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Elton Rogozi

Mosquito trapping in recreational parks in Selangor, Malaysia

FOREWORDS Mosquitoes are important vectors of infectious diseases in human and animals. Female mosquitoes after mating feed on human or warm blooded animals to take a blood meal for the development of their eggs. They mate only once, and after the first batch of eggs, they seek for another warm blood animal or human to take another blood meal for the second batch of eggs development, and so on. But, scientists have GLVFRYHUHGPRVTXLWRVSHFLHVIHHGLQJHYHQRQUHSWLOHV¶EORRGHYHQWRWKHDPSKLELDQV¶ blood like frogs and toads. Feeding on blood behavior is really important for the public health, as they serve as a bridge for liking the pathogen agents with humans or other animals. It is really important to know the feeding behavior, biological cycle of mosquitoes, bio-ecology and reproduction, in order to better draw new techniques on their control. Knowing the attractiveness of mosquitoes from different traps augmented with dry ice or pheromones, with light and suction ventilation systems; provides data on the different mosquito species to study their host preferences, feeding behavior and their role as vector of pathogen agents like viruses, protozoan, bacteria and other parasites in human and higher animals. This study has been performed during a period of six month in the Medical Entomology Unit, Institute for Medical Research, Kuala Lumpur, Malaysia during the year 2010, from May to October. As objectives of the study, we determined three sites or station in the recreational areas around the Selangor State, Malaysia, surrounded by tropical forest close to human settlements as recreational parks. The study has been financed and supported by the Institute for Medical Research as a part of the Diploma in Applied Parasitology and Entomology, Offered at the Institute for Medical Research, Kuala Lumpur, Malaysia, May-October, 2010. We appreciate the support of the Medical Entomology Unit, in the mosquito capturing and identification, as well as the field assistance by this Unit member during the twelve hours of the night, from 6 PM to 6 AM to perform mosquito capturing via CDC light traps baited and augmented with dry ice (CO2), resting catch and the human landing catch during this twelve hours in the field.

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ABSTRACT A study of the mosquito biting activities, species composition, distribution and biodiversity is undertaken in three recreational parks in Selangor: Commonwealth, Sendayu and Sendat. The techniques used to collect the adult mosquito were human landing catch, CDC-light traps baited with dry ice and resting catch. The collection was carried out from 6:00 pm - 06:00 am. Identification of the individuals is done by the standard taxonomical keys. A total of 224 specimens belonging to 26 species and 6 genera all in Culicidae family were trapped during this study. The main genera trapped were Aedes (3 species: Ae. albopictus, Ae. aegypti and Ae. niveus), Anopheles (4 species: An. maculatus, An. karwari, An. introlatus and An. hodgkini), Armigeres (5 species: Ar. durhami, Ar. confuses, Ar. subalbatus, Ar. moultoni and Armigeres sp) Culex (9 species: Cx. vishnui, Cx. pseudovishnui, Cx. quinquefasciatus, Cx. sinensis, Cx. sitiens, Cx. bitaeniorhynchus, Cx. tritaeniorhynchus, Cx. gelidus and Cx. mimulus ) and Mansonia (4 species: Ma. bonneae, Ma. indiana, Ma. annulifera and Ma. uniformis). Biting activities for Aedes peaked at dusk 6:00-7:00 pm and dawn 5:00-6:00 am; Anopheles at 9:00-10:00 pm and 00:00-01:00 am; Armigeres at 6:00-7:00 pm; Culex almost throughout the night and Mansonia: 7:00-8:00 pm and 01:00-02:00 am. The best technique for collecting Aedes and Anopheles is human landing catch; Culex and Mansonia showed a tendency to be caught by CDC-light trap baited with dry ice; Armigeres can be collected with both HLC (human landing catch) and CDC light trap baited with dry ice. Aedes (Ae. albopictus); Anopheles (An. maculatus), Armigeres (Ar. durhami), and Culex (Cx. quinquefasciatus) were present in the three stations; meanwhile Mansonia (Ma. bonneae) only in Commonwealth and Sendat. The similarity quotient is higher between Commonwealth and Sendayu. There is a high diversity, heterogeneous ecosystem or biodiversity for these three recreational parks of Selangor. Key words: Mosquito, trapping technique, biting activity, species composition, Selangor.

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ACKNOWLEDGEMENTS I gratefully would like to thank MTCP (Malaysian Technical Cooperation Program), which provided full funding and financial support for me to attend this course in Malaysia. Warmly acknowledge! I would like to thank IMR (Institute for Medical Research) and the Dean of the DAP&E (Diploma in Applied Parasitology and Entomology) Dr. Lee Han Lim for his support, encourage and taught us the basis of Entomology. Warmly acknowledge! I gratefully would like to thank my supervisor Dr. Rohani Bt. Ahmad for all the support, advices, and guidance on entomology lab work, field work and the way she taught me how to write a thesis and a paper. I would like to thank her for her way of a very kind, polite and gentle communication, that is really so impressive to me. Warmly acknowledge! I would like to thank my Co-supervisors too, Mr. Zamri Ismail, Mr. Rolis Jipun and Ms. Wan Najdah Wan Ali, for their assistance in lab and field work, for the teaching me how to work in a mosquito studying, how to identify them and how to undertake a similar studying independent, also and Medical Entomology staff for their assistance in field trips. I really would like to thank them for the very friendly way they communicated and talked to me! I would like to thank Dr. Sommai, Mr. Ali, Ms. Rani and Ms. Nick, for their hospitality, kindness and warm communication to me. I would like to thank all the staff of the lecturers that have taught me and have support me in knowledge and equipment of the laboratories during all the duration of time of study here, especially Dr. Noor Rain Abdullah, Dr. Nazni, Dr. Shamila, Dr. Khadri. I really would like to thank the Albanian Ministry of Health and the Albanian Foreign Ministry (Ms. Elida Vaso) for their support and for that they trust me to attend this course as a possible and faithfully candidate! I would like to thank Institute of Public Health, Tirana, Albania and Prof. Assoc. Dr. Silva BINO, for her suggestion and her inducement to me to accept and attend this course, for her kindness, advices and the very warm way of communication to me and her assistance she has always given me during all the time that I work in the Institute!

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I would like to thank my colleague Dr. Enkelejda Velo, medical entomologist in the Institute of Public Health for her support, kindness and advices to me and my ODERUDWRU\WHFKQLFLDQV,QD/RFL%DMUDP.DVWUDWLDQG(W¶KHP/ODEDQLIRUWKHLUODEDQG field assistance during the mosquito, tick and small mammal collection! I would like to thank all the student of DAP&E for their friendship to me and for the time we spent here in Malaysia! Finally, I want to thank my parents, my sister Albana Rogozi and my brother Arianit Rogozi, that have supported me so much during the time of staying and studying here, that have blessed and believed in me. Thank them I am here and I had this diploma!

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TABLE OF CONTENTS

)RUHZRUGV«««««««««««««««««««««««««« $EVWUDFW««««««««««««««««««««««««««« $FNQRZOHGJHPHQW«««««««««««««««««««««« 7DEOHRIFRQWHQWV««««««««««««««««««««««« /LVWRISODWHV«««««««««««««««««««««««« /LVWRIILJXUHV«««««««««««««««««««««««« /LVWRIWDEOHV««««««««««««««««««««««««« /LVWRIDSSHQGL[HV«««««««««««««««««««««« 1.0 INTRODUCTION«««««««««««««««««««« 1.$LPVRIWKHVWXG\««««««««««««««««««««« ,PSRUWDQFHRIWKHVWXG\««««««««««««««««««« 2.0 LITERATURE REVIEW««««««««««««««««« ,QWURGXFWLRQWRPRVTXLWRHV&XOLFLGDH ««««««««««««« *HQHUDO%LRORJ\RI0RVTXLWRHV««««««««««««««««. (JJV««««««««««««««««««««..................... /DUYDH«««««««««««««««««««««««« 3XSDH««««««««««««««««««««................... $GXOW««««««««««««««««««««.................... 2.3 Role of mosquitoes in PuEOLF+HDOWK«««««««««««««« 0RVTXLWRHVDVYHFWRU««««««««««««««««««« 9HFWRULDOFDSDFLW\RIPRVTXLWRHV«««««««««««««« 3DWKRJHQVWKDWFDQEHWUDQVPLWWHGE\PRVTXLWRHV«««««««« %LWLQJDFWLYLW\RIPRVTXLWRHV«««««««««««................... 0RVTXLWRDVDQXLVDQFH«««««««««««««.................... 2.4 Mosquito surveillance and entomological studies of vector..................... 2.4.1 Mosquito FROOHFWLRQ««««««««««««««««««« 2.4.2 Other techniquHVXVHGIRUPRVTXLWRFROOHFWLRQ««««««««« 3.0 MATERIALS AND METHODS««««««««««««««« 0HWKRGVIRUFROOHFWLQJPRVTXLWRHV«««««««««««««« 6WXG\VLWH«««««««««««««««««««««««« 0RVTXLWRFROOHFWLRQ«««««««««««««««««««« 5

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3.1.2.1 CDC light traps baited with CO2«««««««««««««« +XPDQODQGLQJFDWFK«««««««««««««««««« 5HVWLQJFDWFK««««««««««««««««««««« 3UHFDXWLRQGXULQJKXPDQODQGLQJFDWFK«««««««««««« 3.2 Mosquito preservation, labeling and traQVSRUWDWLRQ«««««««« 3UHVHUYDWLRQ««««««««««««««««««««««« /DEHOLQJ«««««««««««««««««««««««« 0RVTXLWRLGHQWLILFDWLRQ««««««««««««««««««« 6WDWLVWLFDODQDO\VLVRIWKHGDWD««««««««««««««««« 6LPLODULW\TXRWLHQW«««««««««««««««««««« 'LYHUVLW\LQGH[«««««««««««««««««................... 6LPSVRQGLYHUVLW\LQGH[«««««««««««««««««« 4.0 RESULTS AND DISCUSSION««««««««««««««« 0RVTXLWRSRSXODWLRQVSHFLHVFRPSRVLWLRQDQGELRORJLFDOGLYHUVLW\«« 6SHFLHVFRPSRVLWLRQ««««««««««««««« 7KHVSHFLHVSURSRUWLRQRIWKHPRVTXLWRJHQHUD«««««««« &ROOHFWLRQWHFKQLTXHVIRUWUDSSLQJWKHPRVTXLWRHV««««««« 0RVTXLWRSRSXODWLRQ««««««««««««««««««« 4.1.3 BiologicDOGLYHUVLW\RIPRVTXLWRHV«««««««««««««« %LRORJLFDOGLYHUVLW\RIPRVTXLWRHVLQHDFKVWDWLRQDUHD««««« %LRORJLFDOGLYHUVLW\RIPRVTXLWRHVE\WHFKQLTXHXVHG«««««« 6RPHSLFWXUHVGXULQJPRVTXLWRLGHQWLILFDWLRQ««««««««« '\QDPLFDQGGHQVLW\RIPRVTXLWRSRSXODWLRQ«««««««««« 0RVTXLWRELWLQJDFWLYLW\«««««««««««««««««« %LWLQJDFWLYLW\RIWKHPDLQJHQHUDRIPRVTXLWRFROOHFWHG««««« 4.3.1.1 Biting activity of Aedes JHQXV«««««««««««« 4.3.1.2 Biting activity of Anopheles JHQXV««««««««««««« 4.3.1.3 Biting activity of Armigeres JHQXV««««««««««««« 4.3.1.4 Biting activity of Culex JHQXV««««««««««««««« 4.3.1.5 Biting activity of Mansonia JHQXV««««««««««««« 4.3.&RPSDULVRQEHWZHHQELWLQJDFWLYLW\WLPH«««««« 6WDWLVWLFDODQDO\VLVRIWKHGDWD««««««««««« 6LPLODULW\TXRWLHQW«««««««««««««««««««« 'LYHUVLW\LQGH[««««««««««««««««««««« 4.5 0DSSLQJRIWKHGLVWULEXWLRQRIPRVTXLWRVSHFLHV««««««««« 6

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4.5.1 Mapping of Aedes VSHFLHV««««««««««««««««« 4.5.2 Mapping of Anopheles VSHFLHV««««««««««««««« 4.5.3 Mapping of Armigeres VSHFLHV««««««««««««««« 4.5.4 Mapping of Culex spHFLHV««««««««««««««««« 4.5.5 Mapping of Mansonia VSHFLHV««««««««««««««« 5.0 CONCLUSIONS««««««««««««««. 6.0 RECOMMENDATIONS««««««««««««««««.. 7.0 REFERENCES«««««««««««««««.. 8.0 APPENDIX««««««««««««««««««««««.

