A STUDY ON ISOLATION, CHARACTERISATION AND EFFICACY OF NATURALLY OCCURRING PATHOGENIC BACILLI FROM GOA,

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 () 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 larvae B: 3rd instar Culex quinquefasciatus larvae C: 3rd instar 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. 28 Preliminary screening results following 48 hours of treatment of 3 instar larvae of the 4 test vector species with the Isolates

29 Preliminary screening of the isolates against 3rd instar larvae of Anopheles stephensi

30 Preliminary screening of the isolates against 3rd instar Culex quinquefasciatus larvae

31 Preliminary screening of the isolates against 3rd instar Aedes aegypti larvae 32 Preliminary screening of the isolates against 3rd instar larvae

33 Scanning Electronic Micrographs of the active bacterial isolates

34 Production of a bio-surfactant by Isolate BGUMS101

35 Gel image of PCR amplification using 16S rRNA primers

36 Gel image of PCR amplification using 16S rRNA primers

37 Phylogenetic tree showing the phylogenetic relationships among isolate BGUMS14, BGUMS16, BGUMS101, and their close relatives inferred from 16S rRNA gene sequences from NCBI GenBank. 38 Phylogenetic tree showing the phylogenetic relationships among isolate BIC, C1A, C2A, C2C and their close relatives inferred from 16S rRNA gene sequences from NCBI GenBank. 39 Preliminary screening of culture supernatant of Bacillus sp. against 3rd instar Anopheles stephensi larvae 40 Preliminary screening of culture supernatant of Bacillus sp. against 3rd instar Culex quinquefasciatus larvae 41 Preliminary screening of culture supernatant of Bacillus sp. against 3rd instar Aedes aegypti larvae 42 Preliminary screening of culture supernatant of Bacillus sp. against Anopheles stephensi pupae 43 Preliminary screening of culture supernatant of Bacillus sp. against Culex quinquefasciatus pupae 44 Preliminary screening of culture supernatant of Bacillus sp. against Aedes aegypti pupae 45 Effect of the Crude mosquitocidal toxin (CMT) of the isolates against Anopheles stephensi pupae 46 Effect of the Crude mosquitocidal toxin (CMT) of the isolates against Culex quinquefasciatus pupae 47 Effect of the Crude mosquitocidal toxin (CMT) of the isolates against Aedes aegypti pupae 48 Showing the effect of crude mosquitocidal toxin (CMT) of Isolate 49 101 against the pupae of Anopheles stephensi, Culex 50 quinquefasciatus and Aedes spp. 51 Detection of Cry2(UN) gene 52 Detection of Cry4 (UN) gene 53 Detection of Cry1 (UN) gene 54 Detection of Cyt 2 (UN) gene 55 Detection of BinA gene 56 Detection of BinB gene 57 Detection of MtX toxin gene 58 Detection of Gyrase toxin gene 59 Detection of Surfactin toxin gene 60 61 62 Depicting the death of larvae due to midgut lysis 63 64 Mean number of eggs laid and hatched per treated and control Anopheles stephensi female during each gonotrophic cycle 65 Kaplan-Meier survival curves of the test and control F1 generation Anopheles stephensi females 66 Kaplan-Meier survival curves of the test and control F1 generation Anopheles stephensi males 67A Larval development (L1-L4), pupal formation and adult 67B emergence in the control and treated population of Anopheles stephensi mosquitoes 68 Mean number of eggs laid and hatched per treated and control Aedes aegypti female during each gonotrophic cycle. 69 Kaplan-Meier survival curves of the test and control F1 generation Aedes aegypti females

71A F1 Larval development (L1-L4), pupal formation and adult 71B emergence in the control and treated Aedes aegypti mosquitoes 72 Mean number of eggs hatched per treated and control Culex quinquefasciatus female during each gonotrophic cycle 73 Kaplan-Meier survival curves of the test and control F1 generation Culex quinquefasciatus females 74 Kaplan-Meier survival curves of the test and control F1 generation Aedes aegypti males

75A F1 Larval development (L1-L4), Pupal formation and adult 75B emergence in the control and treated Culex quinquefasciatus mosquitoes

LIST OF TABLES

TABLE NO. TITLE 1 Classification of Mosquitoes 2 Overview of different bio-control agents, strains, activity profile against target species, toxin genes and strain modifications with recombinant technology 3 Lists the available commercial bio-larvicide formulations, their type, potency and field evaluation of these formulations 4 List of primers used for amplification of toxin genes 5 Morphological and Biochemical characteristics of the Ribandar isolates

6 Morphological and Biochemical characteristics of the Miramar isolates

7 Antibiotic sensitivity tests 8 Bio-surfactant production 9 Determination of protein concentrations (mg/ml) using Nanodrop technique 10 Larvicidal activity (% mortality) of Crude mosquitocidal toxin against 3rd instar larvae of Culex quinquefasciatus, Anopheles stephensi, Aedes aegypti and Aedes albopictus. 11 Toxin gene detection by PCR based methods 12 Larvicidal activity in parts per million (ppm) of newly isolated Bacillus spp. against 3rd instar larvae of Anopheles stephensi, Culex quinquefasciatus, Aedes aegypti and Aedes albopictus. 13 Larvicidal activity in parts per million (ppm) of newly isolated Bacillus spp. against 3rd instar larvae of Anopheles stephensi, Culex quinquefasciatus, Aedes aegypti and Aedes albopictus. 14 A comparison of egg hatch, pupation and adult emergence rates in Test and Control population of Anopheles stephensi

