Characterization and expression profiles of Glutathione - S- transferase (GSTs) gene in Anopheles mosquito vector complex Thesis submitted in fulfillment of the requirements for the degree of Doctor of Philosophy in Department of Zoology by K.VANLALHRUAIA Ph.D Registration No: MZU/Ph.D/336 of 29.11.2010 Under the supervision of Dr.G. Gurusubramanian Associate Professor Department of Zoology and Joint-supervision of Dr. N. Senthil Kumar Professor Department of Biotechnology Department of Zoology School of Life Sciences Mizoram University Aizawl, Mizoram CERTIFICATE I certify that the thesis entitled ‘ Characterization and expression profiles of Glutathione - S- transferase (GSTs) gene in Anopheles mosquito vector complex ’ submitted to the Mizoram University for the award of a degree of Doctor of Philosophy in Zoology Department by Mr. K. VANLALHRUAIA is a record of research work carried out by him during the period from 2009 to 2013 under my guidance and supervision, and that this work has not formed the basis for the award of any degree, diploma, associateship, fellowship or other similar titles in this University or any other University or institution of higher learning. Signature of the Supervisor (Dr. G. Gurusubramanian) Signature of the Co-Supervisor (Prof. N. Senthil Kumar) Declaration of the candidate I, K. Vanlalhruaia , a Ph.D scholar in Department of Zoology, Mizoram University, Aizawl, do hereby solemny declare that this thesis hereby submitted by me for the Degree of Doctor of Philosophy at the Mizoram University, Aizawl is my own independent work and has not been previously submitted for any degree, diploma, fellowship or other similar titles in this University or other University or institution of higher learning. (K. Vanlalhruaia) Candidate Acknowledgement First, I would like to thank the Almighty God for his guidance, blessing and care throughout my study. I express my sincere gratitude to my respected supervisor Dr. G. Gurusubramanian, Associate Professor, Department of Zoology, Mizoram University and co-supervisor Prof. N. Senthil Kumar, Department of Biotechnology, Mizoram University for their valuable guidance. My gratitude also goes to Dr. H.T Lalremsanga, Assistant Professor and Mr. Lalhmangaiha, Department of Zoology, Mizoram University and Dr. Zochampuia, M&E Consultant, Directorate of Health Services, Mizoram for their support, help and valuable advise to complete my work. My sincere thanks goes to Mr. B. Vanlalauva, Mamit and those who hosted me in different places of Mizoram for their help, support, advice and hospitality during my field works. My sincere appreciation also goes to all my colleagues Lalrotluanga, Zothansangi, Rita Zomuanpuii, Lalduhawma, Catherine Vanlalruati and Dr. Lamneihthang Vaiphei for their valuable help and suggestions. My heartfelt gratitude also extends to Department of Science and Technology, Government of India, inspire fellowship for financial support. DBT-New Delhi for providing Bioinformatics infrastructure facility. I would also like to thank to Esther Lalhlimpuii for helping me to cross check my thesis. Last but not the least I am forever indebted to my mom and dad, brothers and sister and my wife-Maryne Zothansangi for their love, encouragements and support throughout my research work. K. Vanlalhruaia I CONTENTS Page ACKNOWLEDGEMENT I CONTENTS II LIST OF TABLES VI LIST OF FIGURES VIII I. INTRODUCTION AND REVIEW OF LITERATURE 1-23 1.1 . International and national status of mosquito species 1 1.2 . Status of Malaria and Mosquito management in Mizoram 2 1.3 . Distribution, abundance and breeding habitats 4 1.4 . Environmental factors 6 1.5 . Insecticide resistance 9 1.6 . Target-site resistance 9 1.7 . Insecticide metabolisms 10 1.8 . Glutathione S-transferases 11 1.9 . GST structure 12 1.10. GST classification and organization 13 1.11. GST detoxification functions 14 1.12. Insecticides Detoxification of GST 15 1.13. Regulation of GST expression 17 1.14. P450 monooxygenases 19 1.15. Esterase-Based Resistance 20 1.16. In-silico characterization of GST 21 II II. OBJECTIVES OF THE STUDY 24 III. MATERIALS AND METHODS 26-48 3.1. Study area 27 3.2. Diversity, distribution and population survey of Mosquito 31 3.2.1. Method of sampling and collection of Mosquito 31 3.2.1.1. Larva collection (scoop-net method) 31 3.2.1.2 Adult collection (hand collection and light trap) and preservation 31 3.2.1.3. Identification of mosquito 32 3.2.2. Seasonal Variation and relative abundance 32 3.2.2.1. Distribution and Density of sampled Mosquito 32 3.3. Mosquito Larva and their breeding habitats association 33 3.3.1. Interaction of mosquito species and breeding habitats with water quality 34 3.4. Insecticidal Bioassay 38 3.4.1. Maintenance of mosquito and preparation of test concentrations 38 3.4.2. Larval susceptibility assay 38 3.4.3. Adults susceptibility assay 39 3.5. Biochemical analysis of insecticides treated samples 40 3.5.1. Preparation of mosquito for quantitative enzyme assays 40 3.5.2. Protein