilst Ae. aegypti is entirely domestic, Ae. albopictus has been found breeding both in and around dwellings (Vythilingam et al., 1999). Both species are very adaptable to both tropical and temperate climate (Hawley, 1988). Both species are container breeders and both may be found together (Vythilingam et al., 1999). They are also capable of using a wide range of suitable container habitats. The most typical habitats are artificial containers, tree holes and bamboo stumps near human dwellings (Hawley, 1988). At the beginning of the

20th century, Aedes aegypti was found only in coastal towns (Daniels, 1908;

Leicester, 1908) and by 1920, it had already moved inland and was found in

Kuala Lumpur (Vythilingam et al., 1992).

According to Rudnick et al. (1965), from their studies on dengue studies in

Malaysia between1962-1964 showed that Ae. aegypti was dominant in urban areas, whereas Ae. albopictus is abundant in the suburban, rural and forested areas. Sulaiman et al. (1991), in their study on the distribution and abundance of Ae. aegypti and Ae. albopictus in endemic areas of dengue/dengue haemorrhagic fever in Kuala Lumpur, indicated that Ae. albopictus was more dominant than Ae. aegypti. Many researchers also reported that Ae. aegypti was more common in urban areas (Ho & Vythilingam, 1980; Lee, 1991;

O’meara et al., 1993; Lee, 2000) but the study conducted by Rohani et al.

(2001) indicated that Ae. albopictus was dominant in both rural and urban areas.

Both Ae. aegypti and Ae. albopictus are found in Malaysia, though Ae.aegypti is not an indigenous species (Rudnick et al., 1965).

9 The distribution of Ae. aegypti and Ae. albopictus in Peninsular Malaysia is well established (Lee, 1990) and has been found to overlap. Along with the establishment of Ae. albopictus, a decline in the density of Ae. aegypti has occurred in sites where their distributions overlap (Black et al., 1989; Nasci et al., 1989; Smith et al., 1990; Hobbs et al., 1991; O’Meara et al., 1992; 1993).

The establishment and spread of Ae. albopictus in the U.S is also associated with a reduction in the abundance and the range of the yellow fever mosquito

Ae. aegypti (Hawley 1988; Hanson et al., 1993).

Studies on the dispersion studies of Aedes aegypti conducted by Harrington et al. (2005) showed that in outdoor releases of males and females, the majority of recaptures were made in the house adjacent to their outdoor release location.

The maximum dispersal distance detected was about 556–594 meters for females and 400–456 meters for males, while in indoor releases of females, the majority of recaptured mosquitoes (77%) were collected in the house from which they were released and the maximum dispersal distance detected was about 52 meters from the release site. Other studies on dispersion, conducted by Honório et al. (2003), have shown that Aedes aegypti and Aedes albopictus can be found as far as 800m.

In Southeast Asia, Ae. albopictus has been incriminated as a secondary vector of dengue while Ae. aegypti as the principal vector of the dengue viruses

(Sulaiman et al., 1996). Aedes albopictus inhabits all of Southeast Asia and parts of temperate Asia, where it transmits the dengue fever virus, Dirofilaria immitis (dog heartworm) and other pathogens (Hanson et al., 1993). In addition to its ability to transmit yellow fever and dengue viruses, Ae. albopictus also is a

10 competent laboratory vector of viruses endemic to the United States, including eastern equine encephalitis, La Crosse Encephalitis, St. Louis Encephalitis,

Western Equine Encephalomyelitis Viruses, Eastern Equine Encephalitis Virus and Jamestown Canyon Virus (Shroyer, 1986; Scott et al., 1990; Mitchell,

1991; Mitchell et al., 1992; Grimstad et al., 1997; Moore & Mitchell, 1997).

2.4.1 Biology of Aedes albopictus

All mosquitoes urdergo complete metamorphosis to complete their life cycle. For Aedes sp. they only need clear water, but not necessarily clean water to complete their life cycle (Lee, 1990).

2.4.1.1 Eggs

About 48-72 hours after the females take a blood meal, they begin laying eggs. Aedes albopictus is a container-inhabiting species which lay its eggs in any water-containing receptacle in urban, suburban, rural and forested areas.

The primary immature habitats of this species are artificial containers such as tyres, flower pots, cemetery vases, and even in natural containers such as tree holes, bamboo pots and leaf axils. This mosquito prefers to lay its eggs above the water surface on the dark rounded vertical surface. They deposit them just above the water line on damp substrate, such as mud or leaf or on the inside of tree holes (Service, 1995a). Different from other species, Aedes oviposit their eggs singly and are black in colour. Like other species in the Culicine group, the eggs are elongated and protected by a rigid, proteinaceous shell that minimizes water loss but permits gas exchange. The eggs can withstand desiccation, remain dry for months but still remain viable and hatch when soaked in water

(Service, 1995a). According to Lee (2000), one female can deposit 102 eggs.

11 However newly deposit eggs cannot withstand desiccation (Kettle, 1990). The embryo needs time to develop, hence the eggs needs to be dried slowly (Kettle,

1990). After the embryo is fully developed, it can withstand desiccation for a few months. Several physical factors affect the egg hatching such as water temperature and oxygen pressure.

2.4.1.2 Larvae

Once the eggs hatch, the first instar larvae will emerge. The larvae require water to develop, no larvae can withstand desiccation. All stages of the larval instar (1st, 2nd, 3rd and 4th) are bottom feeders and only use their siphons to breath at the water air interface (Lee, 1990). Depending on the temperature and the availability of food, Ae. albopictus can complete its larval development between 5 to 10 days.

The Ae. albopictus larvae can be identified by several taxonomic characters. A particularly useful characteristic is the nearly complete saddle found in the early instar specimens of Ae. albopictus as well as the late instar larvae. The lateral hairs on the saddle are useful because they can be observed in living specimens without special orientation. The lateral hairs are double in

Ae. albopictus. The four long caudal hairs of the dorsal brush in Ae. albopictus are also a useful character because they can be discerned at very low power.

However, it should not be used as the sole character to identify this species.

Aedes albopictus is an opportunistic container breeder that is capable of utilizing natural as well as artificial container habitats. It has the ability to adapt

12 to an exceptionally wide range of confined water sources. The mosquito is known for its ability to survive in very small collections of water, requiring only a depth of 1/4" to complete its life cycle. Larval habitats of the population discovered included discarded tires, 50 gallon drums, plastic buckets of various sizes, dishpan, plastic drinking cups, crushed aluminium beverage cans and cemetery vases (Lee, 1990; Rohani et al., 2001).

2.4.1.3 Pupae

All pupae are aquatic, comma shaped and dark in colour. They are non- feeding aquatic forms. They spend most of the time at the water –air interface taking in air through the respiratory trumpets. If disturbed they swim up and down in the water in a jerky fashion (Service, 1995a). The life span of the pupae is between 2 to 3 days.

2.4.1.4 Adult

In 24 to 48 hours the pupae will emerge into adults. The adult body is divided into the head, thorax and abdomen. The head bears a pair of compound eyes and antennae as well as mouth parts. A pair of jointed legs is formed on each segment of the thorax. A pair of wings is found on the last 2 thoracic segments. The abdomen is composed of ten segments.

Adults males and females Ae. albopictus are covered with shiny black scales with distinct silver white bands on the palpus and tarsi. Its most striking taxonomic character is the band of silver scales forming a distinct stripe on the dorsal surface of the thorax and head. When they are full grown, the adults emerge from the pupae in the water and after resting on the water surface, they fly away to search for hosts to blood feed. Ae. albopictus is a very aggressive

13 daytime biter with peaks generally occurring during the early morning and late afternoon. It feeds on a large number of hosts including man, domestic and wild animals and this generalized feeding behaviour contributes to its vector potential. Only female adults feed on animal blood while male adults feed on plant juice. A female mosquito has to obtain blood meal for eggs development.

Habitats of the females can be permanent stagnant water, flowing water, temporary stagnant water or containers. Generally male mosquitoes only survive about one week but the females can live up to two to three weeks.

2.5 Other mosquito of medical importance

Other than Aedes, several species are of medical importance because of their habit of biting humans for blood meal such as Anopheles, Culex, Mansonia,

Haemagogus, Sabethes and Psorophora (Service, 1995a; Abu Hassan & Yap,

1999).

2.5.1 Culex quinquefasciatus

Culex quinquefasciatus is a medium size brownish mosquito. This species transmits bancroftian filariasis and is predominantly found in the tropics and temperate regions (Sharma, 2001). Culex quinquefasciatus larvae breed and thrive abundantly in stagnant dirty water (Mak, 1986; Hidayati et al., 2005).

However at times it is also found together with Aedes in clear water. West Nile

Virus has also been isolated from Cx. quinquefasciatus in Mexico (Darwin et al.,

2005); and Lousiana (Marvin et al., 2005).

14 This mosquito is regarded as a nuisance in Malaysia. However, owing to the rapid urbanization and unplanned growth of cities, the risk of urban bancroftian filariasis transmission will also increase since this mosquito is a vector of urban bancroftian filariasis in other countries (Lee, 2005). Culex quinquefasciatus is a night biter. In Malaysia, though urban bancroftian filariasis has been eliminated; cases have been detected in migrant workers from endemic areas. Hence, the re-introduction of urban bancroftian filariasis is possible in the presence of Cx. quinquefasciatus (Lee, 2005).

2.5.2 Culex gelidus

Culex gelidus is a paddy field breeder in the countryside. It is highly zoophilic in nature (CRME, 1989) and prefers to bite large animals such as cattle and pigs rather than humans at night (Miyagi & Toma, 2000). Culex gelidus can be easily recognised by the white scales on the thorax. It rests inside houses, cattle sheds and tents.

According to Lee (2005), Culex gelidus is also found in India, China,

Thailand, Indonesia, Timor and Irian Jaya. It has been reported as a veracious biter of humans indoors and having a preference for larger domestic animals with little preference for human (Colless, 1959) The larval stage can be found in freshwater ground pools, rivers, marshes and containers, dirty water and sometimes with considerable organic matter (Craig et al., 2005).

15 Culex gelidus is primarily a vector of Wuchereria bancrofti, chikungunya virus and getah virus. It is apparently refractive to Dirofilaria immitis (Dog heartworm) and Brugia malayi (Malayan filariasis) (Miyagi & Toma, 2000).

Culex gelidus can also transmit the Japanese enchephalitis virus (JE). The JE disease remains endemic in several countries in Southeast Asia including

Malaysia (Miyagi & Toma, 2000).

2.5.3 Mansonia uniformis

Mansonia uniformis, is a mid-sized mosquito of mottled brownish appearance. Adult Ma. uniformis appears to be active mostly at night, but also bites during the day in or near shelter. They can disperse a few kilometres from their habitats and readily attack humans as well as other animal including birds

(Clements 1999). This mosquito has a highly antropophilic nature and only enter houses to feed (Iyenger, 1938; Wharton, 1962 and Mahapatra et al.,

1995).

The breeding sites characteristics for this species include open swamp forest, neglected rice fields, blocked drains, rivers, canals and neglected ponds in urban and rural areas (Chang, 2000), whereas the main host plants are floating aquatic vegetation such as Eichornia, Salvinia, and swamp grasses.

Mansonia uniformis is primary a vector of Wuchereria bancrofti

(bancroftian filariasis), Brugia malayi (malayan filariasis) and Brugia pahangi

(tropical eosinophilia), chikungunya virus was also isolated from this species

(Chiang & Loong, 1985; Miyagi & Toma, 2000).

16 2.5.4 Subfamily Anophelinae

Anopheles is the only genus in this subfamily which is medically important being the sole vector in the transmission of malaria. Anopheles mosquitoes breed in permanent bodies of fresh water with an abundance of aquatic plants that provide protection from fish and other predators. Eggs supported by floats are laid singly on the water surface. Anopheles mosquitoes can be distinguished from Aedes and Culex mosquitoes in several ways (Plate 2.1), as follows:

Identification of larvae:

1. Absent of siphon

2. Hair no 1 is modified like fan (palmate hair on abdomen)

3. Anopheles larvae float parallel to the surface of the water as opposed to

hanging down at an angle.

Identification of adults:

1. Anopheles have patterned wings,

2. Adults rest on surfaces with their head lower that the abdomen while

Aedes and Culex species rest with the head and abdomen parallel to the

surface

3. The scutellum is rounded, (Culicine scutellum is trilobed).

4. Adult Anopheles females have palps that are almost as long as their

proboscis

Plate 2.1: The characteristics used for identifying mosquitoes of general importance (Source: IMR. Entomological teaching charts)

2.5.4.1 Anopheles peditaeniatus

Anophele peditaeniatus is largely zoophilic and the adults are found abundantly around cattle sheds (Zairi, 1990). In India and Malaysia, adult An. peditaeniatus can always be distinguished by the long hind tarsal pale bands, a long dark mark on vein 5, the line of white scales along the remigium and the bare humeral cross vein (Reid, 1968). Anopheles peditaeniatus is one of the commonest species found in rice field (Zairi, 1990). Aside from being numerous in rice fields, larvae are found in swamps and grassy ponds (Reid, 1953). This species has been recorded in India, Sri Lanka, Myanmar, Thailand, Indochina,

China, Malaysia, Indonesia and the Philippines (Knight & Stone, 1977).

It can be readily infected with Wuchereria bancrofti, Brugia malayi and

Dirofilaria immitis but rapid development of calcified cysts around dead worms indicates poor vector potential (Reid et al., 1962; Wharton et al., 1963).

Furthermore, according to Wharton et al. (1963), the proportion biting man in nature was probably too small to pose a real danger as a vector of human diseases. Therefore it is also not considered as a disease vector in Malaysia

(Zairi, 1990).

19 2.5.4.2 Anopheles sinensis

Anopheles sinensis is generally regarded as a zoophilic and exophilic species although it can also bite human but, only outdoors and after dark (Reid,

1953). Anopheles sinensis is not a vector in Malaysia (Reid et al., 1962), however it is a vector of malaria in other Asian countries such as Japan, China and Korea (Ohmori & Otsuru, 1960; Ho et al., 1962; Kim, 1974). Anopheles sinensis is also a vector of brugian filariasis and bancroftian filariasis (Chiang &

Loong, 1985; Miyagi & Toma, 2000).

The distribution of An. sinensis ranges from Japan and Korea through central and southern China, Taiwan, Hong Kong, Vietnam, Cambodia,

Peninsular Malaysia and Singapore, west ward to the Union of Myanmar

(Burma) and Assam, but it is absent from the rest of India (Knight & Stone,

1977; Zairi, 1990). It breeds in open grass ponds, especially in rice fields (Zairi,

1990).

20 2.6.4.3 Anopheles campestris

In Malaysia, Anopheles campestris is probably the most antropophilic and endophagic of all anopheline mosquitoes (Zairi, 1990). This species was formerly identified as a dark winged form of An. barbirostris (Reid, 1947). In

Malaysia, although 75 species of Anopheles have been recorded, only 9 have shown to be vectors of malaria (Rahman et al. 1997, 2002) including An. campestris. This species can also be a potential vector of filariasis by transmitting Brugia malayi mainly in swampy rice-field terrain (Chiang & Loong,

1985; Miyagi & Toma 2000). The larvae commonly breed in corners of rice fields and burrow pits in coconut plantations, and sometimes found in slightly brackish water (Chow, 1970; Chooi, 1985).

2.5.4.4 Anopheles vagus

Anopheles vagus is a zoophilic species (Wharton, 1953). It is abundant in houses and cow sheds and rest inside cars or small boats. This species is closely related to An. subpictus except that the apical pale band of the palps is usually broader, the subapical dark band narrower and the tip of the proboscis usually has an obvious pale mark (Reid, 1968). It is not considered as a vector in Malaysia, however, it has been found naturally infected by malaria parasite in

India and also considered as a secondary vector of bancroftian filariasis (Lee et al., 1983; Rao, 1984). It is distributed throughout India, Sri Lanka, Andaman

Islands, Myanmar, Thailand, Indo-China, China, Malaysia, Indonesia, Papua

New Guinea, the Philippines and the Marianas Islands (Knight & Stone, 1977).

According to Covell (1944), the larvae are typically found in small freshwater pools and puddles and also in brackish water.

2.5.4.5 Anopheles subpictus

Anopheles subpictus is generally regarded as a zoophilic species but only a small proportion feeds on man (Zairi, 1990). Many studies have reported that An. subpictus is primarily zoophilic, more attracted to bovine than human

(Roy, 1943; Covell, 1944; Collins et al., 1979). It was experimentally infected with malaria parasite but its role in transmission is undelianeated. However, An. subpictus appears to be a malaria vector on the coast of Southeast India

(Panicker et al., 1981) and in Indonesia as a major vector of bancroftian filariasis (Lee et al., 1983). Anopheles subpictus is distributed in India, Nepal,

Pakistan, Afganistan, Iran, Sri Lanka, Myanmar, Thailand, Cambodia, Malaysia,

China, Indonesia, Maldives, Papua New Guinea and the Marianas Islands

(Knight & Stone, 1977). The larvae are found both in fresh and brackish water

(Zairi, 1990). It has also been found together with An. aconitus in rice fields and with An. sundaicus in lagoons (Sundararaman et al., 1957; Soerkirno et al.,

1983). However An. subpictus is confined to the coast in brackish water even though the habitats sometime overlap with An. indefinitus (Zairi, 1990).

2.6 Physical factors

According to WHO (2000), many countries in Asia experienced unusual high levels of dengue and/or dengue haemorrhagic fever in 1998, the activity being higher than in any other year (Andrew et al., 2000). Since laboratory experiments have demonstrated that the incubation period of dengue 2 virus could be reduced from 12 days at 300C to 7 days at 32-350C in Aedes aegypti

(Watts et al., 1987), changes in weather patterns, may be the major contributing factor to the high incidence of the disease.

Temperature, rainfall and relative humidity are physical factors that influence the abundance of the mosquitoes. According to Lee (1990), with no changing seasons in our country’s weather, therefore there is no significant difference in larval numbers throughout the year. However, indoor temperature may provide a suitable condition for Aedes breeding. In general, insects are exceedingly sensitive to temperature and rainfall regiments and tropical and temperate species frequently show great variations in seasonal abundance

(Samways, 1995). In tropical and subtropical climates, Ae. albopictus is abundant all year round; however, in temperate climates such as the

Midwestern United States, Japan and Argentina, the active season for the larval stages is limited to late spring through early fall, with larval abundance greatest in July- August (Mori & Wada, 1978; Toma et al., 1982). The temperature fluctuations affect the mosquito populations and allow Aedes proliferations only between September and April (deGarin et al., 2000).