14A Supplementary table indicating pupation rate and adult emergence rate from egg and from 1st instar larvae of Anopheles stephens respectively 15 A comparison of Fecundity (oviposition) of Anopheles stephensi F1 females in test and control groups

16 Hatchability of eggs produced by F1 Anopheles stephensi females during different gonotrophic cycles 16A Supplementary table of overall egg hatchability in Control vs. Test population of Anopheles stephensi 16B Supplementary table taking number of females of Anopheles stephensi in control and test into account 17A Life span of the F1 generation Anopheles stephensi test female mosquitoes 17B Life span of the F1 generation Anopheles stephensi test male mosquitoes

17C Life span of the F1 generation Anopheles stephensi control female mosquitoes

17D Life span of the F1 generation Anopheles stephensi control male mosquitoes

18 Supplementary table depicting a comparison of mean survival time of test and control females and males of Anopheles stephensi 19 A comparison of egg hatch, Pupation and Adult emergence Rates in Test and Control of Aedes aegypti

19A Supplementary Table indicating Pupation rate and adult emergence rate from egg and from 1st instar larvae of Aedes aegypti respectively

20 A comparison of Fecundity (oviposition) of Aedes aegypti F1 females in test and control groups

21 Hatchability of eggs produced by F1 Aedes aegypti females during different gonotrophic cycles

21A Supplementary table of overall egg hatchability in Control vs. Test population of Aedes aegypti

21B Supplementary table taking number of females of Aedes aegypti in control and test into account 22A Life span of the F1 generation Aedes aegypti Test female mosquitoes

22B Life span of the F1 generation Aedes aegypti Test male mosquitoes

22C Life span of the F1 generation Aedes aegypti control female mosquitoes

22D Life span of the F1 generation Aedes aegypti control male mosquitoes

23 A comparison of mean survival time of test and control females and males of Aedes aegypti 24 A comparison of egg hatch, pupation and Adult emergence rates in Test and Control population of Culex quinquefasciatus

24A Supplementary Table indicating Pupation rate and adult emergence rate from egg and from 1st instar larvae of Culex quinquefasciatus respectively 25 A comparison of Fecundity (oviposition) of Culex quinquefasciatus F1 females in test and control groups 26 Hatchability of eggs produced by F1 Culex quinquefasciatus females during different gonotrophic cycles 26A Supplementary table of ooverall egg hatchability in Control vs. Test population of Culex quinquefasciatus 26B Supplementary table taking number of females of Culex quinquefasciatus in control and test into account 27A Life span of the F1 generation Culex quinquefasciatus Test female mosquitoes

27B Life span of the F1 generation Culex quinquefasciatus Test male mosquitoes 27C Life span of the F1 generation Culex quinquefasciatus control female mosquitoes 27D Life span of the F1 generation Culex quinquefasciatus control male mosquitoes

28 A comparison of mean survival time of test and control females and males of Culex quinquefasciatus

CHAPTER 1: INTRODUCTION

1.1 GENERAL INTRODUCTION ON UNDERSTANDING THE MOSQUITOES

Mosquitoes are which constitute a group of about 3,500 species in the family Culicidae, Order Diptera in Class Insecta and Phylum Arthropoda (Table 1). The word "mosquito" comes from two Spanish words (formed by mosca and diminutive -ito) meaning little [1]. Mosquitoes have a slender body with segments, a pair of wings (the second pair is modified in to balancing organs, halteres), three pairs of long hair like legs, feathery antennae (especially of the males), and elongated mouthparts adapted to probing and sucking [2].

The mosquito life cycle comprises of 4 distinct stages viz., egg, larva, pupae and adult stages. Eggs are mainly laid on the surface of water, which hatch into motile larvae. These larvae feed on aquatic algae and other organic material. Larvae give rise to a non-feeding pupal stage in which adult mosquito develops and emerge in 2 - 3 days at an ambient temperature of 27o C. The adult female mosquitoes of most species have a proboscis which is a tube-like mouth-part. This is used to pierce the skin of a host and feed on blood, which contains components such as protein and iron needed to produce and nourish the eggs [3].

During the bite the mosquito's saliva is transferred to the host, this can cause an itchy rash. In addition, many species of mosquitoes can also ingest pathogens while biting an infected human host which is then transmitted further during a future blood meal leading to mosquitoes being important vectors of diseases such as malaria, yellow fever, Chikungunya, West Nile, dengue fever, filariasis, Zika and other arboviruses [4].

Table 1: Classification of Mosquitoes

Phylum Arthropoda Subclass Pterygota Division Endopterygota Order Diptera Subdivision Superfamily Family Culicidae Subfamily Anophelinae/ / Toxorhynchitinae

1

These mosquito-borne diseases affect over 100 countries in tropical and subtropical regions of the world afflicting not only humans but also [5]. Major diseases transmitted by these vectors are depicted below (Fig. 1).

