NATIONWIDE DISTRIBUTION AND INSECTICIDE RESISTANCE STUDY OF MALAYSIAN MOSQUITO CULEX QUINQUEFASCIATUS SAY BY MOLECULAR AND BIOCHEMICAL TOOLS LOW VAN LUN THESIS SUBMITTED IN FULFILLMENT OF THE REQUIREMENT FOR THE DEGREE OF DOCTOR OF PHILOSOPHY INSTITUTE OF BIOLOGICAL SCIENCES FACULTY OF SCIENCE UNIVERSITY OF MALAYA KUALA LUMPUR 2013 ABSTRACT A nationwide investigation was carried out to (1) determine the distribution of Culex quinquefasciatus and other species of mosquitoes in stagnant water in residential areas, (2) investigate the genetic diversity of Cx. quinquefasciatus, (3) quantify the insecticide susceptibility status of Cx. quinquefasciatus, (4) characterize the biochemical mechanisms of insecticide resistance in Cx. quinquefasciatus, (5) characterize the molecular mechanisms of insecticide resistance in Cx. quinquefasciatus from 13 states and one federal territory in Malaysia. Culex larval surveillance indicated that Cx. quinquefasciatus was the predominant species in residential areas. Several habitat characteristics (i.e., pH, conductivity, salinity, total dissolved solids, elevation and dissolved oxygen) were found to be associated with Culex larvae distribution. In the context of molecular phylogeography, the genetic diversity of Malaysian Cx. quinquefasciatus was extremely low since only three and four haplotypes were revealed by COI and COII, respectively. As for insecticide resistance study of Cx. quinquefasciatus, both WHO larval and adult bioassays exhibited dissimilar trends in susceptibility against DDT, propoxur, malathion and permethrin. Correlations between propoxur and malathion resistance as well as between propoxur and permethrin resistance in larval bioassays were found. In enzyme microassays, elevated levels of α- esterases, β-esterases, mixed function oxidases, glutathione-S-transferase and acetylcholinesterase activities were demonstrated in majority of the populations. Besides, a correlation between α-esterases activity and malathion resistance was also demonstrated. An association between activity of α-esterases and β-esterases and between glutathione-S-transferase and acetylcholinesterase was also demonstrated. In addition, genotyping of insensitive acetylcholinesterase revealed the presence of G119S mutation in the wild populations of Cx. quinquefasciatus, but in heterozygous state and ii at a very low frequency (18 out of 140). Statistical analysis also revealed that malathion resistance was associated with the frequency of ace-1 resistant allele in Cx. quinquefasciatus populations. On the other hand, genotyping of insensitive voltage gated sodium channel revealed the presence of L1014F mutation which was dominated by heterozygous genotype (99 out of 140), followed by three individuals of homozygous genotype. However, no association was found between the frequency of kdr resistant allele and DDT and permethrin resistance. The findings of this study could be utilized in the implementation of strategic measures in vector control programs in Malaysia. Identification of the breeding preferences and population structure of mosquitoes offers an opportunity to elucidate their influence on the current distribution of mosquito species in these study areas and subsequently understand their potential risks in disease transmission. Besides, the bioassay data may also assist local authorities in providing susceptibility baseline and appropriate dosage of larvicide or adulticide to be used during vector control operations. Application of both biochemical and molecular tools provides significant insights into the evolution and adaptation of Malaysian Cx. quinquefasciatus. In conclusion, this study has demonstrated the associations between the Culex distribution and various habitat characteristics. Besides, a phylogenetic relationship among Malaysian Cx. quinquefasciatus was revealed. This study has provided the first evidence for the involvement of target site alterations and detoxification mechanisms in insecticide resistance among Cx. quinquefasciatus populations in Malaysia. iii ABSTRAK Satu kajian di seluruh negara telah dijalankan untuk (1) menentukan taburan nyamuk Culex quinquefasciatus dan spesis nyamuk lain dalam air bertakung di kawasan perumahan, (2) meninjau kepelbagaian genetik Cx. quinquefasciatus, (3) menentukan tahap kerentanan Cx. quinquefasciatus terhadap racun serangga, (4) mengenal pasti mekanisme biokimia kerintangan racun serangga dalam Cx. quinquefasciatus, (5) mengenal pasti mekanisme molekul kerintangan racun serangga dalam Cx. quinquefasciatus dari 13 negeri dan Wilayah Persekutuan di Malaysia. Peninjauan yang dilakukan terhadap larva Culex menunjukkan bahawa Cx. quinquefasciatus merupakan species larva yang utama di kawasan perumahan. Beberapa ciri habitat (pH, kekonduksian, kemasinan, jumlah pepejal terlarut, ketinggian tapak dari aras laut dan oksigen terlarut) didapati berkait dengan taburan larva Culex. Dalam aspek filogeografi molekul, tahap kepelbagaian genetik Cx. quinquefasciatus di Malaysia adalah sangat rendah disebabkan terdapat hanya tiga dan empat haplotip yang dikesan dengan gen COI dan COII. Dalam kajian kerintangan Cx. quinquefasciatus terhadap racun serangga, WHO bioassai pada peringkat larva dan dewasa menunjukkan tahap kerentanan yang berbeza terhadap DDT, propoxur, malathion dan permethrin. Hubungan antara kerintangan propoxur dengan malathion dan propoxur dengan permethrin telah menunjukkan kesan melalui bioassai larva. Dalam kajian mikroassai enzim, peningkatan tahap aktiviti-aktiviti α-esterase, β-esterase, oxidase fungsi campuran, glutation-S-transferase dan asetilkolinesterase telah didapati di kebanyakan populasi. Selain itu, terdapat juga hubungan di antara aktiviti α-esterase dengan kerintangan malathion. Hubung kait di antara aktiviti α-esterase dan β-esterase dan antara glutation- S-transferase dan acetylcholinesterase juga dikenalpasti. Disamping itu, ujian pengesanan alel ace-1 menunjukkan terdapat kehadiran mutasi G119S di populasi Cx. iv quinquefasciatus yang diperolehi dari lapangan tetapi dalam keadaan heterozigot dan berada dalam frekuensi yang rendah (18 daripada 140). Analisis statistik menunjukkan bahawa kerintangan malathion berkait rapat dengan frekuensi alel ace-1 dalam populasi Cx. quinquefasciatus. Selain itu, ujian pengesanan alel kdr juga menunjukkan kewujudan mutasi L1014F yang didominankan oleh genotip heterozigot (99 daripada 140), diikuti dengan tiga individu genotip homozigot. Walau bagaimanapun, tiada hubung kait ditunjukkan antara frekuensi alel kdr dengan kerintangan DDT dan permethrin. Hasil daripada kajian ini dapat digunakan dalam melaksanakan langkah- langkah strategik bagi program kawalan vektor di Malaysia. Pengenalpastian kawasan pembiakan yang sesuai dan struktur populasi nyamuk dapat menjelaskan pengaruh nyamuk ke atas taburan spesiesnya di kawasan-kawasan kajian dan memahami potensi risiko terhadap jangkitan penyakit. Selain itu, data bioassai juga dapat membantu pihak berkuasa tempatan dalam menyediakan maklumat asas mengenai kerentanan dan dos racun serangga yang sesuai semasa operasi kawalan vektor. Penggunaan teknik-teknik biokimia dan molekul dapat mempertingkatkan lagi pengetahuan dalam aspek evolusi dan adaptasi Cx. quinquefasciatus di Malaysia. Kesimpulannya, kajian ini telah menunjukkan hubung kait antara taburan nyamuk Culex dan ciri-ciri habitatnya. Selain itu, kajian ini juga telah menunjukkan hubungan filogenetik Cx. quinquefasciatus dan membuktikan kewujudan perubahan tapak target serta tahap aktiviti enzim dalam kerintangan racun serangga oleh Cx. quinquefasciatus di Malaysia. v ACKNOWLDEGEMENTS First and foremost, I would like to acknowledge and extend my heartfelt gratitude to all those who gave me the possibility to complete this thesis. I would like to thank University of Malaya, the leader in research and innovation for granting me as a registered postgraduate student to commence this thesis. My sincere gratitude also goes to Universiti Malaysia Terengganu, the institution that has sown the seed of research interest in me, particularly in the field of toxicology. I am eternally grateful to my main supervisor, Prof. Dato’ Dr. Mohd Sofian Azirun (Institute of Biological Sciences, Faculty of Science, University of Malaya) for all his guidance and endless support throughout the study period. His extensive networks of research truly help the progression and smoothness of this thesis. Thank you from the bottom of my heart for your commitment. My sincere appreciation also goes to my co-supervisor, Dr. Lim Phaik Eem (Institute of Biological Sciences, Faculty of Science, University of Malaya) who brought me a golden opportunity to set my feet in the University of Malaya. Many thanks for your expertise in phylogenetic analyses and presentation of the research findings. I am deeply indebted to Dr. Chen Chee Dhang (Institute of Biological Sciences, Faculty of Science, University of Malaya) for his scientific discussion. His constant support throughout the experimental works has contributed to the success of this project. I would especially like to thank Assoc. Prof. Dr. Yvonne Lim Ai Lian (Department of Parasitology, Faculty of Medicine, University of Malaya) for providing me a comfortable and well-equipped working environment. Most importantly, thank you for your willingness to share your knowledge and wonderful inspiring thoughts. I am grateful to Dr. Lee Han Lim (Medical Entomology Unit, Institute for Medical Research, vi Kuala Lumpur) for sharing his wealth of experience in the field of medical entomology