2.6.1 Rainfall

Rainfall is the most important factor that affects Aedes breeding (Khim,

2003). Reproduction of Ae.aegypti populations in tropical and subtropical zones occurs all year round and their abundance can either be associated with rainfall regimens (Moore et al., 1978; Chadee, 1991, 1992; Kalra et al., 1997; Micieli &

Campos, 2003) or no asoociation is observed (Shepped et al., 1969, Barrera et al., 1997). Generally, Aedes breeds after rain, not during raining days. With heavy rainfall, water in containers will overflow, and consequently larvae cannot survive in it (Lee & Cheong, 1987). In the study on adult females of Aedes

23 albopictus in Kuala Lumpur, the highest peak can be seen in September, the lowest in May, these situations are closely related to rainfall (Sulaiman & Jeffrey,

1986). According to Chan et al., (1971a) in a study in Singapore there was a few high and low peaks for Aedes albopictus adult female population between

March, June-July and November-December. The larvae and pupae are higher after two months of high peak of adult population.

2.6.2 Temperature

Mosquitoes are sensitive to temperature changes as immature stages in its aquatic environment and as adults. If the water temperature rises, the larvae take shorter time to mature (Rueda et al., 1990) and consequently there is a greater capacity to produce more offspring during the transmission period. Adult female mosquitoes digest blood faster and feed more frequently in warmer climates, thus increasing transmission intensity (Gillies, 1953). However, warming above 340C generally has a negative impact on the survival of vectors and parasites (Rueda et al., 1990).

2.6.3 Relative humidity

High relative humidity can give high hatching rates. With 100% humidity the eggs can hatch on filter papers. It is important to allow slow desiccation of eggs as the embryo takes time to develop prior to the drying process. The low relative humidity also gives negative impact on egg hatching (Horsfall, 1956).

With the tropical weather in this country, the high relative humidity has little impact on eggs development (Manorenjitha, 2005).

24 2.7 Surveillance of mosquitoes

2.7.1 The oviposition trap (ovitrap)

The oviposition trap (ovitrap) is an important component in the monitoring of the mosquito-borne diseases (Bentley & Day, 1989). The ovitrap was first developed in the United States for the surveillance of Ae. aegypti (Fay & Perry,

1965; Fay & Eliason, 1966). This technique was used because ovitrap was a sensitive and efficient technique for detecting the population of Aedes even when the population densities were low. Therefore, a sudden increase of the mosquito population can be detected (Evans & Bevier, 1969; Jacob & Bevier,

1969; Thaggard & Eliason, 1969). It is also safe, economical and environmentally friendly (Chan et al., 1977). Ovitrap surveillance was also found to be an effective and sensitive technique than conventional larval survey (Lee,

1992a; Arunachalam et al., 1999).

Oviposition trap have been used as it is effective and can minimise the disruption of natural and artificial container oviposition habitats (Novak et al.,

1991). Currently, ovitraps are used as a mean of detecting the presence as well as an approximate gauge of the adult population in an area (Ginny & Ren,

2000). It gives an approximate indication of the adult population in an area and act as an early warning signal to pre-empt any impending dengue outbreaks.

Ovitrap also gives very helpful information when used together with the

Geographic Information System (GIS) (Ginny & Ren, 2000). According to Ginny

& Ren (2000), it can be used to estimate fairly well the population of adult mosquitoes in the environment by counting the number of eggs laid on the moist paddle. In this way, a sudden increase of mosquito population can be

25 detected. Changes in the predominance of species breeding (Aedes aegypti to

Aedes albopictus or vice versa) can also be detected.

Ovitraps have also been used to monitor ports for exotic mosquitoes

(Ritchie, 2001), to study mosquito behaviour (Allan & Kline, 1995; Reiter et al.,

1995), to measure the efficacy of control measures (Reiter & Gubler, 1997) and to collect mosquitoes for research purposes such as mark-release recapture

(Reiter et al., 1995) and resistance studies (Canyon & Hii, 1999).

In recent years, ovitrap surveys for monitoring the Aedes aegypti population have found greater acceptability (Santos et al., 2003), as these have been found to be sensitive even at times when vector densities are at low levels

(Chadee et al., 1993; Rawlin et al., 1998; Braga et al., 2000). These traps enable us to establish the Ovitrap Index (OI) and the Egg Density Index (EDI).

Although the ovitrap is a method that is operationally viable, if it remains in the field for more than the maximum of seven days, it becomes a potential breeding site by itself (Santos et al., 2003). Therefore, any ovitrap monitoring should be done in the interval of less than seven days in the field.

According to Yap (1975) Ae. albopictus was the most dominant Aedes species in Penang. Because of its widespread distribution, Ae. albopictus was found associated with numerous container-inhabiting mosquitoes such as

Aedes, Culex, Toxorhynchites and Armigeres but in general even if it is the most abundant species present in shared habitats, it often occurs alone. In fact,

26 Culex quinquefasciatus was also found to be attracted to oviposit in ovitraps

(Rawlins et al., 1998). Selection of oviposition sites by Ae. albopictus are affected by habitat type, light, temperature, and humidity, as well as characteristics of water (biotic and abiotic) and even the oviposition surface

(Bently et al., 1976).

The purpose of vector surveillance is to monitor the total number and density of Aedes mosquitoes (Pang et al., 1991). Therefore, dengue vector surveillance should not just be restricted indoors but should extend outdoors since Ae. albopictus is frequently found outdoors (Lam, 1996). In the study by

Chan et al. (1971b), they found that the most common out-of-door habitats of

Ae. albopictus in Singapore were discarded tin cans, and Chan (1971a) made use of them as convenient ovitraps. The efficacy of plastic, glass and metal ovitraps also has been studied by Bellini et al. (1996) and they found that plastic and glass ovitraps gave similar result, while metal ovitraps collected a significantly lower number of eggs. There have been many modifications to the classical ovitraps, for example, substituting black painted tins or black plastic beakers for glass bottles (Service, 1993); in Thailand, similar traps were used to study Aedes mosquito distribution as in Penang (Yap, 1975); Mogi et al.

(1990) used greenish dark-gray ceramic ant traps that formed circular trough as ovitraps but the concept of ovitrap was still the same i.e, monitoring the Aedes population by providing a place for mosquitoes to lay eggs and the ovitrap helps to identify the mosquito species in an area even at the low adult density.

2.8 Control of vector mosquitoes

2.8.1 Control approaches

Greater efforts have been made to control mosquitoes than any other biting insects. Control measures can be directed either at the immature aquatic stages or the adults or at both stages simultaneously (Service, 1995b). Several control methods such as biological control, genetic control, physical control and chemical control are been applied nowadays. However, according to Yap

(1984) source reduction is the main solution to control mosquitoes but with the lack of cooperation from the community, this method is not an effective approach in our society (Khim, 2003). People forget that the simplest way to control mosquitoes is just by keeping our environment in healthy condition.

Source reduction and environmental management are the best approaches for a long term solution to most pest problems including mosquitoes.

At present, control of mosquitoes in Malaysia relies heavily on the use of insecticides. These chemicals are usually applied in the form of space sprays

(ULV and thermal fogging), especially for the control of vector mosquitoes

(Aedes and Culex species) in urban environments (Yap &Chavasse, 1997, Yap

& Lee, 1999). Strategies for mosquito control especially the control of dengue vectors (Aedes sp.), rely primarily on defensive governmental epidemic prevention measures, which are deemed not successful.

28 Chemical control provides the most immediate and usually the most effective control (Lee & Robinson, 2001). Chemical control is also the most common and widely used throughout the world. Several insecticides are used to control larvae in water and adults either outdoors or indoors. According to Gratz

(1991), chemical control usually is the best approach to control the adult stages.

There is no single control method ever sufficient to lower mosquito level for a long period of time. Nevertheless, vector control is the only option currently available to control dengue outbreak. However, besides source reduction there is no single method that can be applied over a long period time. Integrated control i.e combining biological and insecticidal methods have become an effective approach to control mosquitoes (Service, 1995b).

One of the integrated approaches in controlling mosquitoes is personal protection (Service, 1995b). One cannot be depending on others or solely on the government to prevent man-mosquitoes contact. Household insecticides such as mosquito coils, aerosols, mats, liquid vaporizers and impregnated bed- nets can be used as personal protection (Yap et al., 1994).

However, the use of larvicides such as microbial agents and IGRs to kill mosquito larvae are still limited (Yap et al., 1994). Owing to the heavy use of chemicals, it is important to conduct continuous research to find insecticides that are effective and much safer to human in order to prevent unwanted side effects such as resistance and environmental contamination (Yap & Chung,

1987; Yap et al., 1990a,b). Recent research findings indicate that the dengue virus goes through a transovarial transmission cycle from the infected Aedes

29 female mosquitoes to the larvae via the eggs( Lee & Rohani, 2005). Therefore it is imperative that in dengue vectors control, adulticidal and larvicidal activities should be conducted simultaneously in order to break the transmission cycle.

2.8.2 Household insecticides-mosquito coil

Household insecticide products can be used as a personal protection measure and easily implemented by the community. Household insecticide products are used throughout the world, especially in tropical countries (Jurgen

& Umar, 1999) where individual personal protection from mosquitoes is a must to maintain a certain level of comfort and to avoid the spreading of endemic mosquito-borne diseases. For personal protection against mosquitoes, various household insecticide products (mosquito coils, mats, liquid vaporizers, aerosols and repellents) are used (Yap & Foo, 1984; Yap, 1988). Mosquito coils are the most commonly and widely used personal protection method in Asia,

Africa and South America (Koo, 1994; Yap et al., 1990b). Mosquito coil offers an effective and most economical measures to control or prevent the transmission of vector-borne diseases (Snehalata et. al, 2003).

Mosquito coils are more widely used in Malaysia than any other household insecticide products such as mats and aerosol (Yap & Foo, 1984). The efficacy of mosquito coil does not vary with time due to the continuous and uniform release of a.i throughout the burning period (Mac Iver, 1963, Anonymous, 1987).

When a mosquito coil is burned, the insecticide evaporates with the smoke, which prevents the mosquito from entering the room (Weili et al., 2003).

Mosquito coil was originally made by mixing pyrethrum powder, wood powder

30 and a binder made of plant origin (Toshiji, 1996). It contains organic filters, binders, dyes and other additives capable of smouldering well. The temperature of the burning points of mosquito coil reaches about 7000C (Toshiji, 1996). The pyrethrin or pyrethroid is diffused at the portion when the temperature is about

1600C and 5mm from the burning end. The diffused insecticide in the air exerts biological impact on the mosquitoes.

The study on insecticides efficacy is important to achieve its purpose in protecting the general population from undesired side effects, since insecticides are widely used indoors. Other than cost, the benefit of household insecticides products should be fully considered in order to gain effective protection to persons using this type of protection.

2.8.3 Pyrethroid insecticides in mosquito coil

Pyrethrins are the collective name for a group of six pesticidal compounds derived from flowers in the genus Chrysanthemum. Pyrethrum extracts are relatively low in term of acute toxicity. Several synthetic derivatives of pyrethrin molecules, known as pyrethroids are also widely used in household insecticides. Accounting for about 0.3-0.4% of coil mass, the major active ingredients of the mosquito coil today are pyrethroids (Lukwa & Chandiwana,

1998).

31 Currently, the major insecticides used in household insecticides products belong to the class of pyrethroids. Pyrethroids appear to be the first choice insecticides due to their favourable properties concerning effectiveness and toxicity (Jurgen & Umar, 1999). According to Moretto (1991), the world-wide tendency is to use pyrethroids preferably for the public health control of vectors.

Active ingredients used in mosquito coils are from short-lived knockdown pyrethroids including prallethrin, d-allethrin and d-transallethrin. According to

Yap & Chung (1987), pyrethroids are also known as the 80’s insecticides.

Pyrethroid is more toxic to target organisms (insect pests) but safer or less hazardous against non-target species such as mammals (Ware 1994; Elliot,

1995). According to Barlow & Hadaway (1975), pyrethroids do not change structurally in the environment because of their stability. Pyrethroids are lipophilic and do not dilute in water (Elliot et al., 1978). According to Spiller

(1996), pyrethroids are photolabile, low mammalian toxicity, effective even at very low concentrations and also vaporise readily and efficiently when heated

(Chadwick, 1975).

2.8.4 Factors influencing laboratory efficacy

There are many factors that influence laboratory efficacy testing of mosquito coils including choice of test insect and mosquito culture condition

(Yap & Chong, 1996). The physical conditions are the most important (Mac Iver,

1963). Higher fly activity affect the mortality of mosquitoes (David & Bracey,

1946). The culture condition also affects the test insect response towards the

32 coil emission (Yap & Chong, 1996). According to Yap et al. (1996), the susceptibility of both Ae. aegypti and Cx. quinquefasciatus towards coil decreases with age. A decrease in KT50 values and an increase in post- treatment mortality occurred for female mosquitoes aged 10 days and above.

Their findings also showed that blood-fed mosquitoes were more difficult to knockdown but easier to kill compared to non blood-fed mosquitoes (Yap et al., 1996). The test insects used in this study are non blood-fed female mosquitoes aged 3-5 days. Only female mosquitoes were used as test insects because they bite to take blood meals (Clements, 1999). The choice of test insect is an important determinant as certain species such as the Cx. quinquefasciatus are more tolerant to a wide range of insecticides when compared to Aedes species (Yap & Chong, 1996).

CHAPTER THREE

SURVEILLANCE OF MOSQUITO FAUNA IN AN URBAN AND SUBURBAN

SITE IN PENANG

3.0 INTRODUCTION

Mosquito surveillance is essential for the planning, operation and evaluation of an effective mosquito control programme, whether for the prevention of mosquito borne diseases or to reduce the mosquito population to levels permissible for normal activities without undue discomfort. Surveys are made to identify the species of mosquito present and provide general information on distribution, densities and disease potential.

Various methodologies have been used in mosquito surveillance. Adult biting and larval breeding indices are widely used, while at low population levels, ovitraps are more economic and sensitive than larval surveys (Fay & Eliason,

1966). This type of sampling allows estimation of the average population density through the frequency of the positive sampling units, consequently reducing required operating time and cost.

Developing control system for these pests requires knowledge of their seasonal abundance and general pattern of population growth. The abundance of mosquitoes species has been found to vary according to local meteorological conditions. Several factors contribute to mosquito development such as rainfall, temperature, relative humidity and microhabitats of the breeding places.

34 In order to survey the mosquito fauna and the relation with the meteorological factors in selected areas in Penang, a survey was conducted for both adult and immature stages of several mosquito species. The objectives of this study are:

1.0 To study the biology and life cycle of the mosquitoes,

1.1 To study the seasonal abundance of Aedes using ovitrap technique and

other vector mosquito species in selected urban and suburban areas on

Penang Island using the bare leg catch and cow baited trap techniques,

1.2 To study through routine surveillance, any changes in the vector density,

distribution and other physical parameters relating to the fluctuations of the

vector population, and

1.3 To determine the annual seasonal fluctuation of the vectors.

3.2 MATERIALS AND METHODS

3.1.1 DESCRIPTION OF STUDY SITES

A general survey of Penang state was carried out before the study was conducted. Two areas comprising of a sub-urban and urban site were selected for this study. The sub-urban site was Kampung Pasir Gebu and the urban site was Taman Permai Indah. The choice of the sites was based on the relative abundance of mosquitoes species, the location, (close to the laboratory of

VCRU, USM) and the record of high dengue cases in 2002 and 2003.

3.1.1.1 Kampung Pasir Gebu

Kampong Pasir Gebu is a coastal village situated about 40km north of the island of Penang. This area was chosen for this study because of the availability of suitable habitats for mosquito breeding. The principal crop grown here is rice and coconut palms are also abundant (Plate 3.1). It is close to the sea. The houses are mostly constructed from wood and bricks, fairly close to each other amongst the coconut grooves and scattered on both sides of a small road (Plate 3.2). There are also mangrove swamps along the costal area. Other than that, some natural vegetation consisting of shrubs, clumps of nipah palms and tall grasses or weeds and some floating vegetations such as Hydrilla,

Lemna and Salvinia are common in this area (Plate 3.3). The residents also have livestocks such as chickens, ducks, goats and cattle near the residential area (Plate 3.4).

Plate 3.1: Paddy is the main crop grown in Kampung Pasir Gebu

Plate 3.2: Houses in Kampung Pasir Gebu

Plate 3.3: Natural aquatic plants in Kampung Pasir Gebu

Plate 3.4: Chicken coop built close to the residence houses in

Kampung Pasir Gebu

3.1.3.2 Taman Permai Indah (TPI)

On Penang Island, the study was carried out in Taman Permai Indah,

Sungai Dua which is an urban residential area. This site was chosen based on the highest dengue cases reported in 2002 on Penang Island (MOH, 2003).

This area consists of ten blocks of flat buildings, each separated by a small road and car parks (Plate 3.5, 3.6). A small river flows along the side of the buildings and are surrounded by shrubs, weed and banana plants (Plat 3.7). The drainage (Plate 3.8) and sanitation systems are quite good but rubbish consisting of artificial containers are found scattered throughout the area.

Plate 3.5: Taman Permai Indah, Sg Dua

Source: Google earth

Plate 3.6: Flat buildings in Taman Permai Indah

Plate 3.7: A small river which flows through Taman Permai Indah

Plate 3.8: Concrete drainage in Taman Permai Indah

3.1.2 SAMPLING TECHNIQUES

During the initial stages of field work, adult mosquitoes were collected in

Kampung Pasir Gebu, using two techniques, that is the bare leg catch (BLC) and the animal (cattle) baited trap (CBT), 12 hour all night catches for every two months starting from March 2003 until April 2004. Mosquitoes were collected while they landed on the host to bite or while in the process of biting a human or an animal host. Immature stages were collected using the ovitrap technique.

3.1.2.1 Adult collection

3.1.2.1(a) Bare-leg Catch

The bare leg catch is better known as the landing biting catch (Zairi,

1990; Damar 1997), originally described by Haddow (1954).The field activity consisted of a 12 hour all night catches, commencing at sunset and ending at sunrise. For this technique, three collectors were placed outdoor (rotating at 4 hour intervals) as baits at every early 45 minutes for each hour. Mosquitoes were located using torch lights and collected using vials as they landed on the bare legs as well as on other parts of the body (plate 3.9). All mosquitoes were brought back to the laboratory for identification. The collections were conducted once every two months (frequency: seven collections).

Plate 3.9: The bare leg catch (BLC) technique.

3.1.2.1 (b) Cow-baited trap

The cow-baited trap is based on the technique employed by Reid

(1961) in his comparative studies on the attraction of mosquitoes to human and animal baits (Zairi, 1990; Damar, 1997). The trap consisted of a large cotton mosquito net which measured 2.4m by 2.4m and 1.6m high, with a 1.2m door on one side which was closed by zippers. The net was hung 0.3m above the ground to facilitate the entry of mosquitoes, and a cow, serving as a bait, was tethered inside it (Plate 3.10). A collector entered the net and collected mosquitoes resting on the side of the net. The mosquitoes were collected for 15 minutes at the end of each hour. All mosquitoes were collected using aspirators and were placed in plastic containers or vials plugged with cotton wool. The mosquitoes were grouped together and then transferred to the laboratory for identification. The collections were conducted once every two months

(frequency: seven collections), at the same night BLC were conducted.