Anopheles spp. Culex spp. Aedes spp. Mansonia spp. Malaria Filariasis, West Dengue, Chikungunya, Zika and Filariasis Nile virus and Yellow Fever Japanese encephalitis

Fig. 1: Major mosquito species and the diseases transmitted by them

All mosquitoes require water to complete their life cycle. Mosquitoes inhabit rain puddles and ponds, decomposing material such as wet leaf matter, ditches and marshes, drains, cesspits, septic tanks, cesspools, lakes, rice field, grassy lands, rain pools, wells, curing water, masonry tanks, fountains, tires barrels, coconut shells, buckets, small containers, etc. [6].

Some species of mosquitoes can fly far from their breeding sites, making prevention difficult. However, certain mosquitoes are considered domestic species because they breed around the homes in small, artificial containers such as bird baths and eave troughs. Most mosquitoes are considered a nuisance because the females consume blood from living vertebrates including humans thus affecting quality of life [7].

2

There are 3 subfamilies of the family Culicidae (Fig. 2) which can be distinguished as follows:

1. Anophelinae: Adult females are blood feeders with the maxillary palps of both sexes as long or nearly as long as the proboscis and the dorsal scutellum evenly rounded or strap like. Adults rest with their abdomens tilted forwards at 45° to the surface, larvae have no siphon and each egg has floats [8].

2. Culicinae: The females are blood-feeders with the palps less than half as long as the proboscis and the scutellum is tri-lobed. Adults rest with abdomens about parallel to the surface; larvae have a prominent siphon and the eggs that are deposited as rafts lack floats. Those species that are of veterinary importance all belong to this subfamily [8].

3. Toxorhynchitinae: The adult females do not feed on blood but only on nectar and other plant juices. They are unusually large mosquitoes with metallic colored scales and the proboscis is strongly curved downwards. The larvae are predaceous and feed on other mosquito larvae hence they are natural biological control of mosquitoes especially in the forest areas where species generally abound [8].

3

Fig. 2: Depicts the three subfamilies of mosquitoes [9]

4

1.2. LIFE CYCLE OF MOSQUITO

The life cycle of a mosquito has four distinct stages: Egg, Larva, Pupa, and Adult (Fig. 3)

1.2.1 Egg

Some species of mosquitoes, like Culex quinquefasciatus (common house mosquito), lay their eggs on the surface of stagnant water. Eggs can be laid anywhere in the stagnant water i.e. in cans, bird baths, ditches or puddles, stream margins, man-made habitats such as wells, lintels with water, curing waters, overhead tanks, barrels, discarded tires, rice fields, canals, borrow pits, etc. Mosquitoes prefer water surfaces protected from wind. Some mosquitoes can breed in as little as one centimeter of standing water. Culex mosquitoes usually lay their eggs at night and they stick together due to a special structure on the sides of the eggs and form a raft approximately 64 mm long and 38 mm wide [10], which may contain 100 to 300 eggs.

Anopheles species lay their eggs singly on the water surface. Anopheles eggs have lateral air filled floats. Aedes mosquitoes also lay their eggs singly, and most species select damp soil or foliage touching water.

Aedes eggs have spongy chorion are more resistant to drying out than those of other genera and require low temperatures before the eggs will hatch. Others hatch only when flooded with water above a critical temperature [10].

1.2.2 Larvae

Mosquito larvae resemble little wiggling worms, hence the nickname ‘wigglers.’ At this stage they have no legs or wings. All stages breathe air through spiracle openings as they float on the surface of the water as in case of Anophelines or pierce water film though air tube called siphon in case of Aedes species. They dive down to feed or evade capture. In optimal conditions, they grow very quickly in the larval stage, molting four times over the 6 - 12 days, until they become pupae [10].

5

1.2.3 Pupae

Mosquito pupae are also known as ‘tumblers.’ They are comma shaped and will acrobatically somersault when disturbed. At this stage the adult mosquitoes are metamorphosing and are growing wings and legs. Like larvae, pupae breathe at the surface but they use two tubes on their backs. When they have completed metamorphosis, the adults break through the pupal skin and emerge on the surface of the water [10]. This stage takes 2 - 3 days at ambient temperature 25-30o C.

1.2.4 Adults

Adult mosquitoes rest on the water surface or foliage until they can fly and then the search for food begins. The male mosquitoes are known to make swarms which attract newly emerged females for mating which is once in her entire life time as sufficient sperms are stored in their spermatheca for the entire life time to fertilize all egg batches produced over several gonotrophic cycles. The entire cycle from egg to adult can take less than 10 days for completion, depending on the temperature. A female mosquito can live for several weeks (usually 4 – 6 weeks) depending on the weather and shelter, and if she lives long enough to feed on blood more than once, she has the potential to transmit blood-borne diseases. Since the sexual phase of the malaria parasites takes place in the female mosquito host or the viral pathogens can passage and vigorously develop in mosquitoes, they are considered as their primary hosts [10].

