A STUDY ON ISOLATION, CHARACTERISATION AND EFFICACY OF NATURALLY OCCURRING MOSQUITO PATHOGENIC BACILLI FROM GOA, INDIA A THESIS SUBMITTED TO GOA UNIVERSITY FOR THE AWARD OF DEGREE OF DOCTOR OF PHILOSOPHY (PhD) in ZOOLOGY By JOLEEN SAVIANNE ALMEIDA GOA UNIVERSITY TALEIGAO PLATEAU, GOA AUGUST, 2020 A STUDY ON ISOLATION, CHARACTERISATION AND EFFICACY OF NATURALLY OCCURRING MOSQUITO PATHOGENIC BACILLI FROM GOA, INDIA A THESIS SUBMITTED TO GOA UNIVERSITY FOR THE AWARD OF DEGREE OF DOCTOR OF PHILOSOPHY (PhD) in ZOOLOGY By JOLEEN SAVIANNE ALMEIDA Research guide Co - Guide Dr. Ashwani Kumar Dr. Savita Kerkar Director, ICMR- Vector Control Research Centre Professor, Department of Biotechnology Indira Nagar, Puducherry- 605006 Goa University, Taleigao Plateau, UT, India Goa - 403206 AUGUST, 2020 DECLARATION As suggested by the referees/external examiners, I, Joleen S. Almeida, PhD Research Scholar (as per ordinance OB-9A) of the Department of Zoology, Goa University, have incorporated all the appropriate corrections in the thesis on the relevant pages. Joleen S. Almeida Department of Zoology, Goa University. Place: Goa University Date: DECLARATION I hereby declare that the thesis entitled “A study on isolation, characterization and efficacy of naturally occurring mosquito pathogenic Bacilli from Goa, India”, submitted for the degree of Doctor of Philosophy (PhD) in Zoology to Goa University, is based on studies carried out by me at ICMR-National Institute of Malaria Research, Campal, Panaji, Goa under the supervision of Dr. Ashwani Kumar (Research Guide) and Dr. Savita Kerkar (Co – guide). The work is original and has not been submitted in part or full by me for any other degree or diploma to any other University/Institute. Materials obtained from other sources have been duly acknowledged in the thesis. Joleen S. Almeida Department of Zoology, Goa University. Place: Goa University Date: ACKNOWLEDGEMENT Through my remarkable journey during my PhD, I would like to extend my heartfelt gratitude and appreciation to all the people involved in order to make it a success. I am immensely grateful to my Research Guide Dr. Ashwani Kumar, Director- VCRC, Pondicherry, for his valuable expertise, consistent guidance, constant encouragement and enthusiastic approach to my research work. His ever-helping attitude and immense knowledge as well as and his critical assessment and attention to detail needs appreciation. His inspiring and encouraging words as well as constructive criticism of my work enabled me to reach this final stage. I would like to thank my co-guide Dr. Savita Kerkar Prof. Dept. of Biotechnology, Goa University for her help and guidance and for always making the time to promptly review my progress reports and papers. I wish to express my sincere gratitude to Dr. Ajeet Kumar Mohanty, Research Scientist, ICMR-NIMR, Field Station Goa for his extremely valuable suggestions, continuous monitoring and guidance in order to give my work a proper shape. I wish to thank Dr. R Roy, Prof. and Head, Dept. of Zoology, Goa University as well as the associated teaching and non-teaching staff of the Dept. of Zoology for their prompt action with regards to submission and processing of documents and the conduct of meetings to review my progress. I would like to express my sincere gratitude to VC’s nominee Dr. Sandeep Garg, Prof. and Head, Dept. of Microbiology, Goa University for his constructive comments, suggestions and critiquing. I am very thankful to each and every member of ICMR-National Institute of Malaria Research (NIMR) for their co-operation and willingness to help whenever approached. Mrs. Sushma requires special thanks for helping with maintaining the mosquito larval populations and Mrs. Maria for using her skills in Microsoft Word and Excel to help me with the compilation of this thesis. I thank my parents for their love, support and blessings as well as my family and friends for their encouragement and support. And finally, I am most grateful to God for blessing me with immense strength, patience, motivation and courage for the successful completion of this thesis. TABLE OF CONTENTS SR. CHAPTER CONTENTS PAGES NO NUMBER LIST OF FIGURES LIST OF TABLES 1. 1. INTRODUCTION 1 - 21 1. 1. General Introduction to Mosquitoes 1. 2. Life cycle 1.2.1 Egg 1.2.2 Larvae 1.2.3 Pupae 1.2.4 Adult 1. 3. Major Mosquito Genera and Species 1.3.1 Anophelines (Anopheles) 1.3.2 Culicines (Aedes and Culex) 1.3.3 Mansonia 1. 4. Major Mosquito Borne Diseases 1.4.1 Malaria 1.4.2 Filariasis 1.4.3 Dengue 1.4.4 Chikungunya 1.4.5 Zika 1.4.6 West Nile virus 1.4.7 Japanese encephalitis 1.4.8 Yellow fever 1.5. Control of Mosquitoes 1.5.1 Environmental Management 1.5.2 Chemical Control 1.5.3 Biological Control 1.6. Significance of the thesis and Objectives 2. 2. REVIEW OF LITERATURE 22 - 39 2. 1. Background 2.2. Mosquito Vector control in India 2.3. Bacilli as bio-control agents 2.4. Bioassays, isolation, characterization and identification 2.5. Resistance phenomenon and overcoming resistance 2.6. Commercial Bio-larvicide Formulations 2.7. Future prospects 2.8. Conclusions 3. 