Plate 3.10: The cow baited trap (CBT)

3.1.2.2 Immature collection

3.1.2.2 (a) Ovitrap surveillance

Continuous ovitrap surveillance was conducted biweekly between

March 2003 until March 2004 for a period of 56 weeks in both study

sites. The ovitrap is a simple device consisting of a black painted milk

can with a diameter of 7cm and height 10cm, filled with 250ml tap water

and a hole punctured on each side of the tin, to avoid overflow of water

during heavy rain (Yap & Thiruvengadam, 1979). An oviposition paddle

with two different sides made from hardboard (2cm x 12.5cm x 0.3czm)

is suspended vertically in the ovitrap to provide a suitable surface for

oviposition (Plate 3.11).

One hundred ovitraps were placed randomly outdoors confined to

the immediate vicinity of the houses as described as Lee (1992a,b). All

the ovitraps were collected after 5 days and replaced with fresh ovitraps

and paddles on the following week. The paddles collected were placed in

clear plastic bag and brought back to the laboratory. All the collected

ovitraps from the sites were brought back to the laboratory and its

content poured into plastic trays. Tap water was added into the trays and

all larvae collected were allowed to grow in the laboratory. The paddles

were left to dry under room temperature for at least 24 hours before the

eggs on the paddles were counted under a dissecting microscope as

described by Hornby et al. (1994).

Paddle and water inside the ovitrap

Ovitrap (black painted aluminum can)

Plate 3.11: An ovitrap with a paddle

All larvae present in the ovitraps were counted and identified. Aedes

larvae that hatched from the eggs were identified at the third or fourth instar

stage. The larvae were allowed to grow until adult emergence and used for

the evaluation of the efficacy test of mosquito coils.

Three physical parameters were obtained from the Meteorology Services

Department, namely :

a) Mean temperature (oC)

b) Rainfall (mm) and

c) Relative humidity (%RH)

The following guidelines served as the basis for the selection of locations

for ovitraps (Evan & Bevier, 1969; Jakob & Bevier, 1969):

i. The traps are to be located near other potential breeding

containers except tyres as the tyres are black and often contain

water, therefore ovitraps do not compete well,

ii. The traps are to be located in partial or total shade, not under

direct sunlight and also at suitable resting or breeding areas,

iii. The traps are to be placed at ground level, avoiding disturbance

by children, and

iv. The traps are to be located at the rear or sides of premises than in

front of the yard near the street.

3.1.3 MOSQUITO IDENTIFICATION

All mosquito adults and larvae collected were identified using the key

provided (IMR).

3.1.3.1 Larval identification

All larvae from the ovitraps were individually identified on a slide

under a compound microscope.

Aedes were separated from other species collected. Aedes aegypti

and Ae. albopictus can be differentiated by observing the comb teeth at

the 8th segment of the abdomen and also the spine on the thorax.

The comb scale on the 8th segment of Ae. albopictus abdomen

however has no lateral dentricles and the spine is shorter, ending in

several points (Plate 3.12).

Aedes aegypti has the characteristics of possessing 8-12 strong

comb teeth which have well developed lateral dentricles (Plate 3.13) and

the spine on the thorax is longer, ending in a single point.

Plate 3.12: Comb teeth and thoracic spine of Aedes albopictus (source: IMR)

Plate 3.13: Comb teeth and thoracic spine of Aedes aegypti (source: IMR)

3.1.3.2 Adult identification

For adult identification, Aedes and Culex collected from the

ovitraps were differentiated by the characteristics separating these two

genera.

Aedes is a small medium sized mosquito that is highly

ornamented with patches and spots on lines of snow white scales. They

have one or more broad basal white bands on the tarsi of at least one

pair of legs, one or more tarsal segments completely white, proboscis

entirely dark, fairly slender and straight (except Ae. vittatus with some

yellow scaling).The vertex of Aedes have scales and the scutelum is

broad and flat. Postspiracular bristles are present and the claws of

females toothed (IMR, Entomological teaching charts).

The most useful characteristic to identify Ae. aegypti and Ae.

albopictus is the pattern on the thorax. Aedes albopictus is identified by

the single silver broad line on the thorax (Plate 3.14), whereas Ae.

aegypti is identified by the silvery straight and curved lines (lyre-shaped)

on the thorax (Plate 3.15)

Plate 3.14: Aedes albopictus- single silver broad line on the thorax

(Source: IMR)

Plate 3.15: Aedes aegypti - silvery straight and curved lines (lyre-shaped) on

the thorax (Source: IMR)

54 3.1.4 CULTURE METHODS

Using the standard culture method the mosquitoes were cultured under laboratory conditions at 25 + 2oC and 65+20% RH.

3.1.4.1 Egg hatching

After the eggs on the paddles were counted, the paddles were placed in round rearing tray (diameter 30cm) containing chlorine free water (tap water that has been kept for 24hours) for about 48 hour for hatching. According to

Hornby et al. (1994), mosquito culture can be done at 27OC and 80% RH under laboratory condition. The first instar larvae can be seen after the eggs hatched.

Paddles were left to dry again. This alternating wet and dry procedure was repeated twice before the paddles were cleaned to make sure no more eggs were left on the paddles.

3.1.4.2 Larval rearing

Every larva in the tray was counted and transferred with a pipette into

a new rearing tray containing chlorine-free water. About 200 larvae were in

each rearing tray to avoid cannibalism because of the lack of food (Service,

1995a). Larval food was made from a mixture of dog biscuit, ox liver, yeast

and milk in the ratio of 2:1:1:1 by weight, prepared as a fine powder. About

0.5 to 1.0g of food was added everyday (VCRU, 2003) from day 1 to 5. To

remove scum and debris, the rearing medium was changed every two or

three days, and the larvae were rinsed in a sieve at the same time. For fourth

instar larvae, slices of ox liver were added in the rearing tray. By day 7, the

larvae would have started to pupate.

55 3.1.4.3 Pupal rearing

The pupae were separated from the larvae daily with a pipette and

placed in a paper cup with chlorine-free water. The adult emerged in about

24 hours after pupation. Each adult was identified to species and sexed.

They were then transferred into an adult mosquito cage using an aspirator.

3.1.4.4 Adult Rearing

Adults were fed with 10% sugar solution plus vitamin B-complex soaked in cotton wool. The vitamin was prepared by dissolving 8 tablets of vitamin B complex formulation (Xepa-B Co) in 4 litres of distilled water. The vitamin solution was changed every other day. Once a week, the adults were given blood meal by placing a mouse or rat, confined in small screen cages into the cage for a period of at least 12 hours. The sucrose solution were removed prior to blood feeding and will be placed back into the cages after blood feeding.

3.1.4.5 Egg Collections

About 3 days after blood feeding, eggs are ready to be deposited by the female mosquitoes. A petri dish containing chlorine-free water with a cone of round Whatman No.1 filter paper (diameter 24cm) in it was placed in the adult cage. After the eggs were deposited on the filter paper, the dish was removed and allowed to air dry at room temperature for 24 hours. For hatching of the eggs, the filter paper was immersed into an enamel tray containing chlorine-free water.

56 3.1.5 METEOROLOGICAL DATA

During the study period, all the data on the total rainfall, mean temperature and mean relative humidity were obtained from the Malaysian

Meteorological Services for Bayan Lepas and Butterworth which covers the study sites.

3.1.6 DATA ANALYSIS

To evaluate the distribution and the abundance of the species in the study area, several parameters were considered: i) The ovitrap index (OI) for both location:

Ovitrap Index (OI) = Number of positive ovitrap X 100% Total number of ovitrap examined ii) The mean number of eggs in total ovitraps operating:

Mean number of eggs = Total number of eggs in each surveillance Total number ovitrap examined iii) The correlation between OI, mean number of eggs and adults were also

determined. iv) The correlation between OI and mean number of eggs with the

meteorological parameters (mean temperature, rainfall, lag 1 rainfall, lag

2 rainfall and relative humidity). Lag 1 rainfall refer to rainfall amount one

week before ovitrap collection and Lag 2 rainfall refer to rainfall amount

two weeks before ovitrap collection. v) The percentage and ratio of adults produced from the eggs vi) The sex ratio of adults survived.

57 To evaluate the relationship between the eggs collected and the factors studied (mean temperature, rainfall and relative humidity), the correlation among experimental variables was evaluated using the Pearson correlation coefficient (r) and its significance determined.

All statistical tests were considered significant at the =0.05 probability level. All data were tested for normality distribution and log transformed where appropriate. Data were analyzed using SPSS version 11.5.

3.2 RESULTS

3.2.1 Meteorological condition

Malaysia is a tropical country, where rainfall occurs throughout the year.

The weekly rainfall in both sites were plotted for the period between March 2003 to April 2004. There was no great variation in the temperatures and relative humidity between sites except for the cumulative rainfall during the sampling period. From the statistical test of independent samples t test, it was found that there was no significant difference between the mean temperature and the rainfall in Taman Permai Indah and Kampung Pasir Gebu (p>0.05). Only a slightly significant difference between the relative humidity was observed

(p<0.05). However no extreme values were recorded during the study.

The rainfall in both sites fluctuated with a steady increase in the 13th to

14th weeks of the year. However, the amount of rainfall decreased on the 22nd to

24th week with an increase on the 25th week. For both sites, the rainfall

58 fluctuated through week 25 to 27. The study was divided into the season of lower rainfall and season of high rainfall, in reference to the amount of rainfall.

The highest rainfall recorded was on the 13th week for both locations.

Taman Permai Indah recorded 132.4mm and Pasir Gebu, 119.4mm. During the lower rainfall season the lowest rainfall recorded was 0mm. However no obvious weather changes were recorded in both locations. The mean temperature recorded was between 260C to 29.50C. The relative humidity in the environment was about 70%-90%.

The rainfall, temperature and relative humidity in both sites was plotted in figure 3.1 and 3.2. The correlation among experimental variables was evaluated using the Pearson correlation coefficient (r).

high low high low 140.0 100.0

90.0 120.0 80.0

100.0 70.0 o C/%RH ml 60.0 80.0

50.0 60.0 40.0

40.0 30.0

20.0 20.0 10.0

0.0 0.0 12345678 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

week rainfall temp RH

Figure 3.1: Meteorological conditions obtained from the Malaysian Meteorological Services Department in Bayan Lepas, reflective of Taman Permai Indah, Sg Dua, Penang

high low high low

140.0 100.0

90.0 120.0 80.0

100.0 70.0 C/%RH

ml 60.0 80.0 o 50.0 60.0 40.0

40.0 30.0

20.0 20.0 10.0

0.0 0.0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 week

Figure 3.2: Meteorological conditions obtained from the Malaysian Meteorological Services Department in Butterworth, reflective of Kampung Pasir Gebu

3.2.2 Mosquito species

A rich collection of adult mosquito species was gathered from Kg. Pasir

Gebu, Penaga. A total of 2, 960 mosquitoes, belonging to 4 genera of mosquitoes namely Culex, Anopheles, Mansonia and Aedes were collected using the bare leg catch and cow baited trap techniques. Figure 3.3 shows the composition of adult mosquitoes collected in Kg Pasir Gebu.

A total of 1 780 (60.14%) Anopheles sp., 821 (27.74%) Culex sp., 347

(11.72%) Mansonia sp. and 12 (0.41%) Aedes sp. was collected in this area

(Figure 3.4). Seven species were identified. A total of 675 (22.80%) Culex gelidus was collected making it the most dominant species collected followed by

514 (17.37%) Anopheles peditaeniatus, 383 (12.94%) Anopheles sinensis, 254

(8.58%), Mansonia uniformis, 252 (8.52%) Anopheles subpictus, 192 (6.49%)

Anopheles campestris and 135 (4.57%) Anopheles vagus.

A total of 555 adult mosquitoes were not successfully identified to species. These included Culex sp. representing a total of 146 (4.94%),

Anopheles sp. 304 (10.27%), Mansonia sp. 93 (3.14%) and only 12 (0.41%)

Aedes sp.

600

500

400

300 Total abundance

200

100

s p BLC is sp sp s sp nsi str x. a. e e Ae. gelidus pictus C An. M . sin mp CBT Cx n a An. vagus sub A . c n An. Ma.uniformis A An.peditaeniatus Species

Figure 3.3: Mosquito composition in Kg. Pasir Gebu, Penaga, Pulau Pinang

11.72 0.41 27.74 Culex Anopheles Mansonia Aedes 60.14

Figure 3.4: Total percentage of mosquitoes species collected

64 Other important species included the Anopheles mosquitoes. Known as malaria and filariasis vectors, the Anophelinae group contains a large variety of species distributed all over the world. The main Anopheles species caught in

Kampung Pasir Gebu were An. peditaeniatus, An. campestris, An. vagus and

An. subpictus.

Anopheles peditaeniatus formed 514 (17.37%) of the total anophelines caught in Kampung Pasir Gebu, while An. sinensis made up 383(12.94%) of the total anophelines caught in Kampung Pasir Gebu. Anopheles campestris formed 192, (6.49%), An. subpictus 252 (8.52%) and An. vagus formed 135

(4.57%) from the total mosquitoes caught in this study area.

Meanwhile, Mansonia uniformis, the vector for brugian filariasis comprisied 254 (8.58%) of the total mosquitoes caught in Kampung Pasir Gebu.

3.2.2.1 Bare leg catch (BLC)

Based on the total number of mosquito collected in the present study, the human bait trap, was less preferred by mosquitoes. Only 674 (22.77%) mosquitoes were collected using this method during the study period. This includes 155 (5.24%) Culex, 537 (11.05%) Anopheles, 167 (6.08%) Mansonia and only 12 (0.41%) Aedes (Figure: 3.5 A).

3.2.2.2 Cow baited trap (CBT)

It was found that 1453 (49.09%) out of total 2286 (77.23%) mosquitoes collected using the CBT were identified as Anopheles sp. From this total, Culex sp. made up 666 (22.50%), 167(5.64%) were Mansonia sp. while no Aedes sp. were collected using cattle as a bait. (Figure 3.5 B).

Aedes Culex Mansonia 0.41 5.24 6.08

11.05 Anopheles

Figure 3.5 (A): Percentage of mosquitoes collected using BLC in KPG

Mansonia Culex 5.64 0 22.50

49.09

Anopheles

Figure 3.5 (B): Percentage of mosquitoes collected using CBT in KPG

67 3.2.3 Ovitrap surveillance

All the larvae and eggs collected from the ovitrap were identified and recorded for both locations. The eggs were counted before being cultured in the laboratory.

3.2.3.1 Ovitrap index (OI%)

Figure 3.6 shows the ovitrap index in Taman Permai Indah and Kg Pasir

Gebu. Both locations showed fluctuation patterns with a range of 66-99%. This implicated that both areas have a high Aedes albopictus population. Figure 3.6 indicated the distribution of Aedes albopictus and other mosquitoes present in the ovitraps. Only 6% to 15% of the ovitrap contained other species which were

Cx. quinquefasciatus and Ae.aegypti. Aedes albopictus was found in 84.6% and

93.7% of positive ovitrap in Taman Permai Indah and Kg Pasir Gebu, respectively. The mix population of Ae.aegypti and Cx. quinquefasciatus in

Taman Permai Indah and Kg Pasir Gebu was 15.4% and 6.3%, respectively

(Figure 3.7 A, B) . No significant difference was found between both locations in the total ovitrap index and the mean numbers collected (independent sample t test, p > 0.05).

100

60 Ovitrap Index (%) 50

0 1234 56789 10111213141516171819202122232425262728 weeks

TPI KPG

Figure 3.6: Ovitrap index in Taman Permai Indah (TPI) and Kg Pasir Gebu (KPG) for 14 months of sampling

15.4 %

84.6%

Fig: 3.7 (A)

6.3%

93.7%

Ae. albopictus Mix breeding

Fig: 3.7 (B)

Figure 3.7 (A): The distribution of Aedes albopictus and other mosquitoes present in the ovitraps in Taman Permai Indah.

Figure 3.7 (B): The distribution of Aedes albopictus and other mosquitoes present in the ovitraps in Kampung Pasir Gebu.

70 3.2.3.2 Egg abundance

Figure 3.8 shows the mean number of the total eggs collected in both study areas from 14 months of sampling.

3.2.3.2(a) Taman Permai Indah (TPI)

A total of 29 805 eggs was collected from 14 months of ovitrap sampling.

The highest amount of eggs recorded was in the 13th week yielding 2702 eggs, whereas the lowest was in the 1st week yielding 355 eggs. The amount of eggs seemed to be moderate during the high and low rainfall. However, from the independent sample t test, it was found that there was a significant difference

(p<0.05) between the mean number of eggs collected during the high and low rainfall seasons. However, for the ovitrap index, no significant difference was found both during high and low rainfall seasons (p>0.05) (Appendix D).

3.2.3.2(b) Kampung Pasir Gebu, Penaga

A total of 25 665 Aedes albopictus eggs was collected from this study site. On the 13th week, the maximum number of eggs (2702) was recorded, whereas in the 26th week, a minimum of 300 eggs was recorded. From the first week to the tenth week, there was fluctuation with an irregular increase from week 11th to week 15th. An irregular decrease and increase was recorded in the following weeks. The ovitrap index and mean number of eggs collected was found to be significantly different (independent sample t test, p<0.05) (Appendix

E).

35.00

30.00

25.00 m ean num ber of eggs 20.00

15.00

10.00

5.00

0.00 1 3 5 7 9 111315171921232527 w eeks

eggs in TPI eggs in KPG Figure 3.8: Mean number of mosquito eggs per ovitrap collected in both study areas from 14 months of sampling

3.2.3.3 CORRELATIONS

3.2.3.3 (a) Taman Permai Indah

The ovitrap index (OI) showed a significant correlation with the mean number of eggs (r=0.374), lag 1 and lag 2 rainfall (r=0.377, r=0.506). Figure 3.9 showed the correlation between ovitrap index and mean number of eggs collected in

Taman Permai Indah. The mean number of eggs also correlated with the amount of rain in the study area, the temperature and the percentage of environmental relative humidity using the Pearson Correlation test at 0.05 significance level. Data are shown in Table 3.1. Figure 3.10 showed the correlation between ovitrap index and rainfall in Taman Permai Indah. The correlation between mean number of eggs was highly significant with the lag 1 rainfall and lag 2 rainfall, where r= 0.982 and r=0.771, respectively (Figure

3.11). Meanwhile, a significant negative correlation between the mean number of eggs and mean temperature was also recorded (r=-0.374). The mean number of eggs and relative humidity was also found to be significantly correlated (r= 0.477).