6

Fig. 3: Life cycle of mosquitoes [11]

1.3. MAJOR MOSQUITO GENERA AND SPECIES

1.3.1 Anophelines

There are about 400 species of Anopheles in the world of which ~60 species act as malaria vectors in the world [12]. Out of 58 Anopheles species found in India, 6 species viz. Anopheles culicifacies, An. stephensi, An. minimus, An. sundaicus, An. fluviatilis and An. dirus (An. baimaii) are major vectors of malaria and 3 species viz. Anopheles annularis, An. philipinensis (nivipus) and An. varuna are secondary vectors of malaria [13]. Another species viz. An. subpictus has also been incriminated as a malaria vector in India but its role in transmission has not been established in the country although it has been recently found a vector of malaria in urban areas of Goa [14]. An. culicifacies in rural and An. stephensi in urban area are well known

7 vectors of malaria in our country. Anopheles stephensi breed in wells, overhead tank waters, rain water pools in construction sites, terrace, fountains, tires and small containers [15].

1.3.2 Culicines

(i) Aedes:

Aedes aegypti and Aedes albopictus are the main vectors that transmit viral pathogens which causes diseases like Dengue, Chikungunya and Zika. They breed in and around homes like terraces and gardens in a variety of containers viz., coconut shells, buckets, flower pots, vases, tanks, as well as outdoors like tire dumps, drums or barrels, scrap, discarded containers etc. They also breed in flower vases inside the homes [16].

(ii) Culex:

Culex, a large group of mosquitoes also known as common house mosquitoes, are the principal vectors that spread the viruses that cause West Nile fever, St. Louis encephalitis, and Japanese encephalitis, as well as viral diseases of birds and horses. Culex mosquitoes also transmit the parasitic disease, lymphatic filariasis as well as the bacterial disease, Tularaemia.

Culex mosquitoes are distributed worldwide in tropical and temperate regions, with the exception of extreme northern latitudes. They feed at night on humans and animals and are found both indoors and outdoors [17].

1.3.3 Mansonia:

Mansonia mosquitoes prefer to breed in ponds infested with water lilies and other aquatic vegetation. The larvae of Mansonia mosquitoes have long siphon specialized to pierce the spongy tissue of the plant from which they acquire oxygen. Some species of Mansonia (e.g., M. uniformis) transmit several viral diseases in addition to Brugian filariasis caused by Brugia malayi. For example, Shertalai region of Kerala was very badly affected with Mansonia which used to breed in water hyacinth infested ponds and the area was once highly endemic to Brugian filariasis. Even today, several chronic cases of filariasis can be encountered in this region [18].

8

1.4. MAJOR DISEASES CAUSED BY MOSQUITOES

1.4.1 MALARIA

Malaria remains to be the most vital cause of morbidity and mortality in India, like in many other tropical countries, with about 3-4 million new confirmed cases arising every year in 2018 and 2019 [19]. Malaria is a well-known, oldest chronic and most widespread fatal disease that has plagued mankind for centuries also resulting in economic loss and poverty. In 2018, the worldwide burden of malaria was estimated at 228 million cases (95% Confidence Interval [CI]: 206–258 million) in addition in 2018, there were an estimated 405 000 deaths from malaria globally [20].

Malaria is caused by Plasmodium sp. The discovery of the malarial parasite in human blood was made by Charles Laveran in 1880. Subsequently in 1878, Sir Ronald Ross established the role of mosquitoes in its transmission to man and was awarded Nobel Prize in the year 1902. Falciparum malaria (caused by P. falciparum) is the deadliest of the four types of human malaria, the other three being vivax malaria (P. vivax), and a rather rare and mild ovale malaria (P. ovale), and (P. malariae) and recently identified fifth species is P. knowlesi which is even deadlier than P. falciparum. It was originally identified as a parasite of monkeys and initially caused zoonotic disease, however human to human transmission was suspected in [21].

1.4.1.1 LIFE CYCLE OF THE PARASITE a. Exoerythrocytic cycle:

When an infected Anopheles mosquito bites a human, the Plasmodium parasite is inoculated into human blood in the form sporozoites which is the infective stage. These sporozoites invade the hepatocyte cells in the liver and actively feed on its cytoplasm to form the cryptozoite (Schizont) which is a large spherical form. This form multiplies into thousands of cryptomerozoites by the process of multiple fission called schizogony (exoerythrocytic schizogony). Due to the pressure of cryptomerozoites, the body of cryptozoite as well as the host liver cell ruptures releasing the cryptomerozoites into liver sinusoids. In the case of P. vivax and P. ovale, some of these merozoites remain in the liver cells to form hypnozoites, while others remain in the blood serum and invade RBC to initiate the erythrocytic cycle [22].

9 b. Erythrocytic cycle:

Once a RBC is invaded by a cryptomerozoite, it soon becomes a rounded, disc like structure called trophozoite. As it grows, a contractile vacuole appears in its center. The cytoplasm and nucleus is pushed to a thin peripheral layer and the parasite attains a ring like appearance to represent the signet ring stage. Following the disappearance of the vacuole the parasite assumes an amoeboid shape. The trophozoite actively feeds upon the hemoglobin of RBCs and increases in size till the entire corpuscle gets filled with it. This forms the schizont stage and its cytoplasm contains yellowish-brown pigment granules, the haemozoin which is formed by the decomposition of hemoglobin. The schizont undergoes asexual multiplication termed as schizogony or merogony [22].