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List of Plates

Plate 1. 'LIIHUHQWSLFWXUHVIURP&RPPRQZHDOWKVWDWLRQ«««««««« Plate 2. 'LIIHUHQWSLFWXUHVIURP6HQGD\XVWDWLRQ««««««««««« Plate 3. Different pictures from SenGDWVWDWLRQ«««««««««««« Plate 4. Different pictures during setting the CDC-OLJKWWUDSV«««««« Plate 5. +XPDQODQGLQJFDWFKPHWKRGXVHGGXULQJLQILHOGWULSV««««« Plate 6. Human landing catch method during catching the mosquito into the YLDO«««««««««««««««««««««««««««« Plate 7. 5HVWLQJFDWFKPHWKRGVXVHGGXULQJWKHILHOGWULSV««««««« Plate 8. Pictures of Ae. albopcitus left and Ae. aegypti ULJKW«««««« Plate 9. Pictures of Anopheles maculatus««««««««««««« Plate 10. Left & first pictures Culex quinquefasciatus, right pictures Culex vishnui««««««««««««««« Plate 11. Ventral and dorsal view of Armigeres durhami«««««««« Plate 12. Pictures of Mansonia bonneae««««««««««««««

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List of Figures Page )LJXUH/LIHF\FOHRIPRVTXLWRHV««««««««««««««««.. 14 Figure 2. Eggs of Aedes, Anopheles and Culex««««««««««« 15 Figure 3. Larvae of Aedes albopictus and of Culex spp««««««««.. 16 Figure 4. Pupae of Aedes albopictus and Anopheles spp««««««« 17 Figure 5. Adult of Aedes albopictus and Culex quinquefasciatus««««... 18 24 Figure 6. Different CDC light traps baited or not with CO2««««««« Figure 7. A method of collecting mosquitoes: KXPDQODQGLQJFDWFK««... 25 )LJXUH5HVWLQJFDWFKRUWRUFKOLJKWPHWKRG««««««««««««. 25 )LJXUH*UDYLGWUDSER[HVXVHGWRFROOHFWDGXOWPRVTXLWRHV««««««. 26 Figure 10. Ovitaps XVHGWRFROOHFWQRWDGXOWVEXWWKHLUHJJV««««««« 27 )LJXUH&'&0LQLDWXUH/LJKWWUDS«««««««««««««««.. 27 Figure 12. &RPSRVLWLRQDQGSHUFHQWDJHRIWKHPDLQJHQHUDFDXJKW««« 42 Figure 13. Species composition and percentage of Aedes mosquitoes««... 43 8

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Figure 14. Species composition and percentage of Anopheles PRVTXLWRHV« Figure 15. Species composition and percentage of Armigeres mosquitoes« Figure 16. Species composition and percentage of Culex mosquitoes««« Figure 17. Species composition and percentage of Mansonia PRVTXLWRHV«. )LJXUH0RVTXLWRVSHFLHVFRPSRVLWLRQSHUHDFKVWDWLRQ«««««««. Figure 19. Aedes species biting activity in Commonwealth, Sendayu, Sendat««««««««««««««««««««««««««« Figure 20. Anopheles species biting activity in Commonwealth, Sendayu, 6HQGDW««««««««««««««««««««««««««« Figure 21. Armigeres species biting activity in Commonwealth, Sendayu DQG6HQGDW««««««««««««««««««««««««« Figure 22. Culex species biting activity in Commonwealth, Sendayu, 6HQGDW««««««««««««««««««««««««««« Figure 23. Mansonia species biting activity in Commonwealth, Sendayu, Sendat««««««««««««««««««««««««««« )LJXUH6HODQJRUPDSVKRZLQJWKHWKUHHVWXG\VLWHV««««««««. Figure 25. Distribution of Aedes species in the three study sites in Selangor. Figure 26. Distribution of Anopheles species in the three study sites in 6HODQJRU««««««««««««««««««««««««««... Figure 27. Distribution of Armigeres species in the three study sites in 6HODQJRU««««««««««««««««««««««««««... Figure 28. Distribution of Culex species in the three study sites in Selangor. Figure 29. Distribution of Mansonia species in the three study sites in Selangor««««««««««««««««««««««««««...

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List of tables 7DEOH1RRIPRVTXLWRVSHFLHVFDXJKWUHODWHGWRWHFKQLTXHXVHG«««« Table 2. No of mosquito species caught relatHGWRVWDWLRQDUHD«««««.. 7DEOH6LPLODULW\TXRWLHQWIRUWKHWKUHHDUHDVRIVWXG\««««««««

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List of appendixes Appendix 1. Anopheles JHQXVVSHFLHVELWLQJDFWLYLW\LQ&RPPRQZHDOWK«... Appendix 2. Anopheles genus species biting activLW\LQ6HQGD\X««««.. Appendix 3. Anopheles JHQXVVSHFLHVELWLQJDFWLYLW\LQ6HQGDW««««« Appendix 4. Anopheles JHQXVVSHFLHVELWLQJDFWLYLW\LQ6HODQJRU««««.. Appendix 5. Aedes JHQXVVSHFLHVELWLQJDFWLYLW\LQ&RPPRQZHDOWK«««.. Appendix 6. Aedes JHQXVVSHFLHVELWLQJDFWLYLW\LQ6HQGD\X««««««.. Appendix 7. Aedes JHQXVVSHFLHVELWLQJDFWLYLW\LQ6HQGDW«««««««. Appendix 8. Aedes JHQXVVSHFLHVELWLQJDFWLYLW\LQ6HODQJRU««««««. Appendix 9. Armigeres genus species biting activity in CRPPRQZHDOWK«.. Appendix 10. Armigeres JHQXVVSHFLHVELWLQJDFWLYLW\LQ6HQGD\X««««. Appendix 11. Armigeres genus species biting activity in Sendat«««« Appendix 12. Armigeres JHQXVVSHFLHVELWLQJDFWLYLW\LQ6HODQJRU«««« Appendix 13. Culex JHQXVVSHFLHVELWLQJDFWLYLW\LQ&RPPRQZHDOWK««« Appendix 14. Culex JHQXVVSHFLHVELWLQJDFWLYLW\LQ6HQGD\X«««««« Appendix 15. Culex JHQXVVSHFLHVELWLQJDFWLYLW\LQ6HQGDW««««««.. Appendix 16. Culex genus species biting activity in Selangor«««««... Appendix 17. Mansonia JHQXVVSHFLHVELWLQJDFWLYLW\LQ&RPPRQZHDOWK«. Appendix 18. Mansonia JHQXVVSHFLHVELWLQJDFWLYLW\LQ6HQGD\X««««. Appendix 19. Mansonia JHQXVVSHFLHVELWLQJDFWLYLW\LQ6HQGDW««««« Appendix 20. Mansonia genuVVSHFLHVELWLQJDFWLYLW\LQ6HODQJRU««««.

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1.0 INTRODUCTION Mosquito-borne diseases are not usually considered important problem for public health in the recreational parks in Malaysia, but there should be awareness of their potential and to their vectorial capacity. These diseases are dynamic and their potential, either in the resort, camping areas or their vicinity, can generate adverse publicity that often has a severe economic impact on recreational facilities (Newson, 1977; Rohani, 2008). One of the most complaints from people tries to enjoy the outdoors activity, concern the annoyance caused by the mosquitoes. In addition, recreational park are located close to major natural breeding sites of mosquitoes. Many people try to avoid rustic vacation areas with known mosquito problems and not realizing that these diseases are also transmitted in urban and suburban areas. In response to community concerns about the health and well being of visitors to the recreational park, a study will be undertaken to consolidate and amplify information on distribution and abundance of mosquitoes in these areas (Newson, 1977, Rohani, 2008). Mosquito-borne diseases are a real public health problem worldwide. There are many parasites, viruses, bacteria that can be transmitted by insects, and mosquitoes are the most predominant insects in the way of the transmission to the human being. These pathogens can cause very severe diseases to humans with a high mortality rate. The science that studies the mosquitoes and other insects of public health is called Medical Entomology. It provide basis concepts of entomology like the knowledge on morphology, taxonomy or systematic, bio-ecology, distribution, habitat preferences, that are good and important information for medical entomology to know the whole life cycle of mosquitoes in order to control their incontinent population growth. Identification, distribution, habitat preferences, bio-ecological data and the systematic or taxonomy of mosquitoes play an important role in order to control and prevent infectious diseases caused by them. Seasonality and circadian rhythm of mosquito populations, as well as other ecological and behavioral features, are strongly influenced by climatic factors such as temperature, rainfall, humidity, wind, and duration of daylight (Reiter, 2001). Both seasonal and daily activity patterns of mosquito vectors are required as baseline knowledge to understand the transmission

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dynamics of vector-borne pathogens (Reiter 2001), and have been widely studied for many mosquito species throughout the world (Guimarães et al., 2000 a,b). 1.1 This study aims Monitoring the dynamic and the density of mosquito population in order to determine the necessity for control program measures; Identifying the species composition and the biting activity of these mosquitoes in recreational parks of Selangor; Determination of the biological diversity among the species of mosquitoes; Mapping the presence and the distribution of all the species captured.

1.2 Importance of this studying Why is this studying so important? Why does it serve to the tourists and human population? +RZZLOOLWDIIHFWWRWKH³6WUDWHJLHVRIWKH0RVTXLWRes Control Program´" +RZZLOOLWDIIHFWWRWKH³3XEOLF+HDOWK6HUYLFHV'HSDUWPHQW´" It is very important to know the whole life cycle of the mosquitoes, their bioecology, habitat preferences of them, ecological requirements, biting activity, reproduction, breeding seasons, number of generations etc., in order to have a very good strategy of control program. Knowing these important data of mosquitoes, their control can be more successful. Except optimal temperature and of many pond, pool or dams; humidity is one of the most important ecological factors for the survival of the mosquitoes. It is not just the humidity of the air that cause this successful life of mosquitoes, but the humidity conserved in the low grassy vegetation, tree holes, tree dense vegetation leafs etc., can really be very good places where humidity can be sufficient for their growth in recreational parks near by the jungles.

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2.0 LITERATURE REVIEW 2.1 Introduction to mosquitoes (Culicidae) Mosquitoes belonging to the phylum Arthropoda, class Insecta, subclass Pterygota, order Diptera, suborder Nematocera. There are some 3300 species of mosquitoes belonging to 41 genera, all contained in the family Culicidae of which about 100 are vectors of human diseases. Control measures are generally directed against only one or a few of the most important species and can be aimed at the adults or the larvae. This family is divided into three subfamilies: Toxorhynchitinae, Anophelinae and Culicinae (Service, 1993; Harbach & Kitching, 1998). Mosquitoes have a worldwide distribution (Knight and Stone, 1977). They occur throughout the tropical and temperate regions and extend their range northwards into the Arctic Circle. The only area from which they are absent is Antarctica, and a few islands in pacific. They are found at elevations of 5500m and down mines at depths of 1250 m below sea level (Harbach & Knight, 1980; 1981; 1998; 2005). Mosquitoes are important vectors of several tropical diseases, including malaria, filariasis, and numerous viral diseases, such as dengue, Japanese encephalitis, Chikungunya and yellow fever. In countries with a temperate climate they are more important as nuisance pests than as vectors (Service, 1993; Harbach & Kitching, 1998). 2.2 General Biology of Mosquitoes The mosquito has four distinct stages in its life cycle: eggs, larvae, pupa, and adults (Figure 1). The adult is an active flying insect, while the larvae and pupae are aquatic and occur only in water. Depending on the species, eggs are laid either on the surface of water or are deposited on moist soil or other objects that will often be flooded (Service, 1993). There are 4 instars of larvae, in each of them a molting event occur, where the growth of the larvae body is associated with the molting event. Depending on the species, a female lays between 30 and 300 eggs at a time. Many species lay their eggs directly on the surface of water, either singly (Anopheles) or stuck together in floating rafts (e.g. Culex). In the tropics, the eggs usually hatch within 2±3 days. Some species (e.g. Aedes) lay their eggs just above the water line or

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on wet mud; these eggs hatch only when flooded with water. If left dry they can remain viable for many weeks (Service, 1993).