120 30.00 r = 0.34

100 25.00

80 20.00 eggs means

60 15.00

40 10.00 ovitrap index (OI) index ovitrap

20 5.00

0 0.00 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 OI weeks means egg

Figure 3.9: Correlation between ovitrap index and mean number of eggs collected in Taman Permai Indah (TPI)

Table 3.1: Correlation between several parameters in Taman Permai Indah

Correlations

OVITRAP MEAN DAY LAG 1 LAG 2 INDEX EGGS RAINFALL RAINFALL RAINFALL MEAN TEMP RH OVITRAP INDEX Pearson Correlation 1 .374* .000 .377* .506** -.370 .370 Sig. (2-tailed) . .050 .999 .048 .007 .053 .053 N 28 28 15 28 27 28 28 MEAN EGGS Pearson Correlation .374* 1 .348 .982** .771** -.536** .477* Sig. (2-tailed) .050 . .203 .000 .000 .003 .010 N 28 28 15 28 27 28 28 DAY RAINFALL Pearson Correlation .000 .348 1 .413 .253 -.553* .415 Sig. (2-tailed) .999 .203 . .126 .364 .033 .124 N 15 15 15 15 15 15 15 LAG 1 RAINFALL Pearson Correlation .377* .982** .413 1 .814** -.534** .463* Sig. (2-tailed) .048 .000 .126 . .000 .003 .013 N 28 28 15 28 27 28 28 LAG 2 RAINFALL Pearson Correlation .506** .771** .253 .814** 1 -.666** .499** Sig. (2-tailed) .007 .000 .364 .000 . .000 .008 N 27 27 15 27 27 27 27 MEAN Pearson Correlation -.370 -.536** -.553* -.534** -.666** 1 -.526** TEMPERATURE Sig. (2-tailed) .053 .003 .033 .003 .000 . .004 N 28 28 15 28 27 28 28 RELATIVE HUMIDITY Pearson Correlation .370 .477* .415 .463* .499** -.526** 1 Sig. (2-tailed) .053 .010 .124 .013 .008 .004 . N 28 28 15 28 27 28 28 *. Correlation is significant at the 0.05 level (2-tailed). **. Correlation is significant at the 0.01 level (2-tailed).

120 700.0 r=0.506

600.0 100

rainfall, 500.0 mm 80

OI, % 400.0

300.0

40 200.0

20 100.0

r=0.377 0 0.0 12 3 4 5678910 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 weeks

OI lag 1 rain lag2 rain

Figure 3.10: Correlation between ovitrap index and rainfall in Taman Permai Indah (TPI)

30.00 700.0 r= 0.771

600.0 25.00

500.0 20.00 mean number rainfal of eggs (mm) 400.0 r=0.098 15.00

300.0

10.00 200.0

5.00 100.0

0.00 0.0 123456789101112 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 weeks

mean eggs lag1 rain lag 2 rain

Figure 3.11: Correlation between mean number of eggs and rainfall in Taman Permai Indah (TPI)

3.2.3.3 (b) Kampung Pasir Gebu, Penaga

The correlation between the mean number of eggs collected from the field and rainfall at the site was found to be highly significant (r=0.918). However, no significant correlation was found between mean temperature or relative humidity and mean number of eggs collected. Besides, Kampung Pasir Gebu also showed a significant correlation between the ovitrap and the rainfall at the study area (r=0.387) (Table: 3.2 and Figure 3.12). Figure 3.13 showed the correlation between means number of eggs and rainfall in Kampung Pasir Gebu.

Table3.2 : Correlation between several parameters in Kampung Pasir Gebu Correlations

MEAN DAY LAG1 LAG 2 OI EGGS RAINFALL RAINFALL RAINFALL MEAN TEMP RH OVITRAP INDEX Pearson Correlation 1 .295 .314 .387* .248 .116 .150 Sig. (2-tailed) . .128 .320 .042 .203 .557 .446 N 28 28 12 28 28 28 28 MEAN EGGS Pearson Correlation .295 1 .841** .918** .507** .136 .022 Sig. (2-tailed) .128 . .001 .000 .006 .491 .912 N 28 28 12 28 28 28 28 DAY RAINFALL Pearson Correlation .314 .841** 1 .806** .659* -.112 .410 Sig. (2-tailed) .320 .001 . .002 .020 .730 .185 N 12 12 12 12 12 12 12 LAG 1 RAINFALL Pearson Correlation .387* .918** .806** 1 .661** .036 .166 Sig. (2-tailed) .042 .000 .002 . .000 .855 .398 N 28 28 12 28 28 28 28 LAG 2 RAINFALL Pearson Correlation .248 .507** .659* .661** 1 -.335 .428* Sig. (2-tailed) .203 .006 .020 .000 . .081 .023 N 28 28 12 28 28 28 28 MEAN Pearson Correlation .116 .136 -.112 .036 -.335 1 -.877** TEMPERATURE Sig. (2-tailed) .557 .491 .730 .855 .081 . .000 N 28 28 12 28 28 28 28 RELATIVE HUMIDITY Pearson Correlation .150 .022 .410 .166 .428* -.877** 1 Sig. (2-tailed) .446 .912 .185 .398 .023 .000 . N 28 28 12 28 28 28 28 *. Correlation is significant at the 0.05 level (2-tailed). **. Correlation is significant at the 0.01 level (2-tailed).

120 140.0

r=0.387 120.0 100

100.0 80 rainfall (mm)

80.0 OI, % 60

60.0

40 40.0

20 20.0

0 0.0 12345678910 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 weeks

OI lag1 rainfall

Figure 3.12: Correlation between ovitrap index and rainfall in Kampung Pasir Gebu (KPG)

30.00 500.0

r= 0.918 450.0

25.00 400.0 rainfall, mm means 350.0 20.00 eggs 300.0

15.00 250.0

r=0.507 200.0

10.00 150.0

100.0 5.00

50.0

0.00 0.0 1234567 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 weeks

means egg lag 1 rain lag2 rain

Figure 3.13: Correlation between means number of eggs and rainfall in Kampung Pasir Gebu (KPG)

3.2.3.4 Total larvae hatched

A total of 28 714 mosquito larvae was produced from 29 805 (96.34%) eggs collected in Taman Permai Indah, whereas from the total of 25 665 eggs collected in Pasir Gebu, 22 301 (86.89%) hatched into the larvae stage. This indicated that high number of eggs can be hatched and reared to the larval stages. All the larvae hatched in the laboratory were Ae. albopictus. This might be because only outdoor surveillance was carried out in both study locations.

From this total, the sex ratio (male:female) of adults emerged from the larvae was close to one (Table 3.3). These findings indicated that Ae. albopictus can survive very well in both study areas.

Table 3.3: Total number of eggs collected and total number of Aedes albopictus larvae produced including the sex ratio of adults Aedes albopictus produced from the eggs collected in both study sites.

______

Locality Eggs No of Adults emerged Male:Female

*TPI 29 805 28 714(96.34%) 0.93+0.33

*KPG 25 665 22 301(86.89%) 0.97+0.42

______

*TPI = Taman Permai Indah

*KPG = Kampung Pasir Gebu

3.3 DISCUSSION

Various factors affect mosquito abundance and their species distribution, including climatic influences, vegetation cover and the right type of environmental breeding sites (Igbinosa, 1989). Either in the urban or suburban residential study areas, both provided favourable habitats for mosquito breeding.

Both areas have the availability of wooded plants, hosts for blood supply and numerous containers for breeding purposes. Some aspects of human ecology also greatly influenced mosquito survival (Gilliet, 1971), such as the dense human populated with unstructured development and the availability of breeding containers around their houses for the purpose of water storage (Okogun et al.,

2003). This phenomenon could be observed in both study areas even in the urban residential area (Taman Permai Indah). Mosquitoes generally prefer a cool shaded area for their biting and breeding activities (Evan, 1938; Okogun et al., 2003).

An abundance of plants was available in the study areas which provided the suitable environment for mosquito breeding such as banana axles, hibiscus plants (Rosa sinensis), Padanus sp., and several flower hedges provided shades in front and at the sides of homes. Therefore, these sites provided ready breeding sources for Aedes either with the natural environment or artificial containers around the residential areas.

In Kampung Pasir Gebu, the abundance of various mosquito species of the genera Anopheles, Culex, Aedes and Mansonia was associated with the microhabitats available in this site. Knowledge of the species abundance and diseases relationship in this area is important in disease forecasting and monitoring, since these genera are known to be vectors of human diseases including dengue, arboviruses in general, malaria and bancroftian filariasis

(Johnson, 1979; Monath, 1979). In Kampung Pasir Gebu, the suburban area, the availability of paddy fields, coconut palms, and other aquatic vegetations such as Lemna, Salvinia was advantageous to Mansonia mosquitoes to breed since the environment proved to be the most suitable habitat for either the immature stage or adult stage.

As mentioned, the most predominant Culex species was Culex gelidus.

A total of 675 Cx. gelidus was collected during the study. The distribution of

Culex gelidus and other Culex in the vishnui group is limited to Southeast Asia,

Indochina and India (WHO, 1989). This species has also been incriminated as a

JE vector. It is mainly a paddy field breeder. By understanding the habitat of

Culex mosquitoes, appropriate measures can be taken to minimize the human- mosquito contact by eradicating the breeding areas especially around the houses. Once the Culex population is controlled, the management of JE epidemic will be more effective. The availability of suitable habitats here provided good breeding opportunity for this species.

Human landing catch is the most direct, reliable, and favoured method, because it measures the frequency of human-mosquito contact (Service, 1993).

The abundance of human malaria vectors is also usually estimated by this technique (Ibrahim et al., 2005). According to Macdonald (1957b), this contact is a component of the entomological inoculation rate, which quantifies the dynamics of malaria transmission. However, this method is dependent on the skills and experience of a mosquito collector and the natural human variation in attractiveness to mosquitoes (Ibrahim et al., 2005) plus exposing the collector at risk of infective mosquito bites and contracting arboviruses which is unethically.

Because of these, alternative methods that are less sensitive, specific and reproducible and that are ethically acceptable are used such as the cow baited trap.

Mansonia species show a wide host preference (Reid 1961; Wharton,

1962). Man is not usually the preferred host as they are catholic in their feeding habits (Reid, 1961). Mansonia is also exophilic during the day time (Wharton,

1962). From this study, it is determined that the trap using a cow as bait was the most effective. This favourable result may be due to the fact that the most dominant species among the vector species are zoophilic and highly attracted to cattle. Anopheles campestris and Mansonia uniformis showed little attraction to human. Host selection patterns of anthrophod vectors are important for us to understand host-vector relationship and the dynamics of disease transmission. Some mosquito species exhibit distinct preferences

86 (fixed) in this regard, while others are facultative and exhibit variable feeding patterns (Rao, 1984).

A different situation was observed in the urban residential area in Taman

Permai Indah, where the variability of the environment is not very obvious as in the suburban areas. However, both sites have wooded areas and in addition, human and animal hosts provided the vectors with the sources for the blood meal they need. On the other hand, ponds, river, coconut and paddy field nearby in Kg Pasir Gebu served as the breeding sites of other mosquitoes such as Anopheles, Culex and Mansonia. This is one of the possible reasons, accounting for the mix population in the observed ovitraps. However, the containers were likely to be mainly colonized by Ae. albopictus since this species is the main container breeders. The surveillance indicated that

Ae.albopictus is the principle dengue vector in both sites. This finding is similar to the study by Rawlins et al. (1998) where, Ae. albopictus is found associated with numerous container-inhabiting mosquitoes such as Aedes, Culex,

Toxorhynchites and Armigeres, but in general even if it is the most abundant species present in shared habitats, it often occurs alone. In fact, Culex quinquefasciatus is also found to be attracted to oviposit in ovitraps. Aedes aegypti is strictly domilicary, preferring less vegetation, biting indoors and primarily found indoors, while Ae. albopictus is found commonly outdoors and breeds in all type of natural containers (Sucharit et al., 1978; Foo et al., 1985).

This might explain why Ae. albopictus was much more abundant than Ae. aegypti since the ovitrap surveillance was only done outdoors .

Dengue is a disease associated with the areas, where breeding of Aedes mosquitoes is most prevalent such as in the slum areas (Chan & Counsilman,

1985). However, in this study Aedes is not only associated with the residential areas, but also associated with the numerous natural and artificial containers which provided good habitats for breeding. Even in the urban area, which generally has a cleaner environment with piped water supply, the residents stored water in containers outdoor for the purpose of watering their plants and other activities. Furthermore, the concrete drainage (in Taman Permai Indah) with clear stagnant water can also become a potential habitat for the breeding of Aedes. This finding is supported by the study by Lee (1990), which showed that Aedes required clear but not necessarily clean water. According to Chen et al. (2005) in their study of dengue vector surveillance in urban and settlement areas in Selangor, even in clean residential environment with no water storage, the area still had high Aedes aegypti and Aedes albopictus population, and the only possible breeding habitat is the concrete drainage system outside the houses. The drains have clear stagnant water with fallen tree leaves and other debris. Therefore, the drains served as good artificial breeding habitats for Ae. aegypti and Ae. albopictus (Chen et al., 2005).

The study on the biology and ecology of the eggs gave much valuable information. Egg survey is particularly useful with species which remain in the egg state for many months. Potential larval habitats can be recognized and enumerated without waiting for the larvae to appear. Ovitraps which trapped eggs to reflect adult density are very useful tools for the surveillance of Aedes

88 vector. Although the ovitrap is a method that is operationally viable, if it remains in the field for more than the maximum of seven days, it becomes a potential breeding site by itself (Santos et al., 2003). Therefore, any ovitrap monitoring should be done within an interval of less than seven days in the field.

Rainfall is an important factor which regulates the abundance of outdoor breeding mosquito populations. The wet seasons are associated with higher prevalence of mosquito-borne diseases (Okogun et al., 2003). The onset of rainfall supports the development of additional mosquito breeding sites, hatching of eggs following oviposition, high relative humidity (Igbinosa, 1989), and growth of vegetation cover and cool shaded environment for the development of the aquatic stages and the recruitment of young adults and their survivors (Evan, 1938). In this study, total number of eggs was most abundant in the wet season (high rainfall). A high peak in egg population was observed 1 week after the rainfall peaks. Rain supplied the water for Aedes breeding in artificial or natural containers. Rainfall is either correlated or uncorrelated to insects. As a dengue vector, Aedes albopictus population has always been related with rainfall in Asia including Malaysia (Lo & Narimah, 1984). However, heavy rainfall might give negative impact on the number of larvae or eggs due to excess water in ovitraps (Foo et al., 1985) that flushed out the immature stages, thus preventing any oviposition. Heavy rain accompanied by strong winds might disturb the flight activity of Ae. albopictus females, resulting in difficulties in finding hosts and suitable breeding sites. According to Hornby et al. (1994), in a study in the United States, the egg population remain increasing at the beginning and decreased at the end of the wet seasons due to the

89 occurrence of heavy rain. However, the run-off holes at the sides of the ovitraps lessen the effect of heavy flushing, therefore the decrease in eggs sampling was not too obvious in this study.

The seasonal changes in oviposition also are a consequence of seasonal changes in the weather conditions, and the availability of sites for laying eggs.

The increase in Aedes albopictus oviposition at Taman Permai Indah and Kg.

Pasir Gebu in week 12 until week 15 might be the result of greater female activity, due to an increase in the temperature and lower relative humidity during this month. High survival rate of immature stages at the beginning of the dry season led to a rise in the number of emergent adults.

A higher ovitrap index containing Aedes in the low rainfall season than in high rainfall season might be a consequence of the relative attraction to the ovitrap due to the scarcity of other suitable artificial containers near the ovitrap.

This result is also similar to the findings by Mogi et al. (1990) who observed a higher ratio of ovitraps containing Aedes in the dry than in the wet season.

However the egg population remains low in the dry season, increasing at the beginning and decreasing at the end of the raining season. It was also reported that females of Ae. aegypti lay a few eggs in several containers, a behaviour described as a skip oviposition (Chadee, 1992, Corbet & Chadee, 1993, Reiter et al., 1995; Colton et al., 2003). The possible reason for the lower number of eggs recorded may be the females contribute to a smaller number of eggs in more containers since ovitraps have provided a good habitat to lay their eggs.

When the water temperature rises, the larvae take shorter time to mature

90 (Rueda et al., 1990) and consequently there is a greater capacity to produce more offspring during this season and adult female mosquitoes digest blood faster and feed more frequently in warmer climates, thus increasing the oviposition activity (Gillies, 1953). However without rain, the source for breeding is scarce. However, when rain is abundant, mosquitoes can lay eggs continuously and with ideal temperature a cycle can be completed within one week.

Many researchers have reported in their studies, especially in Southeast

Asian countries including Burma (Khai Ming et al., 1974), Thailand (Watts et al. ,

1987; Thongrungkiat et al., 2003) and Malaysia (Sulaiman et al., 1991), that the seasonal patterns of outbreaks of dengue, coincides with the rainy season. In the study by Foo et al., (1985) in Selangor, it was found that the monthly incidence of dengue for the period 1973-1982, was associated with the monthly rainfall during the first wet season, and concluded that the increased in cases was correlated to the increased in vector abundance. Since Malaysia is a tropical country, the abundance of any species depends more upon the availability of breeding places than seasons. The ovitraps set in the study areas are the breeding habitat for the mosquitoes. Therefore, there is not much variability in the number of eggs collected since the breeding source has existed throughout the sampling period.

91 Egg survival also may depend on a combination of density dependent and density independent selective pressures (Estrada-Franco & Craig, 1995).

Factors that may promote egg loss include desiccation, predation, and freezing

(Estrada-Franco & Craig, 1995). The number of Ae. albopictus eggs that survive low humidity appears to depend upon the development stage of the embryos before they are exposed to dry conditions (Estrada-Franco & Craig, 1995). In the study by Gubler (1970), it was found that Ae. albopictus eggs were highly resistant to dry conditions if they were kept in humid conditions for four days before being exposed to drought. Therefore in this study, the eggs were allowed to dry at high relative humidity (+ 80% RH) to avoid the embryos development from being disturbed. On the filter paper, the eggs were also been kept in a moist condition. As a result, the hatching rate of the eggs is very high.

With the efficient ovitrap surveillance, Ae. albopictus distribution and abundance can be studied but it is more difficult to control because of the wide range of breeding habitats. The seasonal fluctuation of Ae. albopictus abundance described in this study is useful in planning the implementation of control measures. During the wet or dry seasons (high or low rainfall), elimination of containers would help to reduce vector population (mainly eggs and immature stages) (Reiter et al., 1995; Edman et al., 1998). This would help to save time and money in control programme. However, continuous monitoring should be done by local authorities in order to prevent the increase of Ae. albopictus population since the abundance of vectors is associated with outbreaks of dengue. The same concept can be applied in order to control other mosquitoes besides Aedes. With the public participation, the breeding sites can

92 be minimized with the eliminations of containers, bush clearing around home, filling of tree holes and clearing of drainage system. By understanding the mosquito fauna available in the area, we can understand more about the disease and will have a successful control programme. More than that, early precaution or warning can be provided, thus making the public more aware on the control measures that can be implemented, not only by the local authorities but also by themselves.