(i) Schizogony:

The schizont divides by multiple fission to from daughter nuclei which migrate towards the periphery. After sometime the RBC bursts liberating the merozoites and the toxic waste (haemozoin granules) into the plasma of blood. These attack the fresh RBCs and repeat the erythrocytic schizogony process (one cycle is completed within 48 - 72 hours). As a result, the host becomes anemic and its toxin accumulates in the plasma. After about 2 - 5 repetitive cycles the malarial symptoms develop for the first time and the host suffers from chill and fever. This period is known as incubation period post the latent period of about 10 to 15 days since its inoculation in the host [22].

(ii) Formation of gametocytes:

Various factors such as a result of repeated schizogony in the blood stream, due to lack of fresh RBCs in the blood and the resistance from the host the parasite prepares to enter the new host by the formation of gametocytes. Some of the merozoites may form the gametocytes, after entering the RBCs as they neither form trophozoite nor multiply by binary fission but grow slowly and become compact bodies, the gametocytes. The microgametocytes (male) are small in size and with a large centrally placed nucleus. The macro or mega gametocytes (female) are less numerous but larger in size and with a greater amount of dense cytoplasm and a smaller nucleus. They reach the superficial blood vessels and wait for the bite of female Anopheles [23].

10 c. Sexual Life-Cycle in Anopheles

When Anopheles female sucks the blood of a diseased host, the parasite under different stages of development enters its alimentary canal. But only the gametocytes are able to survive, while others are digested. The gametocytes are set free by the rupture of R.B.Cs. and develop further to form gametes.

(i) Development of male gametes:

The nucleus of microgametocyte divides to form 8 haploid nuclei surrounded by a little of cytoplasm and metamorphoses into a male gamete. Each gamete consists of a small body with a nucleus and a cytoplasmic flagellum.

(ii) Development of female gametes:

The nucleus of the macro gametocyte undergoes reduction division forming two nuclei. One protrudes out as a polar body while the forms a reception cone.

(iii) Syngamy or fertilization:

The actively moving male gamete penetrates the macrogamete through the reception cone. The nuclei of the two fuses together forming the synkaryon. An inert and round zygote is formed.

(iv) Ookinete:

Soon the rounded zygote elongates, assumes vermiform appearance and becomes motile to form the vermicule or ookinete. Its anterior is pointed and with this it penetrates the stomach wall to come to lie in the sub-epithelial tissue. It now becomes rounded, secretes a thin membranous cyst to form the oocyst.

(v) Sporogony:

The nucleolus of the fully mature oocyst undergoes multiple fission (mitosis) producing a large number of daughter nuclei which get surrounded by fragments of cytoplasm. These irregular uni-nucleate bodies thus formed are known as sporoblasts which divides repeatedly by mitosis to form form spindle-shaped sporozoites. The rupture of cyst wall causes these sporozoites to

11 be liberated in the body cavity. They now move to different body organs and the salivary gland. The female Anopheles now becomes infective and is able to inoculate the parasite into the blood- stream of healthy persons [22].

The life cycle of Plasmodium in the mosquito and humans is represented in Fig. 4.

Fig. 4: Life cycle of Plasmodium in mosquito and humans [24]

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1.4.2 FILARIASIS

In India, filaria is one of the most important public health problems. It is a chronic disease and consequent social, physical and economical hazards are enormous. In India high microfilarial rates have been recorded in northern and coastal parts of Andhra Pradesh, Bihar, Tamil Nadu and Kerala and Coastal parts of Orissa and eastern parts of Uttar Pradesh. About 650 million people are exposed to the risk factors in 256 endemic districts within 21 States/ UTs [25].

There are fifty-nine species of mosquito vectors that transmit filarial parasites [26]. Among them most important vector is Cx. quinquefasciatus. Cx. quinquefasciatus is widely distributed in the tropical and subtropical latitude [26]. Increased mosquito nuisance in most urban areas is mainly because of Cx. quinquefasciatus in India. Other than Cx. quinquefasciatus, Cx. molestus and Cx. pallens are filarial vectors. An. gambiae, An. flavirostris and An. barbirostris of genus Anopheles also transmits the filaria worm. Mansonioides like M. annulifera, M. uniformis and M. indiana are other important vectors of Brugian filariasis [27].

1.4.3 DENGUE

Dengue is a mosquito-borne viral infection with symptoms including a severe flu-like illness, sometimes causing severe dengue which is a potentially lethal complication. Over the last 50 years the incidence of dengue has increased 30-fold. It is estimated that annually up to 50 - 100 million infections now occur in over 100 endemic countries, putting almost half of the world’s population at risk [28].

The dengue virus (DEN) comprises four distinct serotypes namely DEN-1, DEN-2, DEN-3 and DEN-4 which belong to the genus Flavivirus, family Flaviviridae. These dengue serotypes show extensive genetic variability with distinct genotypes being identified within each serotype. Among them “Asian” genotypes of DEN-2 and DEN-3 are frequently associated with severe disease accompanying secondary dengue infections. In the year 2018 the number of dengue cases in India reported were 101192 with the number of deaths recorded being 172 [29]. Ae. aegypti and Ae. albopictus are two well-known vectors of dengue in India. Ae. aegypti is vector in the urban area and semi-rural, which was originally introduced in India from Africa. It is well adapted to urban environment because of domestic breeding habits and its total dependence on man for a blood meal. Ae. albopictus is a vector of dengue in semi urban and rural area [30].