Figure 1. Schematic Life cycle of mosquitoes. Source: www.mosquitoes.org/LifeCycle.html.

2.2.1 Eggs One factor common to all mosquito species is that eggs are laid in association with free water or on a moist surface. Eggs are white when first deposited, darkening to a black or dark brown within 12-24 hours. Single eggs are about 1/50 inch (0.5mm) long and those of most species appear similar when seen by the naked eye, one exception is the Anopheles spp. (figure 2a) whose eggs have floats attached to each side of the egg (Harbach & Kitching, 2005).

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a. Anopheles spp.

b. Aedes albopictus

c. Culex spp. Figure 2. Eggs of a. Anopheles, b. Aedes and c. Culex mosquito genera.

Eggs are laid singly by some species, such as Aedes albopictus (figure 2b) and others lay eggs together to form rafts such as Culex species (Figure 2c). The incubation period (time between when eggs are laid and when they hatch) may vary considerably among species. Eggs of permanent-water mosquitoes where eggs are deposited on the water surface may hatch in 1-3 days depending on temperature. Floodwater species deposit their eggs on moist soil or another wet substrate and have a wide variation in incubation periods. These eggs will not hatch until submerged by rising water caused by rainfall, melting snow in the spring, or other floodwater. Depending on the species and conditions, these eggs may hatch the next time they are flooded, as soon as ten days, or may not hatch until they are flooded one year or later (Harbach & Knight, 1980, 1981; Jorge, 2001).

2.2.2 Larvae The larvae (wigglers or wrigglers) of all mosquitoes live in water and have four developmental periods or instars. These are called 1st, 2nd, 3rd, and 4th instars with 15

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each succeeding stage larger than the last. At the end of each instar, the larva sheds its skin by a process called molting. The larva is an active feeding stage. Larvae feed on particulate organic material in the water (Figure 3a, 3b). The larvae of most species have breathing and must occasionally come to the surface of the water to get oxygen.

b. Culex spp.

a. Aedes albopictus

Figure 3. a. larvae of Aedes albopictus and b. of Culex spp.

The total length of time that larvae spend in the larval stage depends on the species and the water temperature. Some can develop in as little as 5 or 6 days. Upon maturity the 4th instar larvae molts into the pupal stage (fmel.ifas.ufl.edu/key/ anatomy/larval.shtml; Harbach & Knight, 1980, 1981; Jorge, 2001).

2.2.3 Pupae Unlike most other insects, the mosquito pupa is very active, and, like the larva, lives in water. It differs greatly from the larva in shape and appearance. The pupa has a comma-shaped body divisible into two distinct regions. The front region consists of the head and thorax (cephalothorax) and is greatly enlarged. It bears a pair of respiratory trumpets on the upper surface (Figure 4a, 4b). It must periodically come to the surface to get oxygen. The second region is the abdomen which has freelymovable segments with a pair of paddle-like appendages at the tip. Feeding does not take place during the pupal stage. The pupal stage only lasts for a few days and is the 16

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stage when all the larval tissues change into the adult tissues. The adult emerges directly from the pupal case on the surface of the water (fmel.ifas.ufl.edu/key/ anatomy/pupae.shtml; Harbach & Knight, 1980, 1981; Jorge, 2001).

Aedes albopictus

Anopheles spp.

Figure 4. Pupae of a. Aedes albopictus and b. Anopheles spp.

2.2.4 Adults The adult mosquito (Figure 5) is entirely terrestrial and is capable of flying long distances. Both females and males feed on nectars which they use for energy. Males and females mate during the first 3 to 5 days after they have emerged. Females mate only once. Males generally live for only a week. Only the females feed on blood, which is what is occurring when they are biting. Females evidently gain little nourishment from blood meals but need them in order to develop eggs. Many mosquitoes feed on any warm-blooded bird or mammal. However, some prefer coldblooded animals. Some species also prefer birds and seldom feed on mammals, which is the case with Culex spp. Unfortunately many species feed on a wide range of warm-blooded mammals and humans are often attacked. Once a female has completely engorged, it flies to a shaded environment until her eggs are completely developed, usually 3 to 5 days. Once the eggs are developed the female is called a gravid female and she begins to search for a desirable place to lay her eggs. If a female survives her egg laying activities, she will very soon start searching for another blood meal after which she will lay another batch of eggs. She does not need to mate a second time (Harbach and Knight, 1980, 1981; Jorge, 2001). Generally a female will only live long enough to lay 1 to 3 batches of eggs. 17

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Figure 5. Adult of Aedes albopictus and Culex quinquefasciatus.

Most of mosquito species are actively searching for a blood meal in the evening hours from just before dark until 2 to 3 hours after dark. During the daytime the females normally rest in cooler vegetated areas where the humidity is higher and they are protected from drying out. Females will often bite in the daytime if humans or animals invade the wooded areas where they are resting. However, Aedes albopictus is an aggressive biter which prefers to feed during the daylight hours and is often a nuisance in urban areas (fmel.ifas.ufl.edu/key/ anatomy/adult.shtml; Harbach and Knight, 1980, 1981; Jorge, 2001).

2.3 Role of Mosquitoes in Public Health Mosquitoes are included in the insects that live in human inhabited areas or near them. Living near humans is very important for their life and surviving. As human belonging to the warm blooded animals, they can serve like possible host for mosquito females to take a blood meal before they lay eggs. They need the blood for the development of their eggs. The most preferable hosts for mosquitoes are the animals; bovine, cows, pigs, ships, goats, horses and birds too, man is not a principal PRVTXLWR¶V KRVW KH FDQ EH DFFLGHQWDOO\ D PRVTXito host, but for some mosquito species man can be an important host (Reiter, 2001; RKPBV, 1997). The most important problems that mosquito can cause to human are listed here: annoyance, biting, toxicity, allergic reaction, invade of the host tissue, diseases caused by mosquitoes, contamination of food, fear from them, false parasitosis, toxins and poisons, protection of the host (Reiter, 2001; RKPBV, 1997; http://www.cdc.gov/ ncidoc/diseases/list_mosquitoes.htm). 18

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2.3.1 Mosquitoes as vector The most important pest and vector species belong to the genera Anopheles, Culex, Aedes, Mansonia, Ochlerotatus, Psorophora, Haemagogus, Sabethes etc. Anopheles species, as well as transmitting malaria, are vectors of filariasis (Wuchereria bancrofti, Brugia malayi and Brugia timori) and a few arboviruses. Certain Culex species transmit Wuchereria bancrofti and a variety of arboviruses. Aedes species are important vectors of yellow fever, dengue fever, encephalitis viruses and many other arboviruses, and in a few restricted areas they are also vectors of Wuchereria bancrofti and Brugia malayi. Species in the very closely related genus Ochlerotatus also transmit filariasis and encephalitis viruses. Mansonia species transmit Brugia malayi and sometimes Wuchereria bancrofti and a few arboviruses (Reiter, 2001; RKPBV, 1997). Haemagogus and Sabethes mosquitoes are vectors of yellow fever and a few other arboviruses in Central and South America, while the genus Psorophora contains some troublesome pest species, as well as a few transmitting arboviruses. Many species, although not carriers of any disease, can nevertheless be troublesome because of the serious biting nuisances they cause (Reiter, 2001; RKPBV, 1997; http://www.cdc. gov/ncidoc/diseases/list_ mosquitoes.htm).

2.3.2 Vectorial capacity of mosquitoes Mosquito species have a high vectorial capacity in transporting and transmission of the pathogens to humans and other high animals. They are the most predominant group of insects and arthropods that can serve as vector of many pathogen agents. Different pathogens can live and reproduce inside the vital organs of mosquitoes, as well as they can be fed there. High vectorial capacity of a mosquito means the opportunity that they have to carry, develop and serving like mechanical and infected transporter of different pathogens like viruses, bacteria, parasites etc. The life cycle of a pathogen agent can occur in two or more host and mosquitoes are the principal reservoirs and hosts (Reiter, 2001; RKPBV, 1997; http://www.cdc.gov/ncidoc/diseases/list_ mosquitoes.htm).

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2.3.3 Pathogens that can be transmitted by mosquitoes There are a tremendous number of different infectious and non-infectious diseases that can be transmitted by mosquitoes. These diseases can be classified in viral infectious diseases, parasites infectious diseases, bacterial infectious diseases and other infectious diseases caused by other pathogens agent. These diseases, some of them, have a very high mortality rate to humans. Parasites: are worldwide, they can be carried by mosquitoes, can be fed and reproduced in the mosquito host vital organs, as well as they can be transmitted to other hosts causing severe diseases. Human is involved as host is these cycle, too. From parasites groups we can mention the group of filarial parasites, where the most predominant parasites species present in Malaysia are Brugia malayi, Wurchereria bancrofti and Brugia pahangi (Reiter, 2001; RKPBV, 1997; http://www.cdc.gov/ncidoc/diseases/list_mosquitoes.htm). These parasites can cause filariasis in human if the mosquito is infected. When the infected mosquito is sucking blood in man the filarial worms can get out from the infected mosquito proboscis and can get in the wound or the small hole that the piercing-sucking mouthparts of mosquito proboscis has done until it was sucking a blood meal. The most important vector of filarial worm is the species of Mansonia genus (Reiter, 2001; RKPBV, 1997). Another very important parasite which can be transmitted by mosquitoes is malaria parasite. It is smaller than filarial worm and a very slender parasite. The most important vector of malaria is the species of Anopheles genus (Rohani, 1999). These are a very severe infectious disease that can result in high mortality rate in humans. Viruses: many viruses can be carried and transmitted by mosquitoes to human and other mammals and other animals. Culex is the most predominant species that serve as the main vector of Japanese Encephalitis. Aedes are the most predominant species that serve as the main vector of Chikungunia fever, dengue fever and dengue haemorrhagic fever, which are the most spread viruses in Malaysia (Reiter, 2001; RKPBV, 1997). These viruses can cause a very severe hemorrhagic fever disease. Aedes aegypti and Aedes albopictus are the most predominant vectors in Malaysia (Reiter, 2001; RKPBV).

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Bacteria and other pathogens: there are a lot of different bacteria and other SDWKRJHQDJHQW¶VVSHFLHVZRUOGZLGHWKDWFDQEHFDUULHGWUDQVSRUWHGDQGWUDQVPLWWHG by mosquitoes. In humans these bacteria can cause very severe infectious disease with a high mortality rate (Reiter, 2001; RKPBV, 1997). 2.3.4 Biting activity of mosquitoes Biting activity, the time and the frequency of biting activity depend on the mosquito species, environment conditions, ecological conditions and requirements. Aedes mosquito bites mainly in dusk hours 6 pm to 9 pm and in dawn between 6-8 am. Culex can bites from 9 pm to 11 pm and sometimes during the early hours of morning. Anopheles can bites after 11 pm, as well as the in early hours of the morning like 2-4 am (Reid, 1968; Loong, 1998). Other species of mosquitoes like Mansonia, Amigeres genera etc., have different biting time and frequency (Cheong, et al., 1988, 1984). Biting activity and frequency depend on the gonotrophic cycle of mosquitoes. After the first bite eggs can develop very fast and after 2, 3 or more days depending on the species, food, temperature, humidity (Cheong, et al., 1988; Onyido et al., 2009). Eggs are laid in the water or water surface and then the mosquito can go to feed again for another blood meal. They mate just one time. The shorter the JRQRWURSKLFF\FOHWKHVKRUWHUWKHHJJV¶GHYHORSLQJWLPH7KHQHHGIRUDQRWKHUEORRG meal is higher followed by high frequency of biting activity. Female mosquitoes feed on animals and humans. Most species show a preference for certain animals or for humans (Cheong, et al., 1988; Onyido et al., 2009). They are attracted by the body odors, carbon dioxide and heat emitted from the animal or person. Some species prefer biting at certain hours, for example at dusk and dawn or in the middle of the night. Feeding usually takes place during the night but daytime biting also occurs. Some species prefer to feed in forests, some outside of houses, and others indoors (Cheong, et al., 1988; Onyido et al., 2009). 2.3.5 Mosquito as a nuisance Mosquitoes can cause nuisance and fear to humans. Recreational parks and areas can be very good places where mosquitoes can breed and live. High humidity, hiding 21