93 CHAPTER FOUR

LABORATORY EFFICACY OF HOUSEHOLD INSECTICIDES (MOSQUITO COIL) WITH DIFFERENT ACTIVE INGREDIENTS AGAINST SELECTED VECTOR MOSQUITOES

4.0 INTRODUCTION

Control of mosquitoes in Malaysia relies heavily on the use of insecticides. Owing to the heavy use of chemicals, it is important to conduct continuous research to find insecticides that are effective and much safer to human in order to prevent unwanted side effects such as resistance and human poisoning (Yap & Chung, 1987; Yap et al., 1990b). One of the effective mosquito control measures is personal protection which includes household products such as mosquito coils, mats, liquid vaporizers, aerosols and repellents (Yap & Foo, 1984; Yap, 1988).

Household insecticides products are used throughout the world especially in tropical countries (Koo & Hoo, 1994; Yap et al., 1990b; Jurgen &

Umar, 1999). Household insecticides are used as individual personal protection from nuisance mosquitoes to maintain a certain level of comfort and to avoid the spreading of endemic mosquito borne diseases. Mosquito coils are effective and economical to control or prevent such diseases. Natural pyrethrum and presently the pyrethroids are the main ingredients in mosquito coils (Yap &

Chong, 1996; Yap et al., 1996).

94 A survey on the usage of mosquito coil was conducted in Kg Pasir Gebu in Penang. This study indicated that the residents widely used mosquito coils as personal protection against mosquitoes. The objective thus, was to conduct a laboratory efficacy test on several ingredients of mosquito coils against four species of mosquito vectors.

4.3 MATERIALS AND METHODS

4.3.1 Mosquito strains

The four mosquito species chosen for adult bioassay testing are Ae. aegypti, Ae. albopictus, Cx. quinquefasciatus and An. sinensis. Laboratory and field strains of these mosquitoes species were used throughout the study. The field strain mosquitoes were collected directly from the field using ovitraps or adult collection method such as the CBT and BLC. The laboratory colonies used throughout the study were been established and colonies maintained at the Vector Control Research Unit, School of Biological Sciences, Universiti

Sains Malaysia since the 1980’s. The laboratory strains which are unexposed for many years, served as the control. For all bioassay conducted, only sucrose- fed adult female mosquitoes, aged 2 to 5 days were used. A minimum of 60 mosquitoes were used in each test and three replicates were conducted. One hour before the test, mosquitoes were prepared and acclimatized in clean polyethylene containers with 10% sucrose pad placed on top of the containers.

95 4.1.2 Test chamber

All tests were conducted in a glass chamber measuring 70x70x70 cm.

The glass chamber is made from glass with aluminium frames (Plate 4.1). The test protocol essentially followed SIRIM standard methods (1996) with necessary modifications. A sliding window measuring 18x20 cm in the middle front of the chamber was used to place the coil and introduce the mosquitoes into the chamber. After each trial, the chambers were then thoroughly cleaned and dried for the next test.

4.1.3 Active ingredients.

From a market surveillance, four brands of mosquito coil were chosen

and labelled as A, B, C and D. The active ingredients for each brand are as

follow: (A) 0.04% prallethrin, (B) 0.15% d-trans allethrin, (C)0.30% d-

allethrin, (D)0.30% d-allethrin and a standard Malaysian formulation

containing 0.20% d-allethrin (labelled as E) was used for comparison

purposes. All active ingredients were pyrethroids.

96 4.1.4 Efficacy of mosquito coil test

In the chamber, 0.5 gram of test coil with different active ingredients

fixed on a coil stand was ignited at both ends (Plate 4.2). The time needed

to complete the burning of the coil was recorded. A total of 20 mosquitoes

were introduced into the chamber after the coil was completely burning.

Knockdown of mosquitoes was observed at 0.5 minutes intervals up to 20

minutes. After 20 minutes’ exposure, all mosquitoes were collected and

placed in clean polyethylene containers with 10% sucrose pad placed on

top of the containers. Mortality at 24 hour post-treatment was recorded.

All test procedures were conducted in an air conditioned laboratory

with ambient temperature of 25+ 20C and 70 + 10% relative humidity.

4.1.5 Statistical analysis

Log time probit mortality regression and the knockdown time (KT)

values were analysed using a probit analysis, SPSS software.

Knockdown times (KT50 and KT95 defined as the minutes needed to

knockdown 50% and 95% of mosquitoes, respectively) was determined

by probit analysis using a digital computer programmed with SPSS.

Twenty four hours post treatment mortality was recorded to determine

the susceptibility status of each mosquito species against each active

ingredient. The resistance ratio (RR) is calculated based on the KT50

values of field to the laboratory strains mosquitoes.

Plate 4.1: Glass chamber (70x70x70 cm) used for the test

Plate 4.2: A piece of 0.5g tested coil was ignited at both ends in the glass chamber

4.2 RESULTS

4.2.1 Bioassay

Knockdown Time (KT), which is the time required to knockdown the mosquitoes exposed to the insecticide was evaluated. The KT50 values, which state the time required to knockdown 50% of the mosquitoes and KT95 values which state the time required to knockdown 95% of the mosquitoes was evaluated using probit analysis.

4.2.1.3 Bioefficacy of 0.04% prallethrin

For product A which contains 0.04% prallethrin as the active ingredient, the lab strain showed lower KT values than the field strain for all species tested.

Table 4.1 shows the values of the knockdown time of this product against the test mosquitoes. The KT50 values for prallethrin coils (0.04% w/w) against An. sinensis, Ae. aegypti, Ae. albopictus and Cx. quinquefasciatus (lab strain) were

2.01, 2.01, 4.40 and 5.67 min, respectively, whereas, the KT95 values were

3.95, 3.78, 9.50 and 10.20 min, respectively. For the field strain the KT50 and

KT95 values were higher than the lab strain. The KT50 values, for field strain were 2.21, 2.61, 5.10 and 6.55 min, respectively. The KT95 values, however, increased to 4.63, 7.29, 9.12 and 10.57 min, respectively for the field strain.

This indicated that the lab strain mosquito were more susceptible than the field strain mosquitoes.

Table 4.1: Knockdown time (minutes) and mortality percentage of product A (0.04% prallethrin) against Anopheles sinensis, Aedes aegypti, Aedes albopictus, and Culex quinquefasciatus using the glass chamber method.

Laboratory strain Field strain RR Mosquito species Reg. + SE KT50 KT95 Reg. + SE Mortality KT50 KT95 Reg. + Mortality (CL 95%) (CL95%) % (CL 95%) (CL95%) SE % (24-h) (24-h)

Anopheles sinensis 2.01 3.95 5.61+ 83.30 2.21 4.63 4.85+ 68.30 2.30 (1.88- (3.63- 0.41 (1.86-2.37) (3.94- 0.35 2.13) 4.40) 5.92)

Aedes aegypti 2.01 3.78 6.01+ 43.30 2.61 7.29 3.69+ 50.00 1.30 (1.75- (3.23- 0.43 (2.43-2.79) (6.54- 0.24 2.26) 4.85) 8.35)

Aedes albopictus 4.40 9.50 4.92 + 28.30 5.10 9.12 6.53+ 20.00 1.16 (3.89- (7.61- 0.34 (4.91-5.30) (8.47- 0.42

5.01) 14.27) 9.99)

Culex 5.67 10.20 6.45 + 0.3 15.30 6.55 10.57 7.91+ 13.30 quinquefasciatus 1.16 (5.47- (9.68- (6.36-6.74) (10.11- 0.39 5.86) 10.83) 11.15)

100

Based on KT 95 values, Ae. aegypti was slightly more susceptible than An. sinensis, Ae. albopictus and Cx. quinquefasciatus against 0.04% prallethrin.

Our study showed that, Cx. quinquefasciatus was the most tolerant among these four species. Based on the 24 hour mortality, the result in table 4.1 indicated that An. sinensis was the most susceptible strain in the laboratory as well as the field strain with a percentage mortality of 83.3% and 68.3%, respectively. Aedes aegypti on the other hand, showed a mortality of 43.30% and 50% for laboratory and field strains, respectively. Aedes albopictus on the other hand indicated a mortality of 28.30% and 20% for laboratory and field strain, respectively. Culex quinquefasciatus only exhibited 24 hour mortality of

15.30% and 13.3% for both the laboratory and field stain, respectively. Hence tolerance of mosquito based on the 24 hour mortality in descending order against 0.04% prallethrin was Cx. quinquefasciatus > Ae. albopictus > Ae. aegypti > An. sinensis.

101

4.2.1.4 Bioefficacy of Product B (0.15% d-transallethrin)

The KT50 values for d-transallethrin coils (0.15% w/w) against laboratory strain of An. sinensis, Ae. aegypti, Ae. albopictus and Cx. quinquefasciatus were 0.92, 1.06, 1.68 and 5.19 min respectively (Table 4.2), and the KT50 values for the field strain were 2.06, 2.16, 3.65 and 6.33 min respectively. KT95 were 2.20, 2.14, 4.56 and 8.53 min respectively, whereas the KT 95 of the field strains was 5.46, 7.25, 7.75 and 12.27 min respectively.

Based on the KT50 and KT 95 values, An. sinensis was more susceptible than Ae. aegypti, Ae. albopictus and Cx. quinquefasciatus. Culex quinquefasciatus was the most tolerant among these four species. Percentage mortality 24 hours post-treatment indicated that laboratory and field strains of

An. sinensis was the most susceptible with 100% and 68.30% mortality, respectively. Aedes aegypti showed 93.30% mortality and the field strain showed 50% mortality. Meanwhile, the laboratory strain of Ae. albopictus indicated 68.30% mortality and 33.33% mortality for the field strain. Culex quinquefasciatus appeared to have lowest mortality values when compared with other species. Only 6.67% mortality was recorded after 24-hours post-treatment for the lab strain and 5.53% for the field strain.

102

Table 4.2: Knockdown time (minutes) and mortality percentage of product B (0.15% d-trans allethrin) against Anopheles sinensis, Aedes aegypti, Aedes albopictus and Culex quinquefasciatus using the glass chamber method.

Laboratory strain Field strain RR Mosquito species Reg. + SE KT50 KT95 Reg. + SE Mortality KT50 KT95 Reg. + Mortality (CL 95%) (CL95%) % (CL 95%) (CL95%) SE % (24-h) (24-h)

Anopheles sinensis 0.92 2.20 4.35 + 100 2.06 5.46 3.88+ 68.30 2.24 (0.82-1.01) (1.95- 0.34 (1.16- (3.53- 0.27 2.55) 3.03) 28.02)

Aedes aegypti 1.06 2.14 5.42 + 93.30 2.16 7.25 3.13+ 50.00 2.04 (0.86-1.26) (1.75- 0.45 (1.74- (5.26- 0.24 3.02) 2.89) 13.23)

Aedes albopictus 1.68 4.56 3.79 + 68.30 3.65 7.75 5.03+ 33.33 2.17 (1.10-2.25) (3.28- 0.22 (3.47- (7.17- 0.29

8.43) 3.83) 8.54)

5.19 Culex (4.93-5.45) 8.53 7.63 + 6.67 6.33 12.27 5.72+ 5.33 1.22 quinquefasciatus (7.95- 0.38 (6.01- (11.06- 0.33

9.34) 6.67) 14.10)

103

4.2.1.3 Bioefficacy of Product C (0.30% d-allethrin)

The KT50 values for d- allethrin coils (0.30% w/w) against laboratory strain of An.sinensis, Ae. aegypti, Ae. albopictus and Cx. quinquefasciatus were

0.77, 1.21, 1.60 and 5.65 min respectively and KT95 values are 1.93, 2.27,

3.93 and 10.25 min respectively (table 4.3). For the field strain the KT50 values were 2.83, 2.99, 3.24 and 4.28min, respectively and the KT95 values were 7.15,

6.36, 6.42 and 8.46 min, respectively.

The resistance ratios based on the KT50 value for An. sinensis, Ae. aegypti, Ae. albopictus and Cx. quinquefasciatus were 3.67,2.47,2.00 and 0.70, respectively. Anopheles sinensis were highly resistant. Even though, it was surprising to note that field Cx. quinquefasciatus strain was more susceptible than the laboratory strain, however the mortality percentage of laboratory strain still higher from the field strain, therefore the field strain still more tolerant than the laboratory strain.

104

Table 4.3: Knockdown time (minutes) and mortality percentage of product C (0.30% d- allethrin) against Anopheles sinensis, Aedes aegypti, Aedes albopictus, and Culex quinquefasciatus using the glass chamber method.

Laboratory strain Field strain RR Mosquito species Reg. + SE KT50 KT95 Reg. + Mortality KT50 KT95 Reg. + Mortality (CL 95%) (CL95%) SE % (CL (CL95) SE % (24-h) 95%) (24-h)

Anopheles sinensis 0.77 1.93 4.14 + 100.0 2.83 7.15 4.08+ 100 3.67 (0.82-1.01) (1.70- 0.37 (2.37- (5349- 0.30 2.28) 3.32) 11.85)

Aedes aegypti 1.21 2.27 6.01+ 100.0 2.99 6.36 5.91+ 100 2.47 (0.99-1.42) (1.89- 0.50 (2.62- (5.14- 0.39 3.14) 3.40) 9.42)

Aedes albopictus 1.60 3.93 4.22 + 80.0 3.24 6.42 5.54+ 68.3 2.00 (1.01-2.19) (2.80- 0.24 (3.08- (5.92- 0.36 7.67) 3.40) 7.09)

Culex 5.65 10.25 6.36 + 15.0 4.28 8.46 5.55+ 6.67 0.76 quinquefasciatus (5.40-5.90) (9.60- 0.30 (3.88- (7.42- 0.29 11.11) 4.68) 10.24)

105

4.2.1.4 Boefficacy of Product D (0.30% d-allethrin)

The KT50 values for 0.30% d- allethrin coils (Product D) against the lab strain An. sinensis, Ae. aegypti, Ae. albopictus and Cx. quinquefasciatus were

0.99, 1.49, 1.54 and 5.04 min, respectively. Whereas the KT95 values were

2.20, 2.86, 3.72 and 9.40 min, respectively (Table 4.4), The field strain, however showed higher value than the lab strain with the KT50 values of 2.76,

2.27, 3.64 and 5.61min, respectively and KT95 values of 2.35, 7.80, 6.59 and

10.43 min, respectively(Table 4.4) . Both strain showed 100% mortality for An. sinensis and Ae. aegypti.

106

Table 4.4: Knockdown time (minutes) and mortality percentage of product D (0.30% d-allethrin) against Anopheles sinensis, Aedes aegypti, Aedes albopictus, and Culex quinquefasciatus using the glass chamber method.

Laboratory strain Field strain RR Mosquito species Reg. + SE KT50 KT95 Reg. + Mortality KT50 KT95 Reg. + SE Mortality (CL 95%) (CL95%) SE % (CL 95%) (CL95%) % (24-h) (24-h)

Anopheles sinensis 0.99 2.20 4.37 + 100.00 2.76 2.35 4.54+ 100 2.37 (0.73- (1.66- 0.39 (1.15- (3.95- 0.34 1.25) 3.90) 5.54) 2158.25)

Aedes aegypti 1.49 2.86 5.78+ 100.00 2.77 7.80 3.66 + 100 1.86 (1.07- (2.22- 0.44 (2.47- (6.62- 0.24 1.86) 5.17) 3.05) 9.79)

Aedes albopictus 1.54 3.72 4.30 + 85.00 3.64 6.59 6.36+ 66.70 2.36 (0.94- (2.58- 0.28 (3.29- (5.81- 0.36 2.17) 8.73) 3.97) 7.95)

Culex 5.04 9.40 6.07 + 33.30 5.61 10.43 6.11+ 16.67 1.11 quinquefasciatus (4.78- (8.74- 0.29 (5.26- (9.39- 0.33 5.29) 10.27) 5.97) 12.04)

107

4.2.1.5 Bioefficacy of Product E (0.20% d-allethrin)

This is the Standard Reference Product, containing 0.20% d-allethrin.

The KT50 values for 0.20% d- allethrin coils (Product E) against laboratory strain

An. sinensis, Ae. aegypti, Ae. albopictus and Cx. quinquefasciatus were 0.77 ,

1.78, 1.52 and 5.13 min respectively and 2.31, 2.99, 3.35 and 4.67min respectively for the field strain (Table 4.5). On the other hand, the KT95 values were 2.04, 3.57, 4.88 and 8.80 min respectively for the lab strain and for the field strain the values were 3.86, 7.87, 8.16 and 9.45 min, respectively (Table

4.5). Percentage mortality at 24 hours post-treatment showed that An. sinensis had 100% mortality and Ae. aegypti had 88.3% mortality for both the lab and field strains. However Ae. albopictus showed 55.0% and 50% mortality values while Cx. quinquefasciatus appeared to have the lowest mortality value of

16.7% and 6.67% mortality for lab and field strains respectively. Anopheles sinensis showed lower KT50 and KT 95 values and highest mortality values than

Ae. aegypti, Ae. albopictus and Cx. quinquefasciatus. Therefore this species was the most susceptible strain in comparison to the other three species. Culex quinquefasciatus was the most tolerant among the four species as it showed the highest KT50 and KT95 values and the lowest values for mortality. For the resistance ratio, An. sinensis, Ae. aegypti, Ae. albopictus and Cx. quinquefasciatus showed values of 3.00, 1.68, 2.20 and 0.91 respectively.

108

Table 4.5: Knockdown time (minutes) and mortality percentage of product E (0.20% d-allethrin) against Anopheles sinensis, Aedes aegypti, Aedes albopictus, and Culex quinquefasciatus using the glass chamber method

Laboratory strain Field strain RR Mosquito species Reg. + SE KT50 KT95 Reg. + Mortality KT50 KT95 Reg. + Mortality (CL 95%) (CL95%) SE % (CL 95%) (CL95%) SE % (24-h) (24-h)

Anopheles sinensis 0.77 2.04 3.87 + 100.00 2.31 3.86 3.78+ 100.00 3.00 (0.53-0.96) (1.56-3.42) 0.38 (1.91- (3.21-5.71) 0.25 2.69)

Aedes aegypti 1.78 3.57 5.46 + 88.30 2.99 7.87 7.35+ 88.30 1.68 (1.00-2.50) (2.53-12.57) 0.38 (2.71- (7.22-8.75) 0.53 3.28)

Aedes albopictus 1.52 4.88 3.26+ 55.00 3.35 8.16 4.44+ 50.00 2.20 (0.93-2.19) (3.16- 0.21 (3.16- (6.98-10.14) 0.26 13.400 3.54)

Culex quinquefasciatus 5.13 8.80 7.03 + 16.70 4.67 9.45 5.38+ 6.67 0.91 (4.80-5.45) (8.07-9.85) 0.34 (4.47- (8.81-10.28) 0.29 4.87)

109

4.3 DISCUSSION

Based on the KT50 values, KT95 values and the percentage mortality data, it was observed that An. sinensis was more susceptible than Ae.aegypti,

Ae.albopictus and Cx. quinquefasciatus to all a.i chosen in this laboratory efficacy study. Aedes aegypti was more susceptible than Ae.albopictus. Culex quinquefasciatus was the most tolerant for both the lab and field strains. The test result corresponded well with the mosquito coil biological efficacy studies against Ae aegypti and Cx quinquefasciatus by Yap & Chung (1987) and mat biological efficacy study by Yap et al. (1995). Culex quinquefasciatus was the most tolerant against all a.i tested. The low mortality values and highest KT50 and KT95 values were recorded for this species. These results were also similar to the studies by Ogami et al. (1970) where they reported that Cx. pipien pallens

(a related Cx. pipiens complex species), when tested using coils with d-allethrin had shown a slow knockdown. According to Yap & Chong (1996), Cx. quinquefasciatus was more tolerant to a wide range of insecticides when compared to Aedes species. Yap et al. (1995) stated that a low mortality value also was achieved for this Culex species of mosquito in d- allethrin mat bioefficacy studies.