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1.4.4 CHIKUNGUNYA

Chikungunya is a mosquito-borne viral disease first described during an outbreak in Southern Tanzania in 1952. It is an RNA virus that belongs to the alphavirus genus of the family Togaviridae. The name “chikungunya” derives from a word in the Kimakonde language, meaning “to become contorted” describing the stooped appearance of sufferers with joint pain (arthralgia) [31].

Chikungunya has been identified in over 60 countries in Asia, Africa, Europe and the Americas. The virus is transmitted from human to human by the bites of infected female mosquitoes. Most commonly, the mosquitoes involved are Aedes aegypti and Aedes albopictus, two species which can also transmit other mosquito-borne viruses, including dengue. These mosquitoes can be found biting throughout daylight hours, though there may be peaks of activity in the early morning and late afternoon. Both species are found biting outdoors, but Ae. aegypti will also readily feed indoors. After the bite of an infected mosquito, onset of illness occurs usually between 4 and 8 days but can range from 2 to 12 days. A large outbreak of chikungunya in India occurred in 2006 and 2007. Several other countries in South-East Asia were also affected. Since 2005, India, Indonesia, Maldives, Myanmar and Thailand have reported over 1.9 million cases with 9756 confirmed cases in India in the year 2018 [32].

1.4.5 ZIKA Zika virus is a mosquito-borne flavivirus that was first identified in Uganda in 1947 in monkeys. It was later identified in humans in 1952 in Uganda and the United Republic of Tanzania. Outbreaks of Zika virus disease have been recorded in Africa, the Americas, Asia and the Pacific. From the 1960s to 1980s, rare sporadic cases of human infections were found across Africa and Asia, typically accompanied by mild illness [33]. The first recorded outbreak of Zika virus disease was reported from the Island of Yap in 2007. This was followed by a large outbreak of Zika virus infection in French Polynesia in 2013 and other countries and territories in the Pacific. In March 2015, Brazil reported a large outbreak of rash illness, soon identified as Zika virus infection, and in July 2015, found to be associated with Guillain-Barre syndrome. In October 2015, Brazil reported an association between Zika virus infection and microcephaly. Outbreaks and evidence of transmission soon appeared throughout the Americas, Africa, and other regions of the world. To date, a total of 86 countries and territories have reported evidence of mosquito-transmitted Zika

14 infection. Zika virus is primarily transmitted by the bite of an infected mosquito from the Aedes genus, mainly Aedes aegypti, in tropical and subtropical regions. Aedes mosquitoes usually bite during the day, peaking during early morning and late afternoon/evening. This is the same mosquito that transmits dengue, chikungunya and yellow fever. Zika virus is also transmitted from mother to the foetus during pregnancy, through sexual contact, transfusion of blood and blood products, and organ transplantation [33].

1.4.6 WEST NILE VIRUS

West Nile Virus (WNV) can cause neurological disease and death in people. WNV is commonly found in Africa, Europe, the Middle East, North America and West Asia. WNV is maintained in nature in a cycle involving transmission between birds and mosquitoes. Humans, horses and other mammals can be infected. West Nile Virus (WNV) is a member of the Flavivirus genus and belongs to the Japanese encephalitis antigenic complex of the family Flaviviridae. WN virus is maintained in nature in a mosquito-bird-mosquito transmission cycle. Mosquitoes of the genus Culex are generally considered the principal vectors of WNV, in particular Cx. pipiens. WNV is maintained in mosquito populations through vertical transmission (adults to eggs) [34].

1.4.7 JAPANESE ENCEPHALITIS Japanese encephalitis (JE) is the main cause of viral encephalitis in many countries of Asia. The JE virus is a flavivirus related to dengue, yellow fever and West Nile viruses. The virus exists in a transmission cycle between mosquitoes, pigs and/or water birds Egrets and Paddy Herons. Humans get infected when bitten by an infected mosquito. The disease is predominantly found in rural and peri urban settings. Most JE virus infections are mild (fever and headache) or without apparent symptoms, but approximately 1 in 200 infections results in severe disease characterized by rapid onset of high fever, headache, neck stiffness, disorientation, coma, seizures, spastic paralysis and death. The case fatality rate can be as high as 30% among those with disease symptoms [35]. JEV is generally spread by mosquitoes, specifically those of the Culex type. Five Culex mosquito species are considered as the most prominent species playing a significant role

15 during outbreaks of human JE. They are Culex tritaeniorhynchus, Culex gelidus, Culex pseudovishnui, Culex vishnui and Culex fuscocephala [36].

1.4.8 YELLOW FEVER Yellow fever is caused by a virus (Flavivirus) which is transmitted to humans by the bites of infected Aedes and Haemogogus mosquitoes. Breeding habitats of the mosquito includes around houses (domestic), in forests or jungles (wild), or in both habitats (semi-domestic) [37].

There are 3 types of transmission cycles:

1. Sylvatic (or jungle) yellow fever: Monkeys, which are the primary reservoir of yellow fever, are bitten by wild mosquitoes which pass the virus on to other monkeys. Occasionally humans working or travelling in the forest are bitten by infected mosquitoes and develop yellow fever.