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places in branches, dense leaves trees and the grassy vegetation can be very good places for protecting the mosquitoes from the sun light and high temperatures (Cheong, et al., 1988; Onyido et al., 2009). Many kind of swamps, streamlets, ponds, pools full of water can be very good places for development of the eggs, larvae and pupae mosquitoes life cycle stages. All these optimal conditions in recreational areas are very favorable ecological condition for the development of the whole mosquito life cycle (Cheong, et al., 1988; Onyido et al., 2009). Mosquitoes can be a very high nuisance to human in tourist recreational areas and parks. The mosquito biting activities can inflict the presence and the coming of the tourists in recreational areas. Except transmission of the pathogen agents, mosquitoes can cause parasitosis, false parasitosis, inflammation and the irritation of the human skin, allergies to sensitive people, toxins etc. (Cheong, et al., 1988; Onyido et al., 2009). 2.4 Mosquito surveillance and entomological studies of vector Mosquito surveillance should be a routine part of any mosquito program. A good surveillance program provides information on a list of local mosquitoes (including distribution and population size estimation), and effectiveness of the control strategies being used. Information on the epidemiology of insect-borne diseases is essential if the disease is to be controlled. Entomological, parasitological and clinical studies provide useful information on the characteristics of disease transmission in an area as well as the habits and habitats of the specific vector species. Entomological studies have several important roles to play in vectors of diseases control, including the following (WHO, 1992): i) identification of the vectors responsible for transmission of the disease; ii) provision of basic information on the habits and habitats of vector species for purposes of planning effective control measures; iii) monitoring the impact of control measures (for example, by determining changes in vector population density, rates of infection, susceptibility of vectors to insecticides, and residual effects of insecticides on treated surfaces) and iv) contributing to the investigation of problem areas where control measures prove unsuccessful. Entomological studies must not only be carried out to provide a practical answer to clearly defined control-oriented research questions when data is unavailable or inadequate. Entomological studies are also important in the estimation the expected impact of the various control measures. This helps to decide whether 22

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some measures are more useful than others and whether some control measures are dangerous to implement (AFPM, 2002). 2.4.1 Mosquito collection Collecting and evaluating information on adult mosquitoes is important for decision-making on deployment of appropriate control activities. The association between species of mosquitoes can provide clue to an understanding their biology and their role in the transmission of pathogen. Adult collections are most frequently conducted because adult mosquitoes are generally easier to survey, collect and identify than the immature stages. A trapping device is the most common and the simplest tool for the collection of mosquitoes, mainly for its surveillance, vector relative density, abundance and its control (Cameron & Russell, 2005). The three main collections methods and other methods of mosquito collection use in other studies by other authors are shown in the pictures and paragraphs listed below: a) Light traps are limited to gathering data on density and species composition of nocturnal adult mosquito species that are attracted to light. Some Anopheles and Aedes mosquitoes are poorly attracted to light; therefore light traps are ineffective in collecting these species. Although light traps are generally not recommended for use in collecting these genera, some Aedes and Ochlerotatus are strongly attracted to light traps (e.g., Ae. vexans, Oc. sollicitans and Oc. taeniorhynchus) (WHO, 1975). Because of these behavioral differences, other of adult mosquito collection methods (e.g., resting stations or landing counts) are needed to obtain a valid index of the total population. A variety of light trap types exist. Wide differences in capture efficiency have been noted between species due to differences in their reactions to light. Some species are caught in great numbers while others are rarely taken even though they may be plentiful in the vicinity (e.g., Ae. aegypti mosquitoes). Therefore, to increase their effectiveness, various modifications have been added such as CO2 or dry ice and other components of host odor. The established role of CO2 as a mosquito attractant (Service, 1993; Cameron & Russell, 2005) makes it feasible to use as a standard in the sampling of mosquito population. Vythilingam et al., (1992) reported that light traps supplemented with CO2 showed synergistic effect toward various species of mosquitoes.

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CDC light traps baited with CO2 (dry ice). Traps can be set and fix in metallic stick as holder. %DWWHULHV ZHUH XVHG IRU WKH IXQFWLRQLQJ RI WKH WUDSV¶ OLJKW DQG ventilation system. Insects are attracted to the light during the night and from the smell of the CO2 (dry ice), which imitates the exhaled CO2 from animals, who is a very attractive smell for the mosquitoes. It has been well established that CDC trap without CO2 is not attractive to mosquitoes (Oli et al., 2005). CDC traps augmented with CO2 from dry ice is an efficient trap (Figure 6). However, since it is difficult to obtain dry ice in many remote areas in tropical countries, yeast generated CO2 traps could be used since it is easily available and cheap (Oli et al., 2005).

Figure 6. Different CDC light traps baited or not with CO2.

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b) Human landing catch Landing counts on humans are useful for determining population densities of anthropophagic (human biting) mosquitoes that are not attracted to light traps and for rapid checks of mosquito populations (Figure 7). The use of this method is recommended when complaints or suspicions are not corroborates by light trap collections. This survey technique establishes an index or landing rate by counting the number of mosquitoes landing on the collector during a specific period of time. However, landing count surveillance is time intensive, inconvenient and difficult to standardize (WHO, 1992). This technique may increase the exposure of survey personnel to disease. Therefore, during a mosquito-borne disease outbreak, survey personnel must use personal protective measures, such as wearing head nets and rolling down sleeves, but do not use repellents. All survey personnel should be on any chemoprophylaxis recommended in the area being sampled. If a mosquito-borne disease is present for which no vaccine, chemoprophylaxis or treatment is available (e.g., many viral diseases), this sampling technique should not be used (WHO, 2002).

Figure 7. A method of collecting mosquitoes: human landing catch method.

c) Resting catch Torch-lights are used during the night to see and collect for the female and male mosquitoes (Figure 8). They can be hidden in the tree leafs, tree branches, tree holes, grassy vegetation, rocky crevices and holes and other places where mosquitoes can be surely protected and hiding from nocturnal predators, from low temperatures etc. Then a mechanical aspirator or sucking tubes are used to suck them inside the tube,

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after that a 50X19 mm glass vials is used to catch them inside, after that a piece of cotton is used to plug in the vials.

Figure 8. Resting catch or torch light mosquito collection method.

2.4.2 Other techniques used for mosquito collection Adult sampling. According to Eliningaya & Aneth, 2009, the four sampling methods evaluated were operated in the same time. Human landing catch (HLC), odor-baited trap (OBET), pit shelters (PS) and indoor resting collection (IRC). IRC can be performed in the morning from 6:30 am to 8:30 am of every experimental day in cowshed and indoors using mechanical aspirator as described in entomological manual book (Eliningaya & Aneth, 2009). Pit shelters can be sampled every morning from 7:00 am to 7:30 am, the pit dimensions were as described in entomology manual for collecting outdoor resting PRVTXLWR¶V density (Eliningaya & Aneth, 2009). OBET, the trap is composed of a tent with a either a man or cow whose odors are drawn to a cage trap by a fan via polythene tunnel. OBET dimensions were height 2 meters, length 2 meters and width 1.5 meters (Eliningaya & Aneth, 2009). For HLC, the same man can expose his feet while using mechanical aspirator for collecting landing mosquitoes at each collection site, one collector worked from 6:00 p.m. to 6:00 am, mosquitoes were sorted by an hour interval. OBET used both man and cows: the mosquitoes seeking for hosts were collected in a protected chamber before reaching the host (Eliningaya & Aneth, 2009).

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Gravid Trap Box. This trap looks like a tool box sitting on top of a tray. The tray contains a mixture of fermented grass and straw which has a powerful smell (disagreeable to people) but attractive to mosquitoes. The tool box contains a collecting pod, and a small fan that is powered by four "D" cell batteries (Figure 9). When the mosquitoes are attracted to the water to lay eggs, they pass by the trap opening and are pulled into the collection pod. The mosquitoes are removed in the laboratory for examination and analysis.

Figure 9. Gravid trap boxes used to collect adult mosquitoes. The ovitraps are a small water-containing vessel that looks like a stadium cup with a surface inside it (such as a tongue depressor) for mosquitoes to lay their eggs on (Figure 10). The surface containing the eggs are collected and counted to determine the density of the reproductive mosquito population. These ovitraps will provide initial and ongoing data on mosquito populations.

Figure 10. Ovitap used to collect not adults but their eggs to treat in the lab.

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The Fay Prince trap is a daytime trap that is used to collect the Asian tiger mosquito, Aedes albopicuts. These traps are suspended from low branches and trees and work much like a CDC miniature light trap except it uses contrasting black and white surfaces to attract mosquitoes rather than a light bulb (Figure 11). The trap was designed for mosquito abatement operations and arbovirus survey purposes (Hock, 2004).

Figure 11. CDC Miniature Light trap

These traps are commonly suspended from tree limbs that hang above the ground and are powered by a battery. Traps attract mosquitoes by a light bulb and CO2 that is emitted from the dry ice in a cooler. When the mosquitoes get close to the light they are pulled into the container by a small electric fan where they are captured for analysis.

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3.0 MATERIALS AND METHODS 3.1 Methods for collecting mosquitoes 3.1.1 Study site This study is carried out in three recreational parks of Selangor state in Malaysia. The study sites include these three stations as describes below: 1. Commenwealth Recreational Park, Rawang /DWLWXGHÛ¶ /RQJLWXGHÛ¶ Altitude: 324 ft (feet)

Average humidity: 82.27% $YHUDJHWHPSHUDWXUHÛ&

Plate 1. Different pictures from Commonwealth station. 29

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2. Sedayu Recreational Park, Gombak

/DWLWXGHÛ¶ /RQJLWXGHÛ¶ Altitude: 608 ft (feet)


Plate 2. Different pictures from Sendayu station.

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3. Sungai Sendat Recreational Park, Hulu Yam

/DWLWXGHÛ¶ /RQJLWXGHÛ¶ Altitude: 455 ft (feet)


Plate 3. Different pictures from Sendat station. 31

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3.1.2 Mosquito collection Collection of mosquitoes has been done from 18:00 to 06.00 for the landing and the resting catch and the same period time for the CDC light traps baited with dry ice that have been set in 18:00 until 06:00, so a twelve hour per night collection period is been applied to this study. Mosquito collection was carried out for 4 nights for each station. Collection methods for this research are focused on three main techniques, which are as mentioned below: CDC-light traps baited/augmented with CO2. Human landing catch or bare lag catch and. Resting catch. As mention above, the collection of the adult mosquitoes has been carried out with three simple and most useful and successful methods: CDC-light traps baited with CO2 or dry ice, human landing catch or bare leg catch, and the resting catch. The three collection methods together can realize the collection of a wide variety of PRVTXLWR¶V species, knowing that different mosquito species can be captured with different collection techniques.

3.1.2.1 CDC light traps baited with CO2 (dry ice) A total of twelve CDC light traps baited with dry ice were set for each collection in every capturing night. Traps were set and fix from 6 pm ± 6 am in the whole territory of the station expanded in a similar distance to cover as wider area as SRVVLEOH %DWWHULHV ZHUH XVHG IRU WKH IXQFWLRQLQJ RI WKH WUDSV¶ OLJKW DQG YHQWLODWLRQ system. Traps are set in metallic stick like it is shown in the plate 4.

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Plate 4. Different pictures during setting the CDC-light traps.

3.1.2.2 Human landing catch The second method of collection includes the human landing catch. This method has to leave the legs or arms undressed in order that female mosquitoes to approach to the human body alight to the leg or arm and bite. Then a 50X19 mm glass vials were used to catch them inside, after that a piece of cotton pad is used to plug in the vials. This method is being preceded as it is shown in the plates 5 and 6. It has not been allowed that the mosquito to realize the biting to prevent secondary infection. 33

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Plate 5. Human landing catch method used during the field trips.

Plate 6. Human landing catch method during catching the mosquito into the vial. In plate 5, we show the way how the legs and arms must be bare during the time that the mosquito female to be attracted and to approach to the body site. Plate 6 shows the procedure of catching the mosquito into the vial and isolating it with small cotton wads.

3.1.2.3 Resting catch The third catching method is resting catch. Torch-lights were used during the night to search for the female and male mosquitoes that have been hidden in the tree leafs, tree branches, tree holes and grassy vegetation. Mechanical aspirators or sucking tubes were used to suck them inside the tube during searching time (Plate 7). 34

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Plate 7. Resting catch methods used during the field trips.