The resistance ratio which is a ratio of KT50 values of field strains to laboratory strains indicated that the field strains were more resistant than the laboratory strains for all the a.i tested. It was surprising to note that field Cx. quinquefasciatus strain was more susceptible than the laboratory strain against product C (0.30% d-allethrin). However from the observation of the percentage

110 mortality, the mortality between field and lab strain still indicated that the field strain was more resistant than the lab strain with lower percentage mortality.

Different active ingredients may give different results for knockdown of mosquitoes. The formulation use in this study was 0.04% prallethrin (Product

A), 0.15% d-trans allethrin (B), 2 brands containing 0.30% d-allethrin (C and D) and a Standard Reference Product containing 0.20% d-allethrin. The higher the a.i the more toxic it is to the mosquito species. Owing to the fact that all pesticides are toxic to some degree against target organisms or non-target organisms, the lower amount of chemical used in the field, the better to avoid environmental contamination. Pyrethroids are effective when used at extremely low doses; therefore the final product should have a lesser impact on the public.

Under normal usage, the smoke produced by mosquito coil is not harmful to humans when inhaled (Yap, 1996; Jurgen & Umar, 1999).

Mosquito coil is widely known as an efficient mosquito repellent (Weili et al., 2003). The major active ingredients of the mosquito coil are pyrethoids, accounting for about 0.3-0.4% of coil mass (Lukwa & Chandiwana, 1998). The remaining components of mosquito coils are organic filters, binders and dyes

(Weili et al., 2003). Mosquito coils are final products which may differ depending on the type and/or amount of wood, coconut shells or binders, present in coil

(Jurgen & Umar, 1999). The active ingredient present is a knockdown agent.

The different concentration of active ingredients used in this study ((A)0.04% prallethrin, (B)0.15% d-trans allethrin, 2 brands (C and D) containing 0.30% d- allethrin and a Standard Reference Product (E) containing 0.20% d-allethrin) showed different percentage of mortality, and KT50 and KT95 values for different

111 species of mosquitoes. The amount of active ingredient present in the coils gave different mortality percentage. High concentration of a.i. present may give a quick knockdown but less mortality. Less a.i. present may give a slow knockdown but a better percentage of mortality such as product A and product

B as shown by Cx. quinquefasciatus. In the study by Lukwa & Chandiwana

(1998), formulation containing 0.4% pyrethrins inhibited 92.5% from biting as compared to a 0.3% formulation (80%), which indicated that formulation containing 0.4% pyrethrins is more effective in repelling mosquitoes than a

0.3% formulation. In the study on the bio efficacy of mosquito mat, coil and dispenser formulations containing allethrin group of pyrethroids against mosquito vectors by Amalraj et al. (1996), all the three allethrins tested in mat and coil formulations were equally effective against the three vector mosquitoes, when compared to mat formulations of d-allethrin and bioallethrin, whereas mat formulation of esbiothrin was 156 and 144 times more effective against Cx. quinquefasciatus. It was 162 times more effective against Cx. quinquefasciatus than in the coil formulation. Dispenser containing d-allethrin was the least effective against Cx. quinquefasciatus (KT50: 2.65 min.) and Ae. aegypti (KT50: 4.68 min.) but as effective as coil against An. stephensi. In the present study, it was also found that when coil burnt continuously for 20 minutes the knockdown was consistently above 60% in all the four vector species but mortality was not consistently 100% for An. sinensis and Ae aegypti against all the a.i. tested. This indicated that the a.i. only gave a good knock down effect to the species but not effective in killing the insect. As a conclusion, mosquito coil might result in knock down of mosquito but not killing the mosquito. Mosquito coil is useful as a repellent for personal users.

112 CHAPTER FIVE

SUMMARY AND CONCLUSION

Surveillance on mosquito fauna was carried out in a suburban area in

Penang. It was found that several species were abundant here. By using the cow baited trap and bare leg catch techniques, a total of 1 780 (60.14%)

Anopheles sp., 821 (27.74%) Culex sp., 347 (11.72%) Mansonia sp. and 12

(0.41%) Aedes sp. was collected in this area. Seven species were identified. A total of 675 (22.80%) of Culex gelidus was collected making it the most dominant species collected; followed by 514 (17.37%) Anopheles peditaeniatus,

383 (12.94%) Anopheles sinensis, 254 (8.58%), Mansonia uniformis, 252

(8.52%) Anopheles subpictus, 192 (6.49%) Anopheles campestris and 135

(4.57%) Anopheles vagus, The mosquitoes seemed to be more attracted to animal than human.

Dengue still constitutes a great public health problem in many tropical countries. The vectors, Aedes aegypti and Aedes albopictus are container breeders and are found in the immediate vicinity of human habitations. An outdoor ovitrap surveillance was carried out in an urban (Taman Permai Indah) and suburban areas (Kg Pasir Gebu) in Penang Island for 14 months. Aedes albopictus was the most abundant species and this indicated that it is the dengue vector in the study sites.

113 The seasonal fluctuation of egg population corresponded to the seasonal rainfall pattern in the study sites. The mean temperature and relative humidity did not seem to affect the egg production. Eggs were found in abundance during dry season while in wet months a decrease in egg production was observed. The environmental factors were found to affect Aedes breeding.

During this study, it was found that the residents in Kg Pasir Gebu used mosquito coils widely as a personal protection measure against mosquitoes. A laboratory bio efficacy test on several common brands of mosquito coils against four species of mosquito vectors that were available in this site was conducted.

Laboratory and field strains of Ae.aegypti, Ae albopictus, Cx. quinquefasciatus and An. sinensis were used throughout the study. Five coil formulations were tested agains these mosquitoes. Based on the KT50 and KT95 and mortality, it was found that An. sinensis was the most susceptible whereas Cx. quinquefasciatus was the most tolerant. The lab strain mosquito was also more susceptible than the field strain mosquitoes.

The finding from this study provides a better understanding on the bionomics and insecticide susceptibility of mosquito vectors in Malaysia. Further studies and continuous surveillance should be conducted from time to time in order to find a solution for control of mosquito especially Aedes in the effort to reduce the transmission of dengue or dengue haemorrhagic fever.

114

BIBLIOGRAPHY

Abu H & Salmah MR. 1990. A checklist of the mosquitoes of Malaysia. Journal of Bioscience 1: 29-41.

Abu Hassan A & Yap HH. 1999. Mosquitoes. In: Chong NL, Jaal Z, Lee CY, Yap HH, eds. Urban Pest Control a Malaysian Perspective Penang: Vector Control Research Unit, Universiti Sains Malaysia. pp 26-38.

Abubakar S & Shafee, N. 2002. Outlook of dengue in Malaysia: a century later. Malaysian Journal of Pathology 24: 23-27.

Adanan CR, Jahangir K, Yap HH & Zairi J. 2000. Culex: mosquitoes that spread Japanese encephalitis. In: Ng FSP, Yong HS, eds. Mosquito and mosquito borne disease Kuala Lumpur, Malaysia: Akademi Sains Malaysia. pp 73.

Allan SA & Kline DL .1995. Evaluation of organic infusions and synthetic compounds mediating oviposition in Aedes albopictus and Aedes aegypti (Diptera: Culicidae). Journal of Chemical Ecology 21:1847-1860.

Amalraj DD, Sivagnaname N, Boopathidoss PS & Das PK. 1996. Bioefficacy of mosquito mat, coil and dispenser formulations containing allethrin group of synthetic pyrethroids against mosquito vectors. The Journal of Communicable Diseases 28:85-93.

Anonymous. 1987. Important aspects associated with the performance of insecticides vaporizing mat and coil products. The Wellcome Foundation Ltd, Berkhamsted, England. 10pp.

Andrew KG, Lindsay SW, Canfalonieri UE & Patz JA. 2000. Climate change and vector-borne diseases: a regional analysis. Bulletin of the World Health Organization 78: 1136-1147.

Arunachalam N, Samuel PP, Hiriya J & Gajanana A. 1999. A comparative study on sampling techniques for Aedes aegypti (Diptera: Culicidae) surveillance in Madurai, South India. Tropical Biomedicine 16: 25-29.

Barrera R, Avila J & Navarro J. 1997. Dinamica poblacional de Aedes aegypti (L.) en centros urbanos con deficiencia en el suministro de agua. Acta Biol Venez 17: 23-35.

Barlow F & Hadaway AB. 1975. The insecticidal activity of some synthetic pyrethroids against mosquitoes and flies. PANS 21:233-238.

Barry JB & Williams CM. 1996. The biology of diseases vector. University Press of Colarado.

115 Bellini R, Carrieri M, Burgio G & Bacchi M. 1996. Efficacy of different ovitraps and binomial sampling in Aedes albopictus surveillance activity. Journal of the American Mosquito Control Association 12:632-636.

Bentley MD & Day JF. 1989. Chemical ecology and behavioral aspects of mosquito oviposition. Annual Review of Entomology 34: 401-421.

Bently MD, McDaniel IN, Lee HP, Stiehl B & Yatagai M. 1976. Studies of Aedes triseriatus and Aedes atropalpus (Diptera: Culicidae). Journal of Medical Entomology 13: 112-115.

Black WC IV, Rai KS, Turco BJ & Arroyo DC. 1989. Laboratory study of competition between United States strains of Aedes albopictus and Aedes aegypti (Diptera: Culicidae). Journal of Medical Entomology 26: 260-261.

Braga IA, Gomes AC, Nelson M, Mello RCG, Bergamaschi DP & Souza JMP. 2000. Comparação entre pesquisa larvária e armadilha de oviposição para detecção de Aedes aegypti. Revista Brasileira de Medicina Tropical 33: 347- 353.

Brenda TB, Anthony AJ & Bruce MC. 2000. Genetics of Mosquito Vector Competence. Microbiology and Molecular Biology Reviews 64: 115–137.

Canyon DV & Hii JLK. 1999. Insecticide susceptibility status of Aedes aegypti (Diptera: Culicidae) from town-ville. Australian Journal of Entomology 38:40- 43.

CDC. 2004. Dengue fever. Pg. 1-3. Available from World Wide Web: http://www.cdc.gov/ncidod/dvbid/dengue/

CDC. 2005. Dengue fact sheet. pg. 1-3. Available from World Wide Web: http://www.cdc.gov/ncidod/dvbid/dengue/

Chadee DD. 1991. Seasonal incidence and vertical distribution patterns of oviposition by Aedes aegypti in an urban environment in Trinidad, West Indies. Journal of The American Mosquito Control Association 7: 383-386.

Chadee DD. 1992. Seasonal incidence and horizontal distribution patterns of oviposition by Aedes aegypti in an urban environment in Trinidad, West Indies. Journal of the American Mosquito Control Association. 8:281-284.

Chadee DD, Lakhan A, Ramdath WR & Persad RC.1993. Oviposition response of Aedes aegypti to different concentrations of hay infusion in Trinidad, West Indies. Journal of the American Mosquito Control Association 9: 346-348.

Chadwick PR. 1975. The activity of some pyrethroids, DDT and lindane in smoke from coils for biting inhibition, knockdown and kill of mosquitoes. Bulletin of Entomological Research 65:97-107.

116 Chan KL. 1971. Life table studies of Aedes albopictus (Skuse). In : Proceedings of a symposium on the sterility principle for insect control or eradication. International Atomic Energy Agensy 131-144.

Chan KL, Chan YC & Ho BC. 1971a. Aedes aegypti (L) and Aedes albopictus in Singapore city .1. Distribution and density. Bulletin World Health Organization 44: 617-627.

Chan KL, Chan YC& Ho BC. 1971b. Aedes aegypti (L) and Aedes albopictus in Singapore city .1. Competition between species. Bulletin World Health Organization 44: 643-649.

Chan KL & Counsilman JJ. 1985. Effects of slum clearance, urban redevelopment and vector control on densities of Aedes mosquitoes in Singapore. Tropical Biomedicine 2: 139-147.

Chan KL, Kiat NS & Kok TK. 1977. An autocidal ovitrap for the control and possible eradication of Aedes aegypti. Southeast Asian Journal of Tropical and Public Health 8:56-62.

Chang MS. 2000. Mansonia: mosquitoes that spread filariasis. In: Ng FSP, Yong HS, eds. Mosquito and mosquito borne disease Kuala Lumpur, Malaysia: Akademi Sains Malaysia. pp 81.

Chen CD, Seleena B, Mohd Masri S, Chiang YF, Lee HL, Nazni WA & Sofian Azirun M. 2005. Dengue vector surveillance in urban residential settlement areas in Selangor, Malaysia. Tropical Biomedicine 22:39-43

Chiang GL & Loong KP. 1985. Colonization of mosquito vectors of Brugian filariasis. In: Mak JW & Yong HS eds. Control of Brugian Filariasis WHO Kuala Lumpur: Institute of Medical Research. p 97-102.

Chua KB, Chua IL, Chua IE & Chua KH. 2005. Effect of chemical fogging on immature Aedes mosquitoes in natural field conditions. Singapore Medical Journal 46: 639-644.

Chooi CK. 1985. Status of malaria vectors in Malaysia. Southeast Asian Journal of Tropical and Public Health 16:133-133.

Chow CY. 1970. Bionomics of malaria vectors in the Western Pacific Region. Southeast Asian Journal of Tropical and Public Health 1:40-57.

Clements AN. 1999.The biology of mosquitoes New York: CABI Publishing. Oxford, New York: Oxford University Press. 356 pp.

Clements. 1999. The biology of mosquitoes Volume 2: Sensory Reception and Behaviour New York: CABI Publishing.

117 Colless DH. 1959. Notes on the culicinae mosquitoes of Singapore. VII. Host preferences in relation to the transmission of disease. Annals of Tropical Medicine and Parasitology 53: 259-67.

Collins RT, Jung RK, Anoes H, Sutrisno RH & Putut D. 1979. A study of the coastal malaria vectors Anopheles sundaicus (Rodenwaldt) and Anopheles subpictus Grassi, in south Sulawesi, Indonesia. WHO /VBC/79.740. WHO Geneva, Switzerland. 12pp.

Corbet PS & Chadee DD. 1993. An improved method for detecting substrate preferences shown by mosquitoes that exhibit ‘skip oviposition’. Physiological Entomology 8: 114-118.

Colton YM. Chadee DD & Severson DW. 2003. Natural skip oviposition of the mosquito Aedes aegypti indicated by codominant genetic markers. Medical and Veterinary Entomology 17: 114-118.

Covell G. 1944. Notes on the distribution, breeding places, adult habits and relation to malaria of the anopheline mosquitoes of India and the Far East. Journal of the Malaria Institute of India 5: 399-434.

Craig RW, Scott AR & Peter IW. 2005. Potential distribution of the Asian disease vector Culex gelidus Theobald (Diptera: Culicidae) in Australia and New Zealand: a prediction based on climate suitability. Australian Journal of Entomology 44: 425.

CRME. 1989. Annual Report. Centre For Research in Medical Entomology, Madurai. p 184.

Damar TB. 1997. Anopheles hyrcanus species group in Northwestern Peninsular Malaysia and Nias Island, Indonesia (Bionomics, phylogenetic relationship and vectorial capacity). [PhD desertation]. USM.

Darwin EQ, Todd D, Iidefonso FS, Roman EL, Dolores VO, Lourdes CSG, Magaly AA, Armando EQ, Jose IG, Juan FCC, Hila G, Amelia TdR, Bradley JB, Alan DTB, Barry JB & Robert BT. 2005. West Nile Virus Isolation in human and mosquitoes Mexico. Emerging Infectious Disease 11: 1449-1452.

Daniels CW. 1908. Notes on the mosquitos of the river and coast district of the eastern side of the Peninsular. Studies from IMR. In: Vithlingam I, Chiang GL, Lee HL, & Singh I. 1992. Bionomics of important mosquito vectors in Malaysia. Southeast Asian Journal of Tropical Medicine and Public Health 23: 587-603.

David WAL & Bracey P. 1946. Factors influencing the interaction of insecticidal mists on flying insects. Bulletin of Entomological Research 27:177-190.

118 deGarin A, Bejaran RA, carbajo AE, de Casas & Schweigmann N. 2000. Atmospheric control of Aedes aegypti population in Buenos Aires (Argentina) and its variability. International Journal of Biometeorology 44: 148-156.

Edman J. Scoot T, Costero A, Morrison AC, Harrington LC & Clark GG. 1998. Aedes aegypti (Diptera: Culicidae) movement influenced by availability of oviposition sites. Journal of Medical Entomology 35: 578-583.

Elliot M. 1995. Chemicals in Insect control. In ‘Pyrethrum Flowers. Production, Chemistry, toxicology and Uses. Eds:JE Casida, GB Quidstad, pp 3-31.

Elliot M, Janes NF & Potter C. 1978. The future of pyrethroids in insect control. Annual Review of Entomology 23:443-469.

Estrada-Franco JG & Craig GB Jr. 1995. Biology, disease relationships and control of Aedes albopictus. Washington D. C.: Pan American Health Organization. 49 pp.

Evan AM. 1938. Mosquitoes of the Ethiopian Region II: Anophelini. Oxford University Press, London.

Evans BR & Bevier GA. 1969. Measurement of field populations of Aedes aegypti with the ovitrap in 1968. Mosquito News 29: 347-353.

Fay RW & Eliason DA. 1966. A preferred oviposition site as a surveillance method for Aedes aegypti. Mosquito News 26: 531-535.

Fay RW & Perry AS. 1965. Laboratory studies of ovipositional preference of Ae. aegypti. Mosquito News 25:276.

Foo LC, Tim TW, Lee HL & Fang R. 1985. Rainfall, abundance of Aedes aegypti and dengue infection in Selangor, Malaysia. Southeast Asian Journal of Tropical Medicine and Public Health 16: 560-568.

Gillies MT. 1953. The duration of gonotrophic cycle in Anopheles gambiae and An. funestus with a note on the effiency of hand catching. East African Medical Journal 30:129-135.

Gilliet JD. 1971. Mosquitoes: The World Naturalist. Richardo Clay (The Chaucer Press) ltd.