2. Intermediate yellow fever: In this type of transmission, semi-domestic mosquitoes (those that breed both in the wild and around households) infect both monkeys and people. Increased contact between people and infected mosquitoes leads to increased transmission and many separate villages in an area can develop outbreaks at the same time. This type of outbreak in most commonly seen in Africa.

3. Urban yellow fever: People infected with yellow fever may introduce the virus into heavily populated areas with high mosquito density and where most people have little or no immunity, due to lack of vaccination. This could lead to the occurrence of large pandemics. In these conditions, the virus may be transmitted from person to person by the infected mosquito. Once contracted, the incubation period in the body is 3 to 6 days. The most common symptoms of Yellow fever include fever, muscle pain with prominent backache, headache, loss of appetite, and nausea or vomiting. However it may be asymptomatic in may people. A small percentage of patients, may enter a second, more toxic phase within 24 hours of

16 recovering from initial symptoms. High fever returns body systems, usually the liver and the kidneys are affected. In this phase jaundice (yellowing of the skin and eyes, hence the name ‘yellow fever’) may develop, with additional conditions such as dark urine and abdominal pain with vomiting as well as bleeding from the mouth, nose, eyes or stomach. Half of the patients who enter the toxic phase die within 7 - 10 days.

The main methods to prevent Yellow fever is through vaccination and mosquito control. The yellow fever vaccine is safe and affordable, with a single dose providing life-long immunity against the disease. In situations where vaccination coverage is low or the vaccine is not immediately available mosquito control to prevent yellow fever, is vital [38].

Mosquitoes kill millions of people being vectors hosting many different parasites and pathogens. Hence the need of the hour is to control of these diseases which is possible either by curbing the causative organism or their mosquito vectors. Normally in India anti-parasitic and anti-vector measures are taken in the case of malaria and filariasis throughout the country as per the guidelines of Govt. of India framed by experts and implemented by the NVBDCP. There is much greater emphasis on vector control in order to keep the vector borne disease under check and many tools are selectively deployed. Initially chemical based methods were tried, which led to enormous amount of damage to the environment and created resistance in mosquitoes. This led to the discovery of the biological control methods. New discoveries are needed to provide newer tools for mosquito control.

1.5 CONTROL OF MOSQUITOES

1.5.1 Environment Management

It is the modification of the topography of the area to prevent stagnation of water and enable proper drainage, filling up of landfills, use of expanded polystyrene beads in disused wells, mosquito-proofing of water tanks, etc.

1.5.2 Chemical control

The traditional method of control was the use of insecticides namely pyrethroids, DDT, Permethrin, Temephos, Fenthion and Malathion, which may be sprayed or added to water bodies etc. Widespread use of chemical formulations through highly effective, rapid,

17 adaptable, economical and reliable have caused the subsequent evolution of resistant species as has been reported [39].

The long-term detrimental effects to non-target populations such as honey bees and other insects, and growing concerns about their bioaccumulation in the environment and bio- magnification in the food chain has necessitated the development and use of newer, healthy alternatives. The intensive use of chemical products for controlling insects through the years has confirmed their negative impact on the environment. This can be observed by the seriously damaged natural conditions as well as by the increased resistance to these products by populations [40].

The most successful current vector control strategies are indoor spraying and use of insecticide treated nets (ITN’s)/long lasting insecticide nets (LLINs). Recent introduction of safer vector control agents such as insect growth regulators (IGRs) which are both safe and long-lasting (e.g., pyriproxyfen) and other bio control agents have yet to get the needed scale of utility for vector control [48].

1.5.3 Biological control

The biological and natural control by nature in the form of predators and parasites maintains a status quo or ecological balance amongst all living creatures, this can be exploited by man to control vector populations. There exists literature on entomopathogenous fungi which may be potential bio control agents of mosquito larvae and these include Coelomyces, Culicinomyces and Lagenidium which is a water mold that parasitizes the larval stage of mosquitoes via a motile spore [41]. The predaceous mosquito (Toxorhynchites splendens) is used to control larval populations of Aedes aegypti although this method has limitation of scale of deployment [42].

The tadpole shrimp (Triops spp.), and the mud loach (Misgurnus mizolepis) have been used successfully to reduce populations of larval mosquitoes in temporary aquatic habitats such as ponds along with the use of fishes like Catla catla, Gambusia affinis which have been found to have a high larvivorous potential [43]. Aplocheilus blockii was found to be a good candidate for the control of Anopheles stephensi in wells [44]. There are several other species of top minnows which are predatory to mosquito larvae e.g., Lebistes reticulatus, Aplocheilus panchax, Aplocheilus lineatus, Danio brachydanio rario, Aphanius dispar, etc. Other predators of mosquito larvae include dragonfly naiads, water skaters and adult

18 dragonflies, which eat adult mosquitoes; frogs and tadpoles also effective destroy adult mosquitoes and larvae respectively [45].

The use of entomopathogenic bacteria has been consolidating itself as one of the effective and promising tools in the integrated control of vectors [46]. An important and relevant aspect of biological control is the use of microorganisms as control agents which are environmentally friendly and highly specific to the target organism and these have been successfully deployed against both agricultural pests as well as mosquitoes. Bacterial strains such as Bacillus alvei and Bacillus brevis, as well as Bacillus circulans, Brevibacillus, and Bacillus subtilis have also been reported to produce mosquitocidal toxins [47].