Then a 50X19 mm glass vials have been used to keep them inside, after that a piece of cotton wad have been used to plug in the vials for preventing the mosquitoes to go escape. A 10 minute per hour time is performed from 6 pm - 6 am during all field trips to collect for female and male adult mosquitoes. Most of the collection of mosquitoes have been trapped or caught with the two first methods; CDC light trap baited with dry ice and human landing catch. The third method, resting catch, has not been successful comparing to the other two methods. This can be as a reason of the heavy rain that has fallen to the most night collection of the field trips. The three selected sites have been very wet stations, where the humidity reached above 90% sometimes. 35

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3.1.3 Precaution during human landing catch During the human landing catch, there should be some awareness about the mosquito catching with this method and the time that the mosquito must be allowed to bite. As a possibility of the infectious from an infected mosquito, when it alight and bite to the leg, arm or body, the time that mosquito must be allowed to bite must be as shorter as possible. The mosquito must not be allowed to stay long to the biting site of our body, it must not be allowed to manage to bite or to bite and stick with its proboscis to our skin. As shorter the time the mosquito rest in our body, as lower the possibility for the mosquito to bite and the possibility of an infectious if the mosquito is infected with any kind of viruses or other pathogen agents. Persons whom apply this method should be very careful from the undetectable mosquito biting and the time that mosquito must stay in their arms, legs or body surface or skin during catching of them.

3.2 Mosquito preservation, labeling and transportation 3.2.1 Preservation After the collection of the mosquito specimens with the three methods described above, they were put into small vials covered with a small cotton pad. At the bottom of each vial, a small piece of wet tissue was placed, in order to keep the mosquito specimens alive during the preservation and the transportation to the lab. The wet piece of tissue can provide such humidity, as to keep the specimens alive until the lab. This is because the living specimens can be easily identified. Many of the bristles, hairs and bands can better seen in the living specimen than in the dead one. This is the main reason why we have kept them alive during the time of the preservation and the transportation to the laboratory to proceed with further procedure and the identification purposes. 3.2.2 Labeling After the specimens have been put inside the vials, every vial was labeled with the date, locality or station and time of capturing to better analyze them later. 36

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4.0 RESULTS AND DISCUSSION

4.1 Mosquito population, species composition and biological diversity 4.1.1 Species composition During this study project, undertaken by the Laboratory of Entomology, IMR; a total of 224 specimens of different mosquito species were caught. They belong to 26 species all included in the Culicidae family. Respectively, genus Aedes 3 species: Aedes albopictus, Aedes aegypti and Aedes niveus; Anopheles genus 4 species: Anopheles hodgkini, Anopheles introlatus, Anopheles karwari and Anopheles maculatus; Armigeres genus 5 species: Armigeres confuses, Armigeres durhami, Armigeres moultoni, Armigeres subalbatus and one species unidentified belonging to this genus Armigeres sp. Genus Culex 9 species: Culex bitaeniorhynchus, Culex gelidus, Culex mimulus, Culex pseudovishnui, Culex quinquefasciatus, Culex sinensis, Culex sitiens, Culex tritaeniorhynchus and Culex vishnui. Genus Culiseta only one unidentified species Culiseta sp. Genus Mansonia 4 species: Mansonia bonneae, Mansonia annulifera, Mansonia indiana and Mansonia uniformis. Table 1 shows the number of mosquito per species caught in relation with the method/technique used. Table 2 shows the distribution and the number of mosquito species collected for the three study stations of the Selangor recreational parks. The last column shows the total number per species captured in all the three stations, thus it shows the number per mosquito species captured during all this study in Selangor.

No 1 2 3

Mosquito species Aedes aegypti Aedes albopictus Aedes niveus

No of mosquito individuals caught Total no of per technique/method used mosquito/ species HLC CDC Resting catch 1 1 40 12 52 2 2 39

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4 Anopheles hodgkini 5 Anopheles introlatus 6 Anopheles karwari 7 Anopheles maculatus 8 Armigeres confuses 9 Armigeres durhami 10 Armigeres moultoni 11 Armigeres sp 12 Armigeres subalbatus 13 Culex bitaeniorhynchus 14 Culex gelidus 15 Culex mimulus 16 Culex pseudovishnui 17 Culex quinquefasciatus 18 Culex sinensis 19 Culex sitiens 20 Culex tritaeniorhynchus 21 Culex vishnui 22 Culiseta sp. 23 Mansonia annulifera 24 Mansonia bonneae 25 Mansonia indiana 26 Mansonia uniformis Total no. of species per technique/method of catch Total no. of mosquito per technique/method of catch Total no. of mosquito collected


1

10 5 1 18

13

3

1 3 1 38 35 8 1 2 1 1 1 1 12 8 1 1 1 32 1 1 13 5 1 28

167

53

4

224

3 1 38 23 7 1

12 1 2

1 1 1 1 7 1

3 7

2 1

1 1 11

21 1

1 3

224

HLC = Human landing catch CDC = Light trap baited with dry ice Table 1. Number of mosquito species related to technique used.

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No


Mosquito species

1 Aedes aegypti 2 Aedes albopictus 3 Aedes niveus 4 Anopheles hodgkini 5 Anopheles introlatus 6 Anopheles karwari 7 Anopheles maculatus 8 Armigeres confuses 9 Armigeres durhami 10 Armigeres moultoni 11 Armigeres sp 12 Armigeres subalbatus 13 Culex bitaeniorhynchus 14 Culex gelidus 15 Culex mimulus 16 Culex pseudovishnui 17 Culex quinquefasciatus 18 Culex sinensis 19 Culex sitiens 20 Culex tritaeniorhynchus 21 Culex vishnui 22 Culiseta sp 23 Mansonia annulifera 24 Mansonia bonneae 25 Mansonia indiana 26 Mansonia uniformis Total no of species/station Total no of mosquito/station Total no of mosquit collected

No of. mosquito individuals caught per Station/Locality/Area Commenwealth Sedayu Sendat 1 33 8 11 2 1 3 1 1 13 24 35 4 2 2 1 2 1 1 1 1 6 6 6 1 1 1 1 1 18 14 1 1 11 2 5 1 16 12 9 122 54 48 224

Table 2. No of mosquito species related to station/area. 41

Total no. of mosquito/ species 1 52 2 1 3 1 38 35 8 1 2 1 1 1 1 12 8 1 1 1 32 1 1 13 5 1 28 224

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More than 50% of the all the specimens caught, were trapped and caught in Commonwealth Station Recreational Park. The two other stations have the same number of specimens captured, almost 22-23%, of the total collection. Only four mosquito species captured were present in all the three study sites. A total of 52 specimens Aedes albopictus were captured; 33 were in Commonwealth, 8 were in Sendayu and 11 were in Sendat. A total of 38 individuals of Anopheles maculatus were captured; 1 were captured in Commonwealth, 13 in Sendayu and 24 in Sendat. Armigeres durhami and Culex quinquefasciatus, were also captured in three study sites. 4.1.1.1 The species proportion of the mosquito genera Figure 12 shows five main genera of mosquitoes collected during this study. The most predominant genera collected are genus Aedes (26.0%) and genus Culex (25.0%). Meanwhile, the 3 other genera have a respectively percentage Armigeres (21.0%), Anopheles (19.0%) and Mansonia (9.0%).

9%

25%

26%

19% 21% Aedes

Anopheles

Armigeres

Culex

Mansonia

Figure 12. Composition and percentage of the 5 main genera caught. Figure 13 shows the species composition and the percentage of Aedes mosquitoes. We can easily see that the most predominant species collected is Aedes albopictus (94.0%). The two other species are Aedes aegypti (2.0%) and Aedes niveus (4.0%).

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4% 2%

94% Aedes aegypti

Aedes albopictus

Aedes niveus

Figure 13. Species composition and percentage of Aedes mosquitoes.

2%

7% 2%

89% Anopheles hodgkini Anopheles karwari

Anopheles introlatus Anopheles maculatus

Figure 14. Species composition and percentage of Anopheles mosquitoes. Figure 14 shows the species composition and the percentage of Anopheles mosquitoes. The most predominant species collected is Anopheles maculatus (89%). The three other species are Anopheles introlatus (7.0%), Anopheles karwari (2.0%) and Anopheles hodgkini (2.0%) Figure 15 shows the species composition and the percentage of Armigeres mosquitoes. The most predominant species collected are Armigeres confuses (75.0%) and Armigeres durhami (17.0%). Meanwhile, the 3 other species have a respectively percentage of 4.0% for Armigeres sp and 2.0% for Armigeres subalbatus and Armigeres moultoni. 43

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2% 4% 2% 17%

75% Armigeres confuses Armigeres sp

Armigeres durhami Armigeres subalbatus

Armigeres moultoni

Figure 15. Species composition and percentage of Armigeres mosquitoes.

Figure 16 shows the species composition and the percentage of Culex mosquitoes. The most predominant species collected are Culex vishnui (54.0%), Culex pseudovishnui (20.0%) and Culex quinquefasciatus (14.0%). Meanwhile, the 6 other species have a respectively percentage of 2.0% for each of them: Culex bitaeniorhynchus, Culex mimulus, Culex sinensis, Culex sitiens, Culex tritaeniorhynchus and Culex gelidus.

2% 2% 2% 20%

54% 14% 2% 2% 2% Culex bitaeniorhynchus Culex pseudovishnui Culex sitiens

Culex gelidus Culex quinquefasciatus Culex tritaeniorhynchus

Culex mimulus Culex sinensis Culex vishnui

Figure 16. Species composition and percentage of Culex mosquitoes. 44

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5%

5%

25%

65% Mansonia annulifera

Mansonia bonneae

Mansonia indiana

Mansonia uniformis

Figure 17. Species composition and percentage of Mansonia mosquitoes.

Figure 17 shows the species composition and the percentage of Mansonia mosquitoes. The most predominant species collected are Mansonia bonneae (65.0%) and Mansonia indiana (25.0%). Meanwhile, the 2 other species have a respectively percentage of 5.0% each for Mansonia annulifera and Mansonia uniformis.

4.1.1.2 Collection techniques for trapping the mosquitoes Most of Aedes species were caught with human landing catch method, 77.0% of the individuals of Ae. albopictus or 40/52 were trapped with human landing catch method and only 12/52 or 23.0% with CDC-light trap baited with dry ice. None of them were caught with resting catch method. All Anopheles maculatus were caught with HLC method, only An. kawari was trapped with CDC, but this can be considered as a random capture, as the individuals of the Anopheles genus are not attracted to the light and the smell of dry ice. None of them were caught with resting catch method. Armigeres were caught with both methods HLC (23/35 or 66.0%) and CDC light trap (34.0% of them). They are attracted to human and CDC light and the smell of dry ice. None of them were caught with resting catch method.

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Culex species were caught with all the three trapping methods used for the study. Most of the Culex species were caught with CDC light trap baited with dry ice (7/9 of the Culex species or 78.0%), followed by HLC method (4/9 of Culex species or 44.0%) and resting catch method (2/9 of Culex species or 22.0%). Most of Mansonia species were attracted to CDC light trap baited with dry ice (3/4 of Mansonia species or 75.0%), followed by HLC method (2/4 of Mansonia species or 50.0%) and none of them were caught by resting catch method. Our study showed that the best technique/method for collecting Aedes (Aedes albopictus) and Anopheles (Anopheles maculatus) is human landing catch; Culex and Mansonia showed a tendency to be caught by CDC-light trap baited with dry ice and Armigeres can be collected with both HLC and CDC light trap baited with dry ice.

4.1.2 Mosquito population The most number of mosquito collected in relation with the technique used, belong to HLC (human landing catch) technique. This can be explained with the biting activity of mosquitoes and the attractiveness of human in accordance with them. Almost 80% of Aedes albopictus collected were caught with the HLC method; this shows that the female individuals of this species are attracted to human blood. The other 20% of them has been trapped with CDC light trap. More mosquito species were collected by HLC method rather than CDC or resting catch. The only mosquito species that was trapped with CDC is Culex quinquefasciatus. Generally, all the species of Culex genus are attracted better with CDC light trap baited with CO2, rather than HLC and resting catch. Anopheles maculatus and Armigeres durhami are the two species which have been caught only with HLC method, in a considerable numbers of the first species. Armigeres species can be attracted to the light and the CO2 smell of the traps, as well as all other species, but Anopheles maculatus shows a tendency only attracted to human. The best way to collect Anopheles species is only HLC (human landing catch), CDC light trap baited with dry ice method is not good for Anopheles maculatus collection. In our collection, the only specimen of Anopheles genus trapped with CDC is Anopheles hodgkini.