Ginny TA & Ren JS. 2000. The use of GIS in ovitrap monitoring for dengue control in Singapore. Dengue Bulletin 24. Available from World Wide Web: http://w3.whosea.org/EN/Section10/Section332/Section522_2536.htm

Gratz NG. 1991. Emergency control of Aedes aegypti as a disease vector in urban areas. Journal of the American Mosquito Control Association 7:353- 65

119 Grimstad PR, Kobayashi JF, Zhang M & Craig GB Jr. 1997.Recently introduced Aedes albopictus in the United State: potential vector of La Crosse virus (Bunyaviridae: California serogroup). Journal of the American Mosquito Control Association 5:422-427.

Gubler DJ. 1970. Comparison of reproductive potentials of Aedes (Stegomyia) albopictus Skuse and Aedes (Stegomyia) polynesiensis Marks. Mosquito News 30: 201-208.

Gubler DJ. 1997. Dengue and dengue haemorrhagic fever : Its history and resurgence as a global public health problem. In D.J Gubler and G. Kuno (eds.) Dengue and Dengue Haemorrhagic Fever. University Press, Cambridge. UK. pp. 1-22.

Gubler DJ & Clark GG. 1996. Community involvement in the control of Aedes aegypti. Acta Tropica 61: 169-179.

Guha-Sapir D. & Schimmer B. 2005. Dengue fever: new paradigms for a changing epidemiology.Emerging Themes in Epidemiology 2 (1): Available from World Wide Web: http://www.ete-online.com/content/2/1/1

Haddow AJ. 1954. Studies on the biting habits of African mosquitoes. An appraisal of methods employed, with special reference to the twenty-four hour catch. Bulletin of Entomological Research 45: 199-242.

Hairi F, Ong CH, Suhaimi A, Tsung T W, Azhar MB, A Sundaraj C & Soe MM. 2003. A Knowledge, Attitude and Practices (KAP) Study on dengue among selected rural communities in the Kuala Kangsar District. Asia-Pacific Journal of Public Health 15: 37-43

Hammond WMcD. 1966. History of mosquito-borne haemorrhagic fever. Bulletin World Health Organisation 44: 643-649.

Hammond WMcD.1973. Dengue haemorrhagic fever-Do we know its cause? American Journal of Tropical Medicine and Hygiene 22:82-91.

Hanson SM, Mutebi JP, Craig Jr. GB & Novak RJ. 1993. Reducing the overwintering ability of Aedes albopictus by male release. Journal of the American Mosquito Control Association 9: 78-83.

Harrington LC, Scott TW, Lerdthusnee K, Coleman RC, Costero A, Clark GG, Jones JJ, Kitthawee S, Kittayapong P, Sithiprasasna R & Edman JD. 2005. Dispersal of the dengue vector Aedes aegypti within and between rural communities. American Journal of Tropical Medicine and Hygiene 72: 209- 220

Hawley AW. 1988. The biology of Aedes albopictus. Journal of the American Mosquito Control Association 4: 1-40.

120

Hidayati H, Mohd Sofian A, Nazni WA & Lee HL. 2005. Insecticides resistance development in Culex quinquefasciatus (Say), Aedes aegypti (L.) and Aedes albopictus (Skuse) larvae against malathion, permethrin and temephos. Tropical Biomedicine 22: 45-52.

Ho C, Chou TC, Chen TH & Hsueh AT.1962. The Anopheles hyrcanus group and its relation to malaria in East China. Chinese Medical Journal 81:71-78.

Ho TM & Vithylingam I. 1980. A preliminary survey of Aedes aegypti in Selangor, Peninsular Malaysia. Medical Journal of Malaya 34:409-413.

Hobbs JH, Hughes EA & Eichold II BH. 1991. Replacement of Aedes aegypti by Aedes albopictus in Mobile, Alabama. Journal of the American Mosquito Control Association. 7: 488-489.

Hornby JA, Moore DE & Miller TW Jr. 1994. Aedes albopictus distribution, abundance and colonization in Lee County, Florida, and its effect on Aedes aegypti. Journal of the American Mosquito Control Association 10:397-402.

Honório NA, Costa Silva WD, Leite, PJ, Gonçalves JM, Lounibos LP & Lourenço-de-Oliveira R. 2003. Dispersal of Aedes aegypti and Aedes albopictus (Diptera: Culicidae) in an Urban Endemic Dengue Area in the State of Rio de Janeiro, Brazil. Memórias do Instituto Oswaldo Cruz 98: 191- 198.

Horsfall WR. 1956. Eggs of floodwater mosquitoes. III (Diptera: Culicidae). Conditioning and hatching of Aedes vexans. Annals of the Entomological Society of America. 49:66-71.

Ibrahim D, Diawo D, Jean BH, Yamar BA, Lassana K., Carlo C & Mawlouth D. 2005. Comparison of human-landing catches and odour-baited entry traps for sampling malaria vectors in Senegal. Journal of Medical Entomology. 42: 104-109.

Igbinosa IB. 1989. Investigations on the breeding site preferences of mosquitoes in Ekpoma, Nigeria. Journal of Applied Entomology 9: 327-355.

IMR. Entomological teaching charts. Unpublished.

Inform'ACTION, 2005. Dengue entomological surveillance. http://www.spc.int/phs/ENGLISH/Publications/InformACTION/IA20/dengue- entomological-surv.pdf

Iyenger MOT. 1938. Studies on the epidemiology of filariasis in Tranvencore. Indian Medical Research Memeograph. 30.

Jacob WL & Bevier GA. 1969. Evaluation of ovitraps in the United States. Aedes aegypti eradication program. Mosquito News. 29:650-653.

121 Johnson KM.1979. Anthropod-borne viral encephalitides. In: Beeson, Mc Dermott, Wyngarrden (Eds): Cecil Textbook of Medicine 279. Sanders Coy, London.

Jurgen P & Umar FA. 1999 Household insecticides: evaluation and assessment of inhalation toxicity: a workshop summary. Experimental and Toxicologic Pathology 50: 67-72.

Kalra NL, Kaul SM & Rastogi RM. 1997. Prevalence of Aedes aegypti and Aedes albopictus- vectors of dengue and dengue haemorrhagic fever in North, North-East and Central India. Dengue Bulletin 21:84-92.

Kettle DS. 1990. Medical & Veterinary Entomology. Sydney:Croom Helm.

Khai Ming C, Thain S, Thaung U, Tin U, Myint KS, Swe T, Halstead SB & Diwan AR. 1974. Clinical and laboratory studies on haemorrhagic fever in Burma, 1970-1972. Bulletin of World Health Organization. 51: 227-235.

Khim PC. 2003. Kajian biology Aedes aegypti (Strain VCRU dan strain Bora- bora) dan kerentanannya terhadap insektisid isi rumah (lingkaran nyamuk). [BSc. dissertation]. Universiti Sains Malaysia 100p.

Kim DC. 1974. Epidemiological studies of filariasis in inland Korea. 4: Vector determination of filariasis malayi in Yongju area. Korean Journal of Parasitology 12: 80-81.

Knight KL & A. Stone. 1977. A catalog of the mosquitoes of the world (Diptera:Culicidae) 2nd ed. College park, Maryland: Entomological Society of America.

Knudsen AB. 1995. Global distribution and continuing spread of Aedes albopictus. Parasitologia 37:91-97.

Koo LC & Hoo JHC. 1994. Mosquito coil smoke and respiratory health among Hong Kong Chinese: Results of three epidemiological studies. Indoor Environment 1994; pp. 304-310.

Lam SK. 1993. Two decades of dengue in Malaysia. Tropical Biomedicine 35: 195-200.

Lam SK. 1994. Dengue vaccines. 1st International Congress of Parasitology and Tropical Medicine 1994:46-47.

Lam SK. 1996. Dengue surveillance and early warning system, Persidangan denggi 9-11 Mei 1996. Institute for Medical Research Malaysia.

Lee CY & Robinson WH. 2001. Handbook of Malaysian household and structural pests. Kuala Lumpur: The Pest Control Association of Malaysia.

122 Lee D, Hicks M & Debenham Ml. 1989. The Culicidae of the Australasian region, Vol 7. Entomology monograph No 2, University of Queensland and University of Sydney, 1989. Australian Government Publishing Service.

Lee HL. 1990. Breeding habitats and factor affecting breeding of Aedes larvae in urban towns of Peninsular Malaysia. Journal of Bioscience 1:107-112.

Lee HL.1991. A nationwide resurvey of the factors affecting the breeding of Aedes aegypti (L.) and Aedes albopictus (Skuse) (Diptera: Culicidae) in urban towns of Peninsular Malaysia - 1988-1989. Tropical Biomedicine 8: 157-160.

Lee HL. 1992a. Aedes ovitrap and larval survey in several suburban communities in Selangor, Malaysia. Mosquito Borne Diseases Bulletin 9: 9- 15.

Lee HL. 1992b. Sequential sampling: its application in ovitrap surveillance of Aedes (Diptera: Culicidae) in Selangor, Malaysia. Tropical Biomedicine 9: 29-34.

Lee HL. 2000. Aedes: mosquitoes that spread dengue fever. In: Ng FSP, Yong HS, eds. Mosquito and Mosquito Borne Disease Kuala Lumpur, Malaysia: Akademi Sains Malaysia. p 45.

Lee HL. 2005. Vector of filariasis in Malaysia-A review, Asian Parasitology Vol.3 Filariasis in Asian and Western Pacific Islands. Eds. Kimura E, Rim HJ, Dejian S and Weerasooriya MV. Printed and bound in Japan by FAP Journal Ltd.

Lee HL & Cheong WH. 1987. A preliminary Aedes aegypti larval survey in the suburbs of Kuala Lumpur city. Tropical Biomedicine 4:111-118.

Lee HL & Inder SK. 1993. Sequential analysis of adult Aedes aegypti and Aedes albopictus in Kuala Lumpur city-its potential use in dengue epidemics prediction. Tropical Biomedicine 10: 117-123.

Lee HL & Rohani A. 2005. Transovarial transmission of dengue virus in Aedes aegypti and Aedes albopictus in relation to dengue outbreak in an urban area in Malaysia. Dengue Bulletin 29: 106-111.

Lee VH, Atmosoedjo S, Dennis DT & Suhaepi. 1983. The anopheline (Diptera : Culicidae) vectors of malaria and bancroftian filariasis in Flores Island, Indonesia. Journal of Medical Entomology 20: 577-578.

Leicester GF.1908. The Culicidae of Malaya. Studies from IMR 3: 18-261 In: Vithilingam I., Chiang G.L. Lee H.L. & Singh I. (1992). Bionomics of important mosquito vectors in Malaysia. Southeast Asian Journal of Tropical Medicine and Public Health 23: 587-603

123 Lo EKC & Narimah A. 1984. Epidemiology of dengue disease in Malaysia, 1973-1982. Journal of Malaysian Society of Health 4: 27-35.

Lukwa N & Chandiwana SK. 1998. Efficacy of mosquito coils containing 0.3% and 0.4% pyrethrins against An.gambiae sensulato mosquitoes. The Central African Journal of Medicine 44:104–107.

Macdonald WW. 1956. Aedes aegypti in Malaya. Distribution and dispersal. Annals of Tropical Medicine and Parasitology. 50: 385-398.

Macdonald D. 1957a. Aedes aegypti in Malaya. 3. Experiments on control and eradication. Annual Tropical Medicine and Parasitology. 51:175-186.

Macdonald D. 1957b. The Epidemiology and Control of Malaria. Oxford University Press, Oxford, United Kingdom.

Mahapatra N, Dash A & Hazra RK. 1995. Host selection patterns of twelve mosquito species in Orissa, India. Tropical Biomedicine 12:147-152.

Mac Iver DR. 1963. Mosquito coils. General description of coils, their formulation and manufacture. Pyrethrum Post 7:22-27.

Mak JW. 1986. Problems in filariasis control and the need for human behavior and social economic research. Southeast Asian Journal of Tropical Medicine and Public Health 17:479-485.

Manorenjitha, 2005. The ecology and biology of Aedes aegypti (Linnaeus) and Aedes albopictus (Skuse) (Diptera: Culicidae) and the resistance status of Aedes albopictus (field strain) against organophosphates in Penang, Malaysia. MSc. dissertation. Universiti Sains Malaysia 149p.

Marvin SG Jr, Nasci R, Harry MS, Stephen A, Raymond Ki, Ann MP, Kristen B, Leah C, Dawn C, Sarah L, Viki T & Charles TP. 2005. West Nile Virus- infected mosquitoes, Lousiana 2002. Emerging Infectious Disease 11(9): 1399-1404.

Micieli MV & Campos RE. 2003. Oviposition and seasonal pattern of a population of Aedes (Stegomyia) aegypti (L.) (Diptera: Culicidae) in suptropical Argentina. Memórias do Instituto Oswaldo Cruz 98: 659-663.

Mitchell C. 1991. Vector competence of North and South American strains of Aedes albopictus for certain arboviruses: A review. Journal of the American Mosquito Control Association 7: 446-451.

Mitchell C, Niebylski M, Smith G, Karabatsos N, Martin D, Mutebi JP, Craig GB, Mahler M. 1992. Isolation of Eastern equine encephalitis virus from Ae. albopictus in Florida. Science 257: 526-527.

Ministry Of Health. 2003. Unpublished data.

124 Ministry Of Health. 2005. Unpublished data.

Miyagi I & Toma T. 2000. The mosquitoes of Southeast Asia. In: Ng FSP, Yong HS, eds. Mosquito and mosquito borne disease Kuala Lumpur, Malaysia: Akademi Sains Malaysia. p 1-17.

Mogi M, Khamboonruang C, Choochote W & Suwanpanit P. 1990. Applicability of presence-absence and sequential sampling for ovitrap surveillance of Aedes (Diptera: Culicidae) in Chiang Mai, northern Thailand. Journal of Medical Entomology 27: 509-514.

Monath TP. 1979. Arthropod-borne encephalitides in the Americas. Bulletin of the World Health Organization 57: 513-533

Monath TP. 1988. The arboviruses: Epidemiology and ecology. Vols 1-5 Boca Raton, FL: CRC press.

Mori A & Wada Y. 1978. The seasonal abundance of Aedes albopictus in Nagasaki. Tropical Medicine 20: 29-37.

Moore CG, Cline BL, Ruiz-Tiben, Lee D, Romney-Joseph & Rivera- Correa.1978. Aedes aegypti in Puerto Rico: environmental determinants of larval abundance and relation to dengue virus transmission. American Journal of Tropical Medicine and Hygiene. 27: 1225-1231.

Moore CG & Mitchell CJ. 1997. Aedes albopictus in the United States: Ten-year presence and public health implications. Emerging Infectious Disease 3:329- 334.

Moretto A. 1991. Indoor spraying with the pyrethroid insecticide lambda- cyhalothrin: effects on spraymen and inhabitants of sprayed houses. Bulletin World Health Organization 69:591-594.

Nasci RS, Hare SG & Willis FS. 1989. Interspecific mating between Lousiana strains of Aedes albopictus and Aedes aegypti in the field and laboratory. Journal of the American Mosquito Control Association. 5: 416-421.

Novak RJ, Steinly BA & Webb W. 1991. A new method for monitoring mosquito oviposition in artificial and natural containers. Journal of the American Mosquito Control Association 7:649-654.

Ogami H, Yoshida Y, Katsua Y, Miyamoto J & Kodata T. 1970. Insecticidal activity of a new synthetic chrysanthemic ester, 5-propargylfurfuryl chrysanthemate (Prothrin). BotyuKagaku 35:45-55.

Ohmori Y & Otsoru M. 1960. Malaria studies in Japan after World War II. The research for Anopheles sinensis sibling species group. Japanese Journal of Experimental Medicine 30: 33-65.

125 Okogun RAG, Bethran EBN, Anthony NO, Jude CA & Anegbe CE. 2003. Epidemiological implications of preferences of breeding sites of mosquito species in Midwestern Nigeria. Annals of Agricultural and Environmental Medicine 10: 217-222.

O’Meara GF, Gettman AD, Evans LF Jr. & Scheel FD. 1992. Invasion of cemeteries in Florida by Aedes albopictus. Journal of the American Mosquito Control Association. 8: 1-10.

O’Meara GF, Gettman AD, Evans LF Jr & Curtis GA. 1993. The spread of Aedes albopictus in Florida. Annals of the Entomological Society of America 39: 163-172.

Pang T, Hamimah H & Ramalinggam S. 1991. Demam denggi dan demam denggi berdarah Kuala Lumpur: Dewan Bahasa dan Pustaka.

Panicker KN, Geetha BM, Bheema R, US Visan K. & Suryanarayanamurthy U. 1981. Anoheles subpictus vector of malaria in coastal villages of Southeast India. Current Science 50:69 4-495.

Poovaneswari S. 1993. Dengue situation in Malaysia. Malaysian Journal Pathology 15:3-7.

Rahman WA, Adanan Cr & Abu Hassan A. 1997. Malaria and Anopheles mosquitoes in Malaysia. Southeast Asian Journal of Tropical Medicine and Public Health 28: 599-605.

Rahman WA, Adanan CR & Abu Hassan A. 2002. Species composition of adult Anopheles populations and their breeding habitats in Hulu Perak district, Peninsular Malaysia. Southeast Asian Journal of Tropical Medicine and Public Health. 33: 547-550.

Rao TR. 1984. The Anophelines of India. Indian Council of Medical Research, New Delhi, p518.

Rawlins SC, Martinez R, Wiltshire & Legall GA. 1998. Comparison of surveillance systems for the dengue vector Aedes aegypti in Port of Spain, Trinidad. Journal of the American Mosquito Control Association 14: 131-136.

Reid JA. 1947. A prelimanary note on the Malayan form of Anopheles barbirostris. Medical Journal of Malaya 2:125-127.

Reid JA. 1953. The Anopheles hyracus group in Southeast Asia. Bulletin of Entomological Research 44: 5-76.

Reid JA. 1954. A preliminary Aedes aegypti survey. Medical Journal of Malaya 9:161-168:76-79.

126 Reid JA. 1961. The attraction of mosquitoes by human and animal baits in relation to the transmission of disease. Bulletin of Entomological Research 52: 43-62.

Reid JA. 1962. The Anopheles barbirostris group (Diptera: Culicidae). Bulletin of Entomological Research 53:1-57.

Reid JA. 1968. Anopheline mosquitoes of Malaya and North Borneo. Studies from the Institute of Medical Research Malaysia 32. 520pp.

Reid JA, Wilson T & Ganapathipillai. 1962. Studies on filariasis in Malaya. The mosquito vectors of periodic Brugia malayi in Northwest Malaya. Annals of Tropical Medicine and Parasitology 56: 323-336.

Reid RG, Kristy LG, Charles , Brent SD, Paul CE, Erwin SA, Brent S, Nicholas AO, Eugene EP & Nasci RA. 2001. First isolation of la Crosse Virus from naturally infected Aedes albopictus. Emerging Infectious Diseases 7: 807- 811.