assay 40 3.5.3. Standard ( α-and β-) Napthol assay 40 3.5.4. Naphthyl Acetate assay for Esterase 41 3.5.5. Assay for Glutathione-S-Transferase 41 III 3.6. Preparation and extraction of total RNA from Anopheles species 42 3.6.1. cDNA synthesis from total RNA 43 3.6.2. Reverse Transcriptase (RT)-PCR of Anopheles β-Actin 44 3.6.3. Study the expression of Anopheles Glutathione-S-transferase epsilon-4 gene 45 3.7. In-silico characterization of GST epsilon-4 of mosquito 47 IV. RESULTS 49-100 4.1. Diversity, distribution and population studies of mosquito 50 4.1.1.General and common morphological features of mosquito 50 4.1.1.1. Key features for adult mosquito species – Anopheles 54 4.1.1.2. Key features for adult mosquito species - Culex 58 4.1.1.3. Key features for adult mosquito species - Aedes 61 4.1.1.4. Key features for adult mosquito species - Culiseta 62 4.1.1.5.Key features for adult mosquito species - Toxorhynchites 63 4.1.2. Survey of mosquito population in six districts of Mizoram 70 4.1.3. Diversity and distribution of Mosquito in Mizoram 75 4.1.4. Abundance of mosquito species 75 4.1.5. Seasonal variation of mosquito and abundance in Aizawl 79 4.2. Larval breeding habitats and influences of environmental factors on their breeding 83 4.2.1. Characters of breeding habitats 86 4.2.2. Relationship between mosquito larval abundance and environmental variables 87 4.3. Lethal bio-assay and susceptibility change in relation to level of metabolic resistant enzyme 89 4.3.1 Biochemical assays on insecticides treated samples 91 IV 4.4. Semi-quantitative expression of GSTe4 partial gene 93 4.5. In-silico characterization of mosquito GSTs 94 V. DISCUSSION 101-108 5.1. Diversity and relative abundances of mosquito 102 5.2. Habitats characteristics of mosquito 103 5.3. Effect of environmental factors on mosquito breeding 105 5.4. Insecticides susceptibility status 106 5.5. Expression of GSTe4 gene 107 5.6. In-silico characterization of mosquito GSTe4 108 VI. SUMMARY 109-115 VII. RECOMMENDATION 116 VIII. BIBLIOGRAPHY 118-138 IX. APPENDIX 139-146 Published papers and Communicated 147 Papers Presentation at Seminars / Conferences 147 Tranning/Workshop participated 148 V List of Tables Table 1. Temperature cycling parameters for RT-PCR of Anopheles β-Actin Table 2 . Temperature cycling parameters for expression of Anopheles GSTepsilon-4 gene Table 3. Main identification characters of collected Anopheline mosquitoes. Table 4. Main identification characters of collected Culicidae mosquitoes. Table 5. Distribution and population size of mosquito across six districts of Mizoram. Table 6. Distribution and abundance of collected mosquitoes across six districts in Mizoram 2009-2011. Table 7. Collection of mosquitoes across the six different districts of Mizoram (India) during 2009 to 2011. A - Presence of Adult; L - Presence of larva. Table 8. Distribution and density % of mosquito across six districts in Mizoram 2009-2011. Table 9. Numbers (Range, mean ± S.E.) of mosquitoes sampled in Aizawl District, Mizoram from 2009 to 2011. Table 10. Results of Tukey test with respect to the abundance of immatures of different mosquito species in Aizawl district. Table 11 . Intermediate calculation ANOVA table for mosquito sampled in Aizawl district. Table 12 . Mosquitoes sampled (mean value) in Aizawl district. Table 13. Mosquito larval breeding habitats/characters in respect to different groups of mosquitoes in six different districts of Mizoram (India) during 2009 to 2011. Table 14 . Similarity matrix for the operative taxonomic units calculated using coefficient of association. Table 15 . Average values (mean± SE) of the measured environmental factors (water quality) in Aizawl. Table 16. Linear regression analysis for the abundance mosquito in Aizawl (showing best water quality predictors). Table 17. Insecticidal bioassays (DDT) against field collected Anopheles barbirostris and Anopheles Vagus . VI Table 18. Insecticidal bioassays (deltamethrin) against field collected Anopheles barbirostris and Anopheles Vagus . Table 19. Overall enzyme assays (mean±SE) in insecticides treated samples. Table 20. Correlation between susceptibility tests on different doses of insectides and enzymes (GSTs and esterases) production in treated samples Table 21. GSTe4 protein sequences of mosquitoes retrieved from NCBI database. Table 22 . Amino acid composition (in %) of mosquito GSTe4 computed using Expasy’s ProtParam tool. Table 23 . Parameters of mosquito GSTe4 computed using Expasy’s ProtParam tool. Table 24. Percentage of residues forming alpha, beta, and coil structures of GSTe4 computed by SSCP server. Table 25. Hydrophilic and hydrophobic residues content of GSTe4 using Protprop software. Table 26. Disulphide (SS) bond pattern of pairs predicted by CYS_REC (using primary structure) and identified by Rasmol (using 3D structure modelled). In EDS36584.1, EAA07591.6 and EDS36584.1 GSTe4 sequences. Table 27. Criteria for a good (model) 3D structure based on ProQ score. Table 28.