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4.1.3 Biological diversity of mosquitoes The study showed that the biological diversity of mosquitoes is relatively high. Biological diversity has not to do with the number per mosquito species captured. Even that the number of specimens per species is very low (for some of them it is only 1 specimen per species captured), we can conclude that the biological diversity is high.

4.1.3.1 Biological diversity of mosquitoes in each station/area The number of species captured varies from one station or place to the other. In some of them the number of species captured is high and is some other it is not so high. The number of species captured in Commonwealth Recreational Park is 16 species, Sendayu 12 species and Sendat 11 species. The biological diversity in Commonwealth is higher compared to the two other stations. For more details on the species composition per each station or area, see the figure 18 below:

9 16

12 Commonwealth

Sendayu

Sendat

Figure 18. Mosquito species composition per each station.

Why the species diversity is different in different areas, station or places? There are some reasons or better bio-ecological and ecological factors why the distribution and the presence of the species is like this. Bio-ecological and ecological 47

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factors are the most definitive species requirements for the presence of this species in one place or habitat. These factors have to do with the presence of the suitable or adaptable habitat that a species request in order to live and breed in this area. There is a big list of such ecological factor that influence to the emplacement or presence of one species in one area. This factors can be: habitat, like water, swamps, ponds, streamlets, streams, river etc.; humidity, temperature, shading places, presence of plants and other places where mosquitoes can hide from the high temperature, predators, food sources, flower of the plants for the males and presence of warm blooded animals for females to develop their eggs; interspecific competition among no mosquitoes species or other insects species; intraspecific competition between mosquito species, etc. (Service, 1993). These ecological factors mentioned above, are essential for the presence of a species in a specific area (Service, 1993). Commonwealth has the highest number of mosquito species captured (16 species), UDWKHUWKDQ6HQGDWDQG6HQGD\X%XWWKLVGRHVQ¶WPHDQWKDWWKHELRORJLFDOGLYHUVLW\ or the number of mosquito species is higher in Commonwealth, rather than in the two other stations. This might be due to the weather condition, breeding seasons, mosquito spray used as insecticide and other human activities in the recreational parks (Service, 1993). During our study in Sendat and Sendayu the weather condition has been with KDUG\UDLQDOPRVWGXULQJDOOWKHWLPHDQGWKDW¶VZK\WKHQXPEHURIPRVTXLWRVSHFLHV is lower than in Commonwealth, in which the weather conditions has been better. In better weather condition number of mosquito species, as well as number of individuals per species, is higher than in the rainy or hardy rain conditions. In hardy rainy condition they stay and rest to their resting place, in crevices, tree leaves and branches, rocky etc.

4.1.3.2 Biological diversity of mosquitoes by technique used This means the number of mosquito species captured per technique or method used. In this way we can conclude that which is the best and the better method to be used more efficiently for mosquito collection for study purposes. The number of mosquito species captured by HLC method is 20. This is a relatively high species number compare to the CDC light trap baited with dry ice and resting catch. This is a 48

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recommended method to catch only female mosquito, mainly of the genus Anopheles (Anopheles maculatus), Aedes (Aedes albopictus), Armigeres etc. The number of mosquito species captured by CDC method is 14 and by the resting catch is 3 species. We recommend that the best method to use for the Culex genus study purposes is CDC, rather than RC and HLC.

4.1.4 Some pictures during mosquitoes identification in the laboratory In plates below (from plate 8 to plate 12) there is shown the procedure of the mosquito identification in stereomicroscope to the lab with the established taxonomical keys (Reid, 1968; Entomological Chart, 1997, IMR).

Plate 8. Pictures of A. albopcitus left, and A. aegypti right. 49

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Plate 10. Left & first pictures Culex quinquefasciatus, right pictures Culex vishnui.

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Plate 11. Ventral and dorsal view of Armigeres durhami.

Plate 12. Pictures of Mansonia bonneae. 52

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4.2 Dynamic and density of mosquito population Mosquito dynamic has to do with the 24 hours activity and movement of the individuals of one population for food, water, shading, hiding places, breeding, laying eggs, taking of a blood meal for female mosquito etc. Mosquito density has to do with the number of mosquito individuals present and active in one area. The higher number of the individuals in one population in one area, the higher will the mosquito density be. The study showed that Commonwealth station showed the highest density compared to Sendayu and Sendat. The low density of mosquito population is related most to the weather conditions, like rainy and windy weather. This weather has a negative impact in mosquito daily and nightly activity and movement. They will reduce their activities like looking for food, blood meal for females, breeding etc. In this weather condition the number of mosquitoes caught can be very low and sometimes zero. So, during our field trips to the stations of Sendayu and Sendat, the weather condition has been very unsuitable for mosquito night activity and movement. This is one of the main reasons why the number of mosquito caught were very low. 4.3 Mosquito biting activity The biting activity of mosquito depends on many factors. Biting activity, the time and the frequency of biting activity depend on the mosquito species, environment conditions, ecological conditions and requirements. It is depending mostly on the mosquito species. Different mosquito species have different biting activity time. Sometimes the biting activity is depending on the weather condition too. Bad weather condition can directly influence in the biting activity time of different mosquito species. Determination of the biting activity of one mosquito species is a very important data, because in this way we can determine the time the mosquito species started to feed on blood. This will be a nuisance to the human population that lives in this area or touristic of the recreation park area. This can limit the movement of people or tourists. Furthermore, knowing of the biting activity of mosquito is very important for the program of mosquito control with insecticides. This program must

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be undertaken during the biting activity time of one mosquito species. In this way the control insecticides program can be more efficient.

4.3.1 Biting activity of the 5 main genera collected during this study During our study the number of mosquito species caught were 26, meanwhile the number of the genera caught were 6. The biting activity for 5 main genera like: Anopheles, Aedes, Mansonia, Armigeres and Culex are shown in the paragraphs and figures below.

4.3.1.1 Biting activity of Aedes species

10 pm -1 11 1 p pm m -0 0 00 am -0 1 01 am -0 2 02 am -0 3 03 am -0 4 04 am -0 5 05 am -0 6 am

pm

-1 0

-9

9

8

-8 7

-7 6

pm

4 3.5 3 2.5 2 1.5 1 0.5 0

pm

Bites/man/hourr

The biting activity of Aedes species is shown in the Figure 19. The peak occurs immediately after sun set (6:00-7:00 pm) for all the study sites. Biting activity was very low after 8:00 pm for Sendayu and Commonwealth. However small biting peaked were observed in Sendat 9:00-10:00 pm and 3:00-04:00 am.

Commonwealth

Sendayu

Sendat

Total

Figure 19. Biting activity for Aedes species in Commonwealth, Sendayu, Sendat.

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4.3.1.2 Biting activity of Anopheles genus The biting activity of Anopheles species collected is shown Figure 20 below. Anopheles first bites were recorded as early as 7:00-8:00 pm. The nocturnal activity peaked at 9:00-10:00 pm and 0:00-01:00 am in Sendayu. Biting activity lower in Commonwealth and Sendayu with several irregular peaks at 10:00-11:00 pm, 0:0001:00 am and 3:00-04:00 am. 3 2.5

Bites/man/hourr

2 1.5 1 0.5

10 pm -1 11 1 p pm m -0 0 00 am -0 1 01 am -0 2 02 am -0 3 03 am -0 4 04 am -0 5 05 am -0 6 am

pm 9

-1 0

-9

pm 8

-8 7

6

-7

pm

0

Commonwealth

Sendayu

Sendat

Total

Figure 20. Biting activity for Anopheles species in Commonwealth, Sendayu, Sendat.

4.3.1.3 Biting activity of Armigeres genus The biting activity for Armigeres species is shown in the Figure 21. It started immediately at 6:00 pm with the highest peak occurred at 6:00-07:00 pm. Observation for Armigeres mosquitoes showed that almost no biting activity were recorded after 9:00 pm for Commonwealth, Sendayu and Sendat. Armigeres fed largely in the first two hours of the night.

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Mosquito trapping in recreational parks in Selangor, Malaysia 4.5 4

Bites/man/hourr

3.5 3 2.5 2 1.5 1 0.5

p 10 m -1 11 1 p pm m -0 0a 00 m -0 1 a 01 m -0 2 a 02 m -0 3 a 03 m -0 4 a 04 m -0 5 a 05 m -0 6 am

pm

-1 0

-9

9

8

7

6

-7

-8

pm

pm

0

Commonwealth

Sendayu

Sendat

Total

Figure 21. Biting activity for Armigeres species in Commonwealth, Sendayu and Sendat stations.

4.3.1.4 Biting activity of Culex genus Figure 22 shows that biting activity of Culex mosquitoes. 2.5

Bites/man/hourr

2 1.5 1 0.5

p 10 m -1 11 1 pm pm -0 0a 00 m -0 1 a 01 m -0 2 a 02 m -0 3 a 03 m -0 4 a 04 m -0 5 a 05 m -0 6 am

pm

-1 0 9

8

-9

pm -8 7

6

-7

pm

0

Commonwealth

Sendayu

Sendat

Total

Figure 22. Culex species biting activity in Commonwealth, Sendayu, Sendat. 56

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The mosquitoes came out throughout the night with the highest peak at 8:00-09:00 pm and another smaller peak at 11:00 pm ± 00:00 am, followed by 3:00-04:00 am. Biting activities are almost similar in Commonwealth, Sendayu and Sendat.

4.3.1.5 Biting activity of Mansonia genus

p 10 m -1 11 1 p pm m -0 0a 00 m -0 1 a 01 m -0 2 a 02 m -0 3 a 03 m -0 4 a 04 m -0 5 a 05 m -0 6 am

pm

-1 0

-9

9

8

-8 7

-7 6

pm

1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0

pm

Bites/man/hourr

Figure 23 shows the nocturnal activity of Mansonia species in Commonwealth, Sendayu and Sendat. Mansonia species bite quite early at 7:00-08:00 pm for all the study sites. High biting activity was recorded between 8:00-09:00 pm in Commonwealth and Sendat, before decreasing to minimum at 11:00 pm ± 0:00 am. The biting activity rose again at 1:00-02:00 am, before going down to another minimum at 2:00-03:00 am and rose again at 3:00-04:00 am. The study also showed that Mansonia species bite all through the night with several irregular peaks for all the study sites.

Commonwealth

Sendayu

Sendat

Total

Figure 23. Biting activity for Mansonia species in Commonwealth, Sendayu, Sendat.

4.3.2 Comparison between biting activity time of mosquito genera The same mosquito genus is expected to have the same biting activity in one area. But, sometimes this is not the same for the same areas even for different areas. Different areas have different conditions, ecological factors that influence are 57

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different, and so the time when the mosquito goes to have a blood meal will depend directly from these factors. The study showed that the biting activity time for the same genus in three different areas is almost the same, or in other words each genus have almost the same biting activity time. This cannot be 100% the same for all of the three areas because different areas have different ecological factors that influence directly and indirectly to the activity and biting activity of mosquitoes and the individuals of the same species have not the same requirements in different areas. The study also showed that the species of the each genus have almost the same activity and biting activity in different areas, this cannot be 100% the same for different areas, as this is explained above with the influence that ecological factors have in the general activity of mosquitoes. As we can see from graphs the peak biting activity coincide with the main peak for all Selangor regions. Even for the three stations or areas the peak of the biting activity for one species of mosquito in one area coincides with the peak or peaks biting activity of the same species in different areas.

4.4 Statistical analysis of the data 4.4.1 Similarity quotient Similarity quotient or similarity index was used to compare the species diversities of two different sites. Similarity index for Commonwealth, Sendayu and Sendat is present in Table 3. Similarity quotient Commonwealth Sendayu Sendat

Commonwealth 100.00 42.85% 40.00%

Sendayu 100.00 38.095%

Sendat 100.00

Table 3. Similarity quotient for the three study areas.