Reiter P, Amador MA, Anderson RA & Clark GG. 1995. Short report: dispersal of Aedes aegypti in an urban area after blood feeding as demonstrated by rubidium-marked eggs.The American Journal of Tropical Medicine and Hygiene. 52: 177-179.

Reiter P & Gubler DJ. 1997. Surveillance and control of urban dengue vectors. In: Dengue and dengue haemorrhagic fever. Gubler DJ and Kuno, G. (eds.). Fort Collins CO:CAB International. pp 425-453.

Ritchie SA. 2001. Effect of some animal feeds and oviposition substrates on Aedes oviposition in ovitraps in Caims, Australia. Journal of the American Mosquito Control Association 17: 206-208.

Rohani A, Abdullah AG, Ong YF, Saadiyah I, Zamree I & Lee HL. 2001. Survey of mosquito larvae distribution in container habitats collected from urban and rural areas in major towns of Malaysia. Tropical Biomedicine 18: 41-49.

Roy DN. 1943. The role of Anopheles subpictus Grassi as a carrier of malaria. Journal of the Malaria Institute of India 5: 117-121.

Rudnick A, Tan EE, Lucas JK & Mohammed O. 1965. Mosquito-borne haemorrhagic fever in Malaysia. British Medical Journal 1: 1269-1272.

Rueda LM, Patel KJ, Axtel RC & Stinner RE. 1990. Temperature-dependent development and survival rates of Culex quinquefasciatus and Aedes aegypti (Diptera: Culicidae). Journal of Medical Entomology 27: 892-898.

Samways MJ. 1995. Insect Conversation Biology, Chapman & Hall, London, 358p.

127 Santos SRA, Melo-Santos MAV, Regis L & Albuquerque CMR. 2003. Field evaluation of ovitraps consociated with grass infusion and Bacillus thuringiensis var. israelensis to determine oviposition rates of Aedes aegypti. Dengue Bulletin 27: 156-162.

Scott T, Lorenz L & Weaver S. 1990. Susceptibility of Ae. albopictus to infection with eastern equine encephalomyelitis virus. Journal of the American Mosquito Control Association. 6: 274-278.

Service MW. 1993. Mosquito Ecology: Field Sampling Methods United Kingdom: Elsevier Science Publishers LTD.

Service MW. 1995a. Medical entomology for students. Chapman and Hall. Liverpool. pp. 278.

Service MW. 1995b. Can we control mosquitoes without pesticides: A summary. Journal of the American Mosquito Control Association 11:290-293.

Sharma VP. 2001. Health hazards of mosquito repellents and safe alternatives. Current Science 80:341-342.

Shepped PM, Macdonald WW, Tonn RJ & Grabs B. 1969. The dynamics of an adult population of Aedes aegypti in relation to dengue haemorrhagic fever in Bangkok. The Journal of Animal Ecology 38: 661-702.

Shroyer DA. 1986. Aedes albopictus and arboviruses: A concise review of the literature. Journal of the American Mosquito Control Association. 2: 424-428.

Singh S. 2000. Surveillance of dengue fever. In: Ng FSP, Yong HS, eds. Mosquitoes and mosquito-borne diseases Kuala Lumpur, Malaysia: Academy of Sciences Malaysia. p 149-160.

SIRIM. 1996. Specification for mosquito coil part 2: Methods for the evaluation of biological efficacy- Glass chamber Method. MS 23: Part 2.

Skae FM. 1902. Dengue fever in Penang. British Medical Journal 2:1581-1582.

Smith CEG. 1956. The history of dengue in tropical Asia and its probable relationship to the mosquito Aedes aegypti. Journal of Tropical Medicine and Hygiene 59:243-252.

Smith JP, Loyless TM & Mulrennan JA Jr. 1990. An update on Aedes albopictus in Florida. Journal of the American Mosquito Control Association 6: 318-320.

Snehalatha KS, Ramaiah KD, Vijay Kumar KN & Das PK. 2003. The mosquito problem and type and costs of personal protection measures used in rural and urban communities in Pondicherry region, South India. Acta tropica 88:3-9.

128 Soekirno M, Bang YH, Sudomo M, Tjokorda PP & Fleming GA. 1983. Bionomics of Anopheles sundaicus and other anophelines associated with malaria in coastal areas of Bali, Indonesia. WHO/VBC/83.885.

Spiller NJ. 1996. Are active ingredient cocktails of any value in mosquito coils. In: Yap HH ed. Proceedings of the International Seminar on Recent Development of Mosquito coils: Efficacy, Toxicology, Environmental Impact and Legislation. 5-7 dec. 1996: Penang:VCRU, School of Biology, USM. pp 12-18.

Sucharit S, Tumrasvin W, Vutikes S & Viraboonchai S. 1978. Interaction between larvae of Ae. aegypti and Ae. albopictus in mixed experimental populations. Southeast Asian Journal of Tropical Medicine and Public Health 9: 93-97.

Sulaiman S. 2000. Anopheles: mosquitoes that spread malaria. In: Ng FSP, Yong HS, eds. Mosquito and mosquito borne disease Kuala Lumpur, Malaysia: Akademi Sains Malaysia. p 63.

Sulaiman S & Jeffery J. 1986. The ecology of Aedes albopictus (Skuse) (Diptera: Culicidae) in a rubber estate in Malaysia. Bulletin of Entomological Research 76: 553-557.

Sulaiman S, Pawanchee ZA, Arifin A & Wahab A. 1996. Relationship between Breteau and House Indices and cases of dengue/dengue hemorrhagic fever in Kuala Lumpur, Malaysia. Journal of the American Mosquito Control Association. 12: 494-496.

Sulaiman S, Pawanchee ZA, Jeffery J, Ghauth I & Busparani V. 1991. Studies on the distribution and abundance of Aedes aegypti (L.) and Aedes albopictus (Skuse) (Diptera: Culicidae) in an endemic area of dengue/ dengue hemorrhagic fever in Kuala Lumpur. Mosq-Borne Diseases Bulletin. 8: 35-39.

Sundararaman S, Soeroto RM & Siran M. 1957. Vectors of malaria in Mid Java. India Journal of Malariology 11:321-338.

Teng AK & Singh S. 2001. Epidemiology and New Initiatives in the Prevention and Control of Dengue in Malaysia. Dengue Bulletin 25: 7-14

Thaggard CW & Eliason DA. 1969. Evaluation of ovitraps in the U.S. Aedes aegypti eradication program. Mosquito News 29: 608.

Thongrungkiat S, Jirakanjanakit N, Apiwathnasorn C, Prummongkol S & Samung Y. 2003. Comparative susceptibility to oral infection with dengue viruses among local strains of Aedes aegypti (Diptera: Culicidae) collected at different seasons of the year. Journal of Vector Ecology. 28:166-70

129 Toma T, Sakamoto S & Miyagi. 1982. The seasonal appearance of Aedes albopictus on Okinawajima, the Ryukyu Archipelago, Japan. Mosquito News 42: 179-185.

Toshiji N. 1996. Historical perspective of mosquito coil.In: Efficacy, Toxicology, Environmental Impact and Legislation Proceedings of the International Seminar on Recent Development of Mosquito coils; 1996 December 5-7. Pulau Pinang: Vector Control Research Unit, Universiti Sains Malaysia. p 1- 6.

Urdaneta L, Herrera F, Pernalete M, Zoghbi N, Rubio-Palis Y,Barrios R, Rivero J, Comach G, Jimenez M & Salcedo M. 2005. Detection of dengue viruses in field-caught Aedes aegypti (Diptera:Culicidae) in Maracay, Aragua state, Venezuelaby type-specific polymerase chain reaction. Infection, Genetics and Evolution: Journal of Molecular Epidemiology and Evolutionary Genetics in Infectious Diseases 5: 177-184.

VCRU. 2003. Laboratory culture of selected household insect pests. In: The 12th Annual Workshop on Biology and control of vectors & Urban Pests. 2-4 September 2003. Universiti Sains Malaysia, Pulau Pinang, Malaysia, pp5-9.

Vythilingam I. Chiang GL, Lee HL & Inder Singh K. 1992. Bionomics of important mosquitoes vectors in Malaysia. Southeast Asian Journal of Tropical Medicine and Public Health. 23: 587-603.

Vythilingam I, Novi S & Seleena P. 1999. Ovipositional response of Aedes aegypti and Aedes albopictus in different types of water. Tropical Biomedicine 16: 23-28.

Ware GW. 1994. The pesticide Manual: A World Compendium. Bath, UK: bath Press. 1341 pp 10th ed.

Watts DM, Burke DS, Harrison BA, Whitmire RW, Nisalak A. 1987. Effect of temperature on the vector efficiency of Aedes aegypti for dengue 2 virus. The American Journal of Tropical Medicine and Hygiene 36: 143-152.

Weili L, Junfeng Z, Jamal HH, Juliana J, Zailina H & Bernard DG. 2003. Mosquito coil emissions and health implications. Environmental Health Perspective 11: 1454-1460.

Wharton RH. 1953. The habits of adult mosquitoes in Malaya. III: Feeding preferences of anophelines. Annals of Tropical Medicine and Parasitology 57: 235-254.

Wharton RH. 1962. The biology of Mansonia mosquitoes in relation to the transmission of filariasis in Malaya. Bulletin WHO. II. Institute for Medical Research Federation of Malaya. pp 114.

130 Wharton RH, Laing ABG & Cheong WH. 1963. Studies on the distribution of malaria and filariasis among aborigines in Malaya. Annals of Tropical Medicine and Parasitology 57: 235-254.

WHO. 1989. Geographical Distribution of Artropod-borne Diseases and Their principal Vectors. WHO/VBC/89.967. World Health Organization. Geneva, Switzerland.

WHO. 2000 . Dengue in the WHO Western Pacific Region. Weekly epidemiological record, 1998. 73: 273–277.

WHO. 2002. Dengue and dengue haemorrhagic fever. WHO Fact Sheet. 117. Wigglesworth VB. 1950. The principles of insect physiology. E. P. Dutton: New York.

Yap HH. 1975. Distribution of Aedes aegypti (Linnaeus) and Aedes albopictus (Skuse) in small town and villages of Penang Island, Malaysia-an ovitrap survey. Southeast Asian Journal of Tropical Medicine and Public Health 6:519-524.

Yap HH. 1984. Vector control in Malaysia-present status and future prospects. Journal of Malaysian Society of Health 4: 7-12.

Yap HH. 1988. Household Pests and household Insecticide Usage in Malaysia. MACA Newspaper 1:11-12.

Yap HH. 1995. Proceeding of the International Seminar on Recent Development of Mosquito Coils: Efficacy, Toxicity, Environmental Impact and Legislation. Vector Control Research Unit, School of Biological Science, Penang. 93pp.

Yap HH. 1996. Proceeding of the International Seminar on Recent Development of Mosquito Coils: Efficacy, Toxicity, Environmental Impact and Legislation. Vector Control Research Unit, School of Biological Science, Penang. 93pp.

Yap HH & Chavase DC. 1997. Chemical methods for the control for the control of vectors and pests of public health importance. WHO/CTD/WHOPES/97.2. WHO, Geneva, Switzerland. 129pp.

Yap HH & Chong ASC. 1996. Comparative efficacy of mosquito coils with different active ingredients under laboratory and field conditions. Efficacy, Toxicology Environmental Impact and Legistation Proceedings of the international Seminar on Recent Development of Mosquito coils; 1996 December 5-7. Pulau Pinang: Vector Control Research Unit, Universiti Sains Malaysia. p 7-11.

Yap HH, Chong NL, Foo AE & Lee CY. 1994. Dengue vector control:present status and future prospects. Kao-Hsiung-I-Hsueh-Tsa-Chih 10:S102-108.

131 Yap HH &Chong NL, Itam Sulaiman, Loh PY, Sidhu HS. 1990a. Perspektif- Kawalan biologi terhadap nyamuk. Perantara (Jun):10-14.

Yap HH & Chung KK. 1987. Laboratory bioassays of mosquito coil formulations againts mosquitoes of public health importance in Malaysia. Tropical Biomedicine 4:13-18.

Yap HH & Foo AES . 1984 Household pests and household insecticides usage on Penang Island, Malaysia: A questionnaire survey. Bulletin of Public Health society 16: 2-8.

Yap HH, Lee CY, Chong NL, Rohaizat B & Tan HT. 1995. Laboratory bioassay of Malaysian standard mosquito mat formulation against Aedes aegypti (L.) and Culex quinquefasciatus (Say) using two test methods. Journal of Bioscience 6:86-93.

Yap HH, Lee CY, Chong NL, Yahaya AM, Baba R & Awang AH. 1996. Performance of mosquito coils containing transfluthrin against Culex quinquefasciatus (Say) in an urban squatter environment. Tropical Biomedicine 13: 101-103.

Yap HH & Lee YW. 1999. Safe use of public health and household insecticides. In: Proceeding on the Conference on “Managing Risks in The Use of Chemicals for Agriculture and Public Health” 16-17 August 1999, Kuala Lumpur.

Yap HH, Tan HT, Yahaya AM, Rohaizat B, , Loh PY & Chong NL. 1990b. Field efficacy of mosquito coil formulation containing d-allethrin against indoor mosquitoes especially Culex quinquefasciatus Say. Southeast Asian Journal of Tropical Medicine Public Health 21:558-563.

Yap HH, Thiruvengadam V & Yap KH. 1978. A preliminary field evaluation of ULV synergised pyrethrins for control of Aedes aegypti (Linnaeus) and Aedes albopictus (Skuse) in urban area of West Malaysia. Pyrethrum Post 14:98-102.

Yap HH & Thiruvengadam V. 1979. Relative abundance of Aedes aegypti (Linnaeus) and Aedes albopictus (Skuse) in different habitat-an ovitrap survey conducted in Georgetown, Penang, Malaysia. Medical Journal of Malaysia 34:76-79.

Zairi J. 1990. Studies on the ecology of the coastal Anopheline mosquitoes of Northwestern Peninsular Malaysia Ph.D. dissertation. University of Liverpool 129p.

132 APPENDICES

Appendix A: Test of Normality (TPI)

Kolmogorov-Smirnov(a) Shapiro-Wilk Statistic df Sig. Statistic df Sig. mean temperature .098 28 .200(*) .944 28 .139 sampling day rain .349 28 .000 .424 28 .000 lag 1 sampling rainfall .130 28 .200(*) .897 28 .010 lag 2 sampling rainfall .203 28 .005 .686 28 .000 RH .139 28 .176 .947 28 .165 means egg .236 28 .000 .800 28 .000 ovitrap index .105 28 .200(*) .965 28 .454 * This is a lower bound of the true significance. a Lilliefors Significance Correction

Tests of Normality

a Kolmogorov-Smirnov Shapiro-Wilk Statistic df Sig. Statistic df Sig. LOGDAY .185 15 .178 .964 15 .754 LOGMEAN .187 15 .167 .907 15 .123 LOGRAIN2 .103 15 .200* .982 15 .982 *. This is a lower bound of the true significance. a. Lilliefors Significance Correction

133

Appendix B: Test of Normality (KPG)

Kolmogorov-Smirnov(a) Shapiro-Wilk Statistic df Sig. Statistic df Sig. mean temperature .148 28 .120 .927 28 .053 sampling day rain .348 28 .000 .497 28 .000 lag 1 sampling rainfall .142 28 .157 .899 28 .011 lag 2 sampling rainfall .178 28 .024 .780 28 .000 RH .117 28 .200(*) .968 28 .531 means egg .237 28 .000 .757 28 .000 ovitrap index .076 28 .200(*) .972 28 .626 * This is a lower bound of the true significance. a Lilliefors Significance Correction

Tests of Normality(KPG)

Kolmogorov-Smirnov(a) Shapiro-Wilk Statistic Df Sig. Statistic df Sig. LOGDAY .109 12 .200(*) .968 12 .887 LOGMEAN .182 12 .200(*) .932 12 .397 LOGRAIN2 .276 12 .012 .788 12 .007

* This is a lower bound of the true significance. a Lilliefors Significance Correction

134

Appendix C: Independent sample t test for the total ovitrap index and the mean number of eggs collected between TPI and KPG

Group Statistics

Std. Error LOCATION N Mean Std. Deviation Mean ovitrap index TPI 28 80.7857 10.19207 1.92612 KPG 28 78.0357 12.14871 2.29589 mean egg TPI 28 2.1326 .67503 .12757 KPG 28 2.0665 .52243 .09873

Independent Samples Test

Levene's Test for Equality of Variances t-test for Equality of Means 95% Confidence Interval of the Mean Std. Error Difference F Sig. t df Sig. (2-tailed) Difference Difference Lower Upper ovitrap inde Equal variance .820 .369 .918 54 .363 2.7500 2.99684 -3.25830 8.75830 assumed Equal variance .918 52.416 .363 2.7500 2.99684 -3.26247 8.76247 not assumed mean eggs Equal variance 3.808 .056 .409 54 .684 .0660 .16131 -.25740 .38942 assumed Equal variance .409 50.804 .684 .0660 .16131 -.25786 .38989 not assumed

135

Appendix D: Independent sample t test, for ovitrap index and means of eggs collected during the high and low rainfall seasons in Taman Permai Indah

Independent Samples Test

Levene's Test for Equality of Variances t-test for Equality of Means 95% Confidence Interval of the Mean Std. Error Difference F Sig. t df Sig. (2-tailed) Difference Difference Lower Upper ovitrap indexEqual variances .373 .547 .549 26 .588 2.1429 3.90306 -5.88001 10.16572 assumed Equal variances .549 25.796 .588 2.1429 3.90306 -5.88310 10.16882 not assumed mean eggs Equal variances 9.906 .004 3.271 26 .003 .7159 .21883 .26604 1.16568 assumed Equal variances 3.271 20.869 .004 .7159 .21883 .26060 1.17113 not assumed

Appendix E: Independent sample t test, for ovitrap index and means of eggs collected during the high and low rainfall seasons in Kampung Pasir Gebu.

Independent Samples Test

Levene's Test for Equality of Variances t-test for Equality of Means 95% Confidence Interval of the Mean Std. Error Difference F Sig. t df Sig. (2-tailed) Difference Difference Lower Upper ovitrap index Equal variances .962 .336 2.851 26 .008 11.6429 4.08433 3.24741 20.03831 assumed Equal variances 2.851 23.811 .009 11.6429 4.08433 3.20967 20.07604 not assumed mean eggs Equal variances .986 .330 2.463 26 .021 .4463 .18118 .07389 .81875 assumed Equal variances 2.463 25.251 .021 .4463 .18118 .07335 .81929 not assumed

136

LIST OF PUBLICATIONS AND SEMINARS

137

138

139

140

Rozilawati H, Zairi J & Adanan CR. 2007.Seasonal abundance of Aedes albopictus in selected urban and suburban area in Penang, Malaysia. Tropical Biomedicine 24(1): 83-94.

Abstract:

141