3. MATERIALS AND METHODS 40 - 63 3.1. Sample Collection 3.2. Screening for Mosquito Pathogenic Bacilli 3.3. Isolation of Mosquito Pathogenic Bacilli 3.4. Maintenance of Mosquito Colony 3.5. Preliminary Toxicity Assay 3.6. Morphological and Biochemical Characterization of the Isolates 3.6.1. Colony Characteristics 3.6.2. i) Gram Staining ii) Endospore Staining 3.6.3. Scanning electron microscopy (SEM) 3.6.4. Biochemical Characterization and Optimization of growth conditions 3.6.5. Bio surfactant production 3.7. Crude protein extraction and determination of concentration by Nanodrop technique 3.8. DNA extraction and PCR amplification 3.8.1 DNA extraction 3.8.2 PCR amplification and Nucleotide BLAST 3.8.3 Phylogenetic Tree construction 3.9. Large Scale Production of Active Isolates 3.9.1 Mass Production of Culture 3.9.2 Lyophilization 3.10. Harvesting of Culture Supernatant and Crude Mosquitocidal Toxin (CMT) 3.11. Toxin Studies 3.12. Main Bioassays 3.12.1. Calculations of LC50 and LC90 values 3.13. Effect of sub-lethal dose of Bacilli/toxin on the survival and fecundity of F1 generation of mosquito species 4. 4. RESULTS 64 - 153 4.1. Sampling, Isolation and Screening of soil samples for mosquito-pathogenic bacilli 4.2. Maintenance of Mosquito Colony 4.3. Preliminary toxicity testing of Bacillus isolates 4.4. Morphological and biochemical characterization of new isolates and reference strains i) Colony characteristics ii) Gram staining and endospore staining iii) SEM imaging iv) A. Biochemical characterization B. Antibiotic sensitivity tests v) Bio surfactant production 4.5. Crude protein extraction and determination of concentration by Nano drop technique 4.6. i) Molecular identification of the new isolates ii) Phylogenetic analysis 4.7. Analysis of Culture supernatant and Crude Mosquitocidal Toxin (CMT) for larvicdal and pupicidal activity A) Culture supernatant i) Larvicidal activity ii) Pupicidal activity B) Crude Mosquitocidal Toxin (CMT) i) Larvicidal activity ii) Pupicidal activity 4.8. Toxin Studies 4.9. i) Main Bioassay tests ii) Mode of action of the larvicidal bacteria 4.10. Effect of sub-lethal dose of the bacilli/toxin 5. 5. DISCUSSION 154 - 167 6. 6. SUMMARY AND CONCLUSIONS 168 - 171 7. 7. BIBLIOGRAPHY 172 - 191 8. 8. APPENDIX 192 – 197 9. 9. PUBLICATIONS AND PRESENTATIONS 198 - 199 LIST OF FIGURES FIGURE NO. FIGURE 1 Major mosquito species and the disease transmitted 2 Depicts the three subfamilies of mosquitoes 3 Life cycle of mosquitoes 4 Life cycle of Plasmodium in mosquito and humans 5A Diagrammatic representation of Bti spore crystal complex 5B Diagrammatic representation of protein inclusions which are characteristic of Bacillus thuringiensis israelensis 6A Diagrammatic representation of mode of action of Bti toxin 6B Cartoon showing death of mosquito larvae due to toxin action of bio- larvicide 7 Sampling sites 8 Flowchart of cyclic maintenance of mosquito colony 9 Scaling – up the bacterial cultures 10 Main Bioassays for testing mosquito pathogenic activity 11 Pictorial representation of the protocol to study effect of sublethal dose on the test vector species 12 Ribandar Salt pan Sampling site 13 Miramar beach sampling site 14 Screening of soil sample using Method 1 using Nutrient broth (NB) 15 Screening of soil sample using Method 2 using Nutrient Yeast Sporulating Medium (NYSM) 16 Screening of soil sample using Method 3 (Dhindsa et al., 2002) 17 Curca Mangrove and Salt pan sampling site 18 Screening of soil samples by Method # 2 using Nutrient Yeast Sporulating Medium (NYSM) and testing against mosquito larvae 19 Screening of soil samples by Method # 3 (Dhindsa et al., 2002) and testing against mosquito larvae 20 Preliminary activity of the Isolates when grown in different media for 96 hours against A: 3rd instar Anopheles stephensi larvae B: 3rd instar Culex quinquefasciatus larvae C: 3rd instar Aedes aegypti larvae on 24 hours of treatment 21 Percentage mortality caused by the isolates (BGUMS14, BGUMS16, BGUMS101) to the 3rd instar Anopheles stephensi larvae following varied time of incubation (24-144 hrs.) in NYSM 22 Percentage mortality caused by the isolates (BGUMS14, BGUMS16, BGUMS101) to the 3rd instar Culex quinquefasciatus larvae following varied time of incubation (24-144 hrs.) in NYSM 23 Percentage mortality caused by the isolates (BGUMS14, BGUMS16, BGUMS101) to the 3rd instar Aedes aegypti larvae following varied time of incubation (24-144 hrs.) in NYSM 24 Percentage mortality of the vector species with Isolate BGUMS14 at varying salt concentrations 25 Percentage mortality of the vector species with Isolate BGUMS16 at varying salt concentrations 26 Percentage mortality of the vector species with Isolate BGUMS101 at varying salt concentrations rd. 27 Preliminary screening results following 24 hours of treatment of 3 instar larvae of the 4 test vector species with the Isolates rd.