$VZHFDQVHHIURPWKHFDOFXODWLRQVRIWKH6RUHQVHQ¶VIRUPXODLQWKH7DEOHWKH similarity quotient is higher between Commonwealth and Sendayu (42.85%), 58

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compared with the Commonwealth and Sendat (40.00%) and between Sendayu and Sendat (38.095%). In general, the similarity quotients between the three stations compare to each other, have almost same values with a small difference of 2-3% from each similarity quotient. 4.4.2 Diversity index A diversity index were used to analyze our data is the Simpson's diversity index. , with values near zero corresponding to highly diverse or Note that heterogeneous ecosystems and values near one corresponding to more homogeneous ecosystems. 6RDFFRUGLQJWR6LPSVRQWKHYDOXHRI³'´PXVWEHEHWZHHQDQGLQFOXGLQJWKH YDOXHVDQG$FFRUGLQJWRRXUGDWDDQGFDOFXODWLRQVWKH6LPSVRQ¶VGLYHUVLW\LQGH[ is D=0.1135. So, this value is near zero value and we can conclude that this values near to zero corresponding to highly diverse or heterogeneous ecosystem. In other words this mean the biodiversity for the three study sites in Selangor is high; the number of species present in each area is high. Heterogeneous ecosystem means that the ecosystem has a high composition of mosquito species.

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0DSSLQJRIWKHPRVTXLWRVSHFLHV¶GLVWULEXWLRQ Figure 27 shows the map of Selangor region and the location of the three study sites: Commonwealth, Sendayu and Sendat.

LEGEND:

Sendat Sendayu Commonwealth

Figure 24. Selangor map showing the three study sites.

4.5.1 Mapping of Aedes species Figure 28 shows a map of the location of the three stations and the distribution or the presence of the species of Aedes species. 60

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Legend:

0-1 2-5 6-10

Red symbol = Aedes aegypti Blue symbol = Aedes niveus Red symbol = Aedes albopictus

11-20

> 20

Figure 25. Distribution of Aedes species in the three study sites in Selangor.

As we can see from the map A. albopictus is present in the three stations of the study. As it can be seen from the size of the symbols, Aedes ablopictus has a higher density of caught (52 individuals in total) in comparison with Aedes niveus (2 individuals) and Aedes aegypti (1 individual), which are present, respectively in Sendat and in Sendayu.

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4.5.2 Mapping of Anopheles species Figure 26 shows a map of the location of the three stations and the distribution or the presence of Anopheles species.

Legend:

0-1 2-5 6-15

Red symbol = Anopheles hodgkini Black symbol = Anopheles karwari Blue symbol = Anopheles introlatus

16-25

Green symbol = Anopheles maculatus

Figure 26. Distribution of Anopheles species in the three study sites in Selangor. We can see from the map that Anopheles maculatus is present in the three stations of the study. As it can be seen from the size of the symbols, Anopheles maculatus were more caught (38 individuals in total) in comparison with three other species. Anopheles introlatus is present only in Sendat with 3 individuals; Anopleles karwari and Anopheles hodgkini are present only in Sendayu with only one individual respectively. 62

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4.5.3 Mapping of Armigeres species Figure 27 shows a map of the location of the three stations and the distribution or the presence of the species of Armigeres genus. We can see from the map that Armigeres confuses is present only in the station of Commonwealth and high number was collected here (35 individuals). As it can be seen from the size of the symbols, Armigeres confuses has a higher density in comparison with four other species. Armigeres durhami is present in the three stations, but with a low density of 4, 2 and 2 individuals for Commonwealth, Sendayu and Sendat.

Legend:

0-1 2-5

Purple symbol = Armigeres subalbatus Red symbol = Armigeres moultoni

16-25

Black symbol = Armigeres sp Blue symbol = Armigeres durhami Green symbol = Armigeres confuses

Figure 27. Distribution of Armigeres species in the three study sites in Selangor. 63

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4.5.4 Mapping of Culex species Figure 28 shows a map of the location of the three stations and the distribution or the presence of the species of Culex genus. We can see from the map that Culex vishnui is present in the station of Commonwealth and Sendayu with higher density.

Legend:

0-1 6-10 11-15

Purple symbol = Culex tritaeniorhynchus Red symbol = Culex sitiens Black symbol = Culex sinensis Blue symbol = Culex mimulus

16-20

Orange symbol = Culex pseudovishnui White symbol = Culex vishnui

Green symbol = Culex gelidus Yellow symbol = Culex bitaeniorhynchus Brown symbol = Culex quinquefasciatus

Figure 28. Distribution of Culex species in the three study sites in Selangor.

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As it can be seen from the size of the symbols, Culex vishnui has a higher density in comparison with eight other species. Culex pseudovishnui is present in the same stations where Cx. vishnui is, but with a lower density compared to Cx. vishnui of 6 individuals for Commonwealth and 6 individuals for Sendayu. Cx. quinquefasciatus is present in the three stations, but it has a very low density, 6 individuals in Commonwealth and represented by 1 individual in the two other stations. Six other species of this genus have a very low density represented by 1 individual each. Cx. gelidus, Cx. mimulus, Cx. tritaeniorhynchus, Cx. bitaeniorhynchus and Cx. sitiens are represented only in Commonwealth by 1 individual. Cx. sinensis is represented only by 1 individual in Sendayu station.

4.5.5 Mapping of the Mansonia species Figure 29 shows a map of the location of the three stations and the distribution or the presence of Mansonia species. We can see from the map that Mansonia bonneae is present in the station of Commonwealth and Sendat. As it can be seen from the size of the symbols, Mansonia bonneae has a higher density in comparison with 3 other species in Commowealth station. Mansonia indiana is present only in Sendayu. Mansonia annulifera is present only in Commonwealth and Mansonia uniformis is present only in Sendat.

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Legend:


0-1 2-5

Red symbol = Mansonia annulifera Black symbol = Mansonia uniformis

6-11

Blue symbol = Mansonia indiana Green symbol = Mansonia bonneae

Figure 29. Distribution of Mansonia species in the three study sites in Selangor.

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5.0 CONCLUSIONS A total of 26 mosquito species belong to 6 genera were collected and identified. The study showed that the best collection technique for Anopheles maculatus and Aedes albopictus were human landing catch. Armigeres, Culex and Mansonia species were attracted to these used techniques: human landing catch and CDC light trap baited with dry ice. Only Culex species were collected using resting catch. Anopheles, Culex and Mansonia species bite throughout the night with several irregular peaks. The biting activity of Armigeres and Aedes species showed similar trends. The highest biting activities for Anopheles and Culex species were at dusk 8:00-9:00 pm, Aedes and Armigeres were at dawn 6:00-7:00 pm and Mansonia was at dusk too at 7:00-8:00 pm. The study showed that the similarity quotient is higher between Commonwealth and Sendayu stations. There is a high diversity index in the three study areas. Thus, high number of mosquito species was recorded. This might be due to the heterogeneous ecosystem for all the three recreational parks of Selangor.

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6.0 RECOMMENDATIONS

The present study will observe a good bionomic study for mosquito species if it is carried out for one year with 5-7 days sampling time per month. In addition to this, if diurnal activities of the mosquito species are conducted, a complete bionomic study will be produced.

For future study of PRVTXLWR¶V population it is better to include larval survey activities in the study areas. This will provide a basic and a very good data for the whole biological cycle of mosquito species.

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7.0 REFERENCES AFPM. (2002). Armed Forces Pest Management Board: Technical Guide No 43. Guide to pest surveillance during contingency operations. Defense Pest Management Information Analysis Centre. Walter Reed Army Medical Centre, Washington DC 20307-50001. Cameron Webb & Richard C. Russell, 2005. A comparison of four commercially available adult mosquito traps. Institute of Clinical pathology and Medical Research. Centers for Disease Control and Prevention (2005). Mosquito Borne Disease. National Centre for Infectious Disease, Centers for Disease Control, Atlanta, Georgia. http://www.cdc.gov/ncidoc/diseases/list_mosquitoes.htm. Cheong, W.H., Chiang, G.L., Loong, K.P., Mahadevan, S. and Samarawickrema, W.A., (1988). Biting activities cycles of some mosquitoes in the Bengkoka Peninsula, Sabah state with notes on their importance. Tropical Biomedicine 5: 27-21. Cheong, W.H., Loong, K.P., Mahadevan, S., Mak, J.W. and Kan, S.K.P. (1984). Mosquito fauna of the Bengkoka Peninsular, Sabah, Malaysia. Southeast Asian Journal of Tropical Medicine and Public Health 15: 19-25. Colinvaux, Paul A. (1973). Introduction to Ecology. Wiley. ISBN 0-471-16498-4. Eliningaya, J., Kweka and Aneth, M., Mahande, 2009. Comparative evaluation of four mosquitoes sampling methods in rice irrigation schemes of lower Moshi, northern Tanzania. Malar J. 2009; 8: 149. Published online 2009 July 6. doi: 10.1186/1475-2875-8-149. Guimarães, A.E., C. Gentile, C.M. Lopes, and R. Pinto de Mello. 2000a. Ecology of mosquitoes (Diptera: Culicidae) in areas of Serra do Mar State Park, State of São Paulo, Brazil. III. Daily biting rhythms and lunar cycle influence. Mem. Inst. Oswaldo Cruz 95: 753-760. Guimarães, A.E., R. Pinto de Mello, C.M. Lopes, and C. Gentile. 2000b. Ecology of mosquitoes (Diptera: Culicidae) in areas of Serra do Mar State Park, State of São Paulo, Brazil. I. Monthly frequency and climatic factors. Mem. Inst. Oswaldo Cruz 95: 1-16. Harbach, R.E. and Kitching, I.J., 1998. Phylogeny and classification of the Culicidae (Diptera). Systematic Entomology 23: 327-370. 69

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Harbach, R.E. and Kitching, I.J., 2005. Reconsideration of Anopheline mosquito phylogeny (Diptera: Culicidae: Anophelinae) based on morphological data. Systematics and Biodiversity 3: 345-374. Harbach, R.E DQG .QLJKW ./ 7D[RQRPLVWV¶ *ORVVDU\ RI 0RVTXLWR Anatomy. Plexus Publishing, Inc. Marlton, NJ, 415 pp. +DUEDFK 5( DQG .QLJKW ./ &RUUHFWLRQV DQG DGGLWLRQV WR WD[RQRPLVWV¶ glossary of mosquito anatomy. Mosquito Systematics 13: 201-217. Hock, W., John. CDC Miniature Light Trap -- Model 512. instr_512_cdcminiature.doc, Friday, December 03, 2004. IMR (Institute for Medical Research), Division of Medical Entomology, 1997. Entomological charts for teaching. Jorge, R., Rey. 2001. The Mosquito. HTTP://EDIS.IFAS.UFL.EDU/IN652. Knight, K. and Stone, A., 1977. A Catalog of the mosquito of the World (Diptera: Culicidae). Thomas Say Foundation, Vol. 6. College Park, Maryland, 611 pp. Loong, K.P., Chiang, G.L., Yap, H.H., (1998). Field study of the bionomics of Anopheles maculatus DQGLW¶VLQPDODULDWUDQVPLVVLRQLQ0DOD\VLD Southeast Asian Journal of Tropical Medicine and Public Health 19: 724-728. Mosquito Larvae Anatomy - Florida Medical Entomology Laboratory. fmel.ifas.ufl.edu /key/anatomy/larval.shtml. Newson, H.D., 1977. Arthropod problems in recreational areas. Annual Reviewers Entomology. 22: 333-353 pp. Oli, K., Jeffery, J. and Vythilingam, I., 2005. A comparative study of adult mosquito trapping using dry ice and yeast generated carbon dioxide. Tropical Biomedicine 22(2): 249±251. Onyido, V., Ezike, N., Ozumba, E., Nwosu, O., Ikpeze, M., Obiukwu & E., Amadi: Crepuscular Man-Biting Mosquitoes of a Tropical Zoological Garden in Enugu, South-Eastern Nigeria. The Internet Journal of Parasitic Diseases. 2009, Volume 4, No 1. Rancangan Kawalan Penyakit Bawaan Vektor (1997). Vector Borne Disease Control Programme, Ministry of Health, Malaysia. Reid, J.A., (1968). Anophelinae mosquitoes in Malaya and Borneo. Stud. Inst. Med. Res. Malaysia, 520 pp. Reiter, P. 2001. Climate change and mosquito-borne disease. Envir. Hlth. Perspect. 109: 141-161.

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Rohani, A., Chan S.T., Abdullah, A.G., Tanrang, H. and Lee, H.L. (2008). Species composition of mosquito fauna in Ranau, Sabah, Malaysia. Tropical Biomedicine 25 (3): 232-236. Rohani, A., Lokman Hakim, S., Hassan, A.R., Chan, S.T., Ong, Y.F., Abdullah, A.G. and Lee Han Lim. (1999). Bionomic of Anopheles balabacensis baisas, the principal malaria vector, in Ranau, Sabah. Tropical Biomedicine 16: 31-38. Service M.W., 1993. Mosquito ecology field sampling methods. 2. London, UK: Elsevier Applied Science. Simpson, E. H. (1951). "The Interpretation of Interaction in Contingency Tables". Journal of the Royal Statistical Society, Ser. B 13: 238±241. Sørensen, T. (1948) A method of establishing groups of equal amplitude in plant sociology based on similarity of species and its application to analyses of the vegetation on Danish commons. Biologiske Skrifter / Kongelige Danske Videnskabernes Selskab, 5 (4): 1±34. The Life Cycle of the Mosquito. www.mosquitoes.org/LifeCycle.html. Vythilingam I., Chiang G.L. and Chan S.T. (1992). Evaluation of CO2 and 1-octen3-ol as mosquito attractants. Southeast Asian Journal of Tropical Medicine and Public Health 2:328-331. WHO 2002. Management of uncomplicated malaria and the use of antimalaria drugs for the protection of travellers. World Health Organization. Division of Control of Tropical Diseases. Geneva. :+2 (QWRPRORJLFDO ILHOG WHFKQLTXHV IRU PDODULD FRQWURO 3DUW /HDUQHU¶V Guide. World Health Organization. Geneva. World Health Organization (1975). Manual on practical entomology in malaria. Part II. Methods and Techniques. Geneva. WHO.

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8.0 APPENDIX Genus Anopheles

Time 6 -7 pm 7 -8 pm 8 -9 pm 9 -10 pm 10-11 pm 11pm-00 00-01 am 01-02 am 02-03 am 03-04 am 04-05 am 05-06 am

Total No. of HLC* Anopheles genus in Commonwealth/man/hour Trip 1 Trip 2 Trip 3 Trip 4 Total Bite/man/h Bite/man/h Bite/man/h Bite/man/h bite/man/h

1/3

0.34

Appendix 1. Anopheles species biting activity in Commonwealth.

Time 6-7 pm 7-8 pm 8-9 pm 9-10 pm 10-11 pm 11- 00 am 00-01 am 01-02 am 02-03 am

Total No. of HLC Anopheles genus in Sendayu/man/hour Trip 1 Trip 2 Trip 3 Trip 4 Total Bite/man/h Bite/man/h Bite/man/h Bite/man/h bite/man/h 1/3 1/3 1/3 1/3 1/3

1/3 2/3

2/3

72

1/3 1/3 1/3

0.34 0.34 0.34 0.34 0.34 0.65

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03-04 am 1/3 04-05 am 05-06 am *HLC = Human landing catch Appendix 2. Anopheles species biting activity in Sendayu.

Time 6 -7 pm 7 -8 pm 8 -9 pm 9 -10 pm 10-11 pm 11 pm ± - 00 am 00-01 am 01-02 am 02-03 am 03-04 am 04-05 am 05-06 am

0.34

Total No. of HLC Anopheles genus in Sendat/man/hour Trip 1 Trip 2 Trip 3 Trip 4 Total Bite/man/h Bite/man/h Bite/man/h Bite/man/h bite/man/h 6/5 1/4

1/6

1.2 0.27 0.5

1/5

2/5

0.3

1/5 1/5 2/5

1/3

0.25 0.3 0.3

2/5 3/5

3/5

1/5

1/3

Appendix 3. Anopheles species biting activity in Sendat.

Time 6-7 pm 7-8 pm 8-9 pm 9-10 pm 10-11 pm

Total No. of HLC Anopheles genus in Selangor/man/hour Trip 1 Trip 2 Trip 3 Trip 4 Total Bite/man/h Bite/man/h Bite/man/h Bite/man/h bite/man/h 1/3 1/3 3/10 1/3

6/5 2/5 3/5 1/3

1/6 3/5 1/3 73

0.88 0.29 0.45 0.34

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11 pm ± - 00 am 00-01 am 01-02 am 02-03 am 03-04 am 04-05 am 05-06 am


1/3

1/5 3/8 1/5 2/5 1/3

3/8

0.31

2/3

2/6

1/5

1/3

0.41 0.2 0.3 0.34

Appendix 4. Anopheles species biting activity in Selangor.

Genus Aedes


Total No. of HLC Aedes genus in Commonwealth/man/hour Trip 1 Trip 2 Trip 3 Trip 4 Total Bite/man/h Bite/man/h Bite/man/h Bite/man/h bite/man/h 8/6 12/5 1.82 3/6 1/5 0.36 1/3

Appendix 5. Aedes species biting activity in Commonwealth.

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Total No. of HLC Aedes genus in Sendayu/man/hour Trip 1 Trip 2 Trip 3 Trip 4 Total Bite/man/h Bite/man/h Bite/man/h Bite/man/h bite/man/h 3/5 1/5 0.4 1/4 1/5 0.22

Time 6-7 pm 7-8 pm 8-9 pm 9-10 pm 10-11 pm 11 pm ± - 00 am 00-01 am 01-02 am 02-03 am 03-04 am 1/4 04-05 am 05-06 am Appendix 6. Aedes species biting activity in Sendayu.

0.25

Total No. of HLC Aedes genus in Sendat/man/hour Trip 1 Trip 2 Trip 3 Trip 4 Total Bite/man/h Bite/man/h Bite/man/h Bite/man/h bite/man/h 1/4 4/5 2/3 3/6 0.56 2/4 1/6 0.3

Time 6 -7 pm 7 -8 pm 8 -9 pm 9 -10 pm 10-11 pm 11 pm ± - 00 am 00-01 am 01-02 am 02-03 am 03-04 am 04-05 am 05-06 am Appendix 7. Aedes species biting activity in Sendat. 75

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Total No. of HLC Aedes genus in Selangor/man/hour Trip 1 Trip 2 Trip 3 Trip 4 Total Bite/man/h Bite/man/h Bite/man/h Bite/man/h bite/man/h 1/4 7/10 10/9 16/16 0.87 3/8 3/6 3/16 0.3 1/3

1/4

0.34

0.25

Appendix 8. Aedes species biting activity in Selangor.

Genus Armigeres

Time 6 -7 pm 7 -8 pm 8 -9 pm 9 -10 pm 10-11 pm 11 pm ± - 00 am 00-01 am

Total No. of HLC Armigeres genus in Commonwealth/man/hour Trip 1 Trip 2 Trip 3 Trip 4 Total Bite/man/h Bite/man/h Bite/man/h Bite/man/h bite/man/h 8/3 9/5 2.13 1/3 2/3 1/5 0.36

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01-02 am 02-03 am 03-04 am 04-05 am 05-06 am Appendix 9. Armigeres species biting activity in Commonwealth.

Time 6-7 pm 7-8 pm 8-9 pm 9-10 pm 10-11 pm 11 pm ± - 00 am 00-01 am 01-02 am 02-03 am 03-04 am 04-05 am 05-06 am

Total No. of HLC Armigeres genus in Sendayu/man/hour Trip 1 Trip 2 Trip 3 Trip 4 Total Bite/man/h Bite/man/h Bite/man/h Bite/man/h bite/man/h 1/5

Appendix 10. Armigeres species biting activity in Sendayu.

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Total No. of HLC Armigeres genus in Sendat/man/hour Trip 1 Trip 2 Trip 3 Trip 4 Total Bite/man/h Bite/man/h Bite/man/h Bite/man/h bite/man/h 1/6

0.17

Appendix 11. Armigeres species biting activity in Sendat.

Time 6 -7 pm 7 -8 pm 8 -9 pm 9 -10 pm 10-11 pm 11 pm ± - 00 am 00-01 am 01-02 am 02-03 am 03-04 am

Total No. of HLC Armigeres genus in Selangor/man/hour Trip 1 Trip 2 Trip 3 Trip 4 Total Bite/man/h Bite/man/h Bite/man/h Bite/man/h bite/man/h 8/3 9/5 2.13 1/3 2/3 3/16 0.27

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04-05 am 05-06 am Appendix 12. Armigeres species biting activity in Selangor.

Genus Culex


Total No. of HLC Culex genus in Commonwealth/man/hour Trip 1 Trip 2 Trip 3 Trip 4 Total Bite/man/h Bite/man/h Bite/man/h Bite/man/h bite/man/h

2/3

6/5 1/5 3/5

1.2 0.2 0.63 0.67

1/5

0.38 0.34

2/3 2/3 3/6 2/3 1/3

0.67 0.34

Appendix 13. Culex species biting activity in Commonwealth.

Time 6-7 pm 7-8 pm 8-9 pm

Total No. of HLC Culex genus in Sendayu/man/hour Trip 1 Trip 2 Trip 3 Trip 4 Total Bite/man/h Bite/man/h Bite/man/h Bite/man/h bite/man/h

1/5 79

0.2

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9-10 pm 10-11 pm 11 pm ± - 00 am 00-01 am 01-02 am 02-03 am 03-04 am 04-05 am 05-06 am


2/3

1/3 1/3

1/5

0.36 0.34

Appendix 14. Culex species biting activity in Sendayu.


Total No. of HLC Culex genus in Sendat/man/hour Trip 1 Trip 2 Trip 3 Trip 4 Total Bite/man/h Bite/man/h Bite/man/h Bite/man/h bite/man/h

1/4

Appendix 15. Culex species biting activity in Sendat.

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Total No. of HLC Culex genus in Selangor/man/hour Trip 1 Trip 2 Trip 3 Trip 4 Total Bite/man/h Bite/man/h Bite/man/h Bite/man/h bite/man/h

2/3 2/3

3/6

7/10 1/5 3/5 1/5

0.7 0.2 0.63 0.43

3/6

1/5

0.36 0.5

3/6 2/3 2/7

0.67 0.29

Appendix 16. Culex species biting activity in Selangor.

Genus Mansonia

Time 6 -7 pm 7 -8 pm 8 -9 pm 9 -10 pm 10-11 pm 11 pm ± - 00 am 00-01 am

Total No. of HLC Mansonia genus in Commonwealth/man/hour Trip 1 Trip 2 Trip 3 Trip 4 Total Bite/man/h Bite/man/h Bite/man/h Bite/man/h bite/man/h 2/3

0.67

1/3

0.34

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01-02 am 02-03 am 03-04 am 04-05 am 05-06 am


5/6 1/5 1/2

0.83 0.2 0.5

Appendix 17. Mansonia species biting activity in Commonwealth.

Time 6-7 pm 7-8 pm 8-9 pm 9-10 pm 10-11 pm 11 pm ± - 00 am 00-01 am 01-02 am 02-03 am 03-04 am 04-05 am 05-06 am

Total No. of HLC Mansonia genus in Sendayu/man/hour Trip 1 Trip 2 Trip 3 Trip 4 Total Bite/man/h Bite/man/h Bite/man/h Bite/man/h bite/man/h

Appendix 18. Mansonia species biting activity in Sendayu.

82

2/5 2/5

0.4 0.4

1/5

0.2

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Total No. of HLC Mansonia genus in Sendat/man/hour Trip 1 Trip 2 Trip 3 Trip 4 Total Bite/man/h Bite/man/h Bite/man/h Bite/man/h bite/man/h

Time 6 -7 pm 7 -8 pm 8 -9 pm 9 -10 pm 10-11 pm 11 pm ± - 00 am 00-01 am 01-02 am 1/5 02-03 am 03-04 am 04-05 am 05-06 am Appendix 19. Mansonia species biting activity in Sendat.

0.2

Total No. of HLC Mansonia genus in Selangor/man/hour Trip 1 Trip 2 Trip 3 Trip 4 Total Bite/man/h Bite/man/h Bite/man/h Bite/man/h bite/man/h

Time 6 -7 pm 7 -8 pm 2/3 8 -9 pm 9 -10 pm 1/3 10-11 pm 11 pm ± - 00 am 00-01 am 01-02 am 5/6 1/5 02-03 am 03-04 am 1/2 04-05 am 05-06 am Appendix 20. Mansonia species biting activity in Selangor. 83

2/5 2/5

0.5 0.4 0.34

1/5

0.2 0.55 0.2 0.5

1/5