The current biological control strategy in use worldwide is the use of Bacillus sphaericus and Bacillus thuringiensis as larvicidal bacteria for mosquito control [48]. Their use has been widely documented with over three decades of research in the field, insecticidal activity is due to the production of crystal toxins associated with sporulation and vegetative growth [49].

(i) Bacillus thuringiensis israelensis:

Bacillus thuringiensis (or Bt) is a Gram-positive, soil-dwelling bacterium. It is the most commonly used biological pesticide with the subspecies israelensis widely used for the control of mosquitoes. Bacillus thuringiensis crystals have the largest spectrum of activity covering larvae of the genera Culex, Aedes, and Anopheles belonging to family Culicidae and also to members of family Simuliidae [50]. They have no effect on vertebrates and non- target invertebrates which is the most promising factor being exploited for their use.

(ii) Lysinibacillus sphaericus (reclassified - previously known as Bacillus sphaericus):

It is a Gram-positive, mesophilic, rod commonly found in soil. Some strains of L. sphaericus are known to target mosquitoes by the production of Bin (Binary) toxins which are the most potent toxin produced during sporulation. It is composed of two proteins of 51.4kDa and 41.9kDa respectively that are co crystallized during sporulation. Certain strains of Lysinibacillus sphaericus can synthesize mosquito larvicidal proteins of 100kDa and 30.8kDa (Mtx toxins) during vegetative growth. Highly mosquitocidal strains such as Bacillus sphaericus 2297 and 2362 produce both Bin and Mtx toxins [51].

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Various aspects such as natural sources of mosquito pathogenic bacteria, their isolation and characterization, Bacillus species used as bio-control agents and their mode of action, the currently available formulations, vector targets as well as the future prospects of Bacilli based vector control will be dealt with in Chapter 2.

1.6 SIGNIFICANCE OF THE THESIS

Although a variety of mosquito pathogenic bacteria have been isolated from various geographic zones around the world, there is a pressing need to explore and deploy indigenous strains in vector control programs due to restrictions imposed on the use of imported strains and the prohibitive cost of imported formulations. In addition, efforts are on to isolate more potent bacterial agents from nature locally which could be developed into an ideal biolarvicide having broad spectrum of activity at very low concentrations without developing resistance in the target mosquito species

In view of the above, the following objectives were proposed for study:

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OBJECTIVES

1. To isolate, screen, identify and characterize new promising isolates of mosquito pathogenic Bacillus spp. from Goa. 2. To determine larvicidal potential (dose-mortality response) of new isolates against 4 mosquito vector species viz., Anopheles stephensi (vector of Malaria), Culex quinquefasciatus (vector of Filariasis), Aedes aegypti and Aedes albopictus (vectors of Dengue and Chikungunya) 3. To carry out main bioassays with promising new isolates against four test vector species 4. To characterize larvicidal toxins and study 16S rRNA profiles of the new isolates 5. To study the effect of sub-lethal doses of bacilli/toxins on fecundity and survival of F1 generation of vector species.

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CHAPTER 2: REVIEW OF LITERATURE

2.1 BACKGROUND

Mosquitoes are associated with nuisance biting and transmission of pathogens to humans and other vertebrates resulting in significant morbidity and mortality due to the difficulty of controlling them [52]. The most important disease vectors belong to the subfamily Anophelinae (Anopheles mosquitoes) which transmits malaria; Culicinae i.e. Culex species transmit filariasis, West Nile virus, Japanese encephalitis and Aedes mosquitoes which primarily transmit dengue, chikungunya, yellow fever and Zika. These diseases account for > 17% of all infectious diseases, causing 7, 00,000 deaths annually with 80% of the population of the world in both developed as well as developing countries at risk of one or more vector-borne diseases [53]. In recent years, changes in public health policy and social factors as well as reports of resistance in both vector mosquitoes and the pathogens transmitted by them has caused a resurgence in the incidence of mosquito borne diseases [54].

2.2 MOSQUITO VECTOR CONTROL IN INDIA Vector control is a key strategy to control these diseases. In India, vector control is primarily based on the use of LLINs i.e., Long-lasting insecticide treated nets in addition to indoor residual spraying (IRS) of insecticides in rural areas along with anti-larval operations are practiced in urban areas [55]. These guidelines of integrated management of vectors have been framed and advocated by the National Vector Borne Diseases Control Programme [18]. Larval control may be particularly valuable in regions where the eradication or elimination of vector borne diseases is being targeted, by reduction of mosquito larval populations before emergence to the adult stage [56].

However, with regards to mosquito control strategies, chemical control agents still play a major role. Insecticides applied with the aim of eliminating mosquitoes have given rise to other serious problems [57]. Not only have mosquitoes developed resistance, but these insecticides also pose threat to humans, animals and the ecosystem as a whole. Chemical insecticide exposure among humans has been linked to immune dysfunction, neurological disorders, and various forms of cancer, birth defects, liver damage and infertility [58]. These ill effects have led to the discovery of alternatives to these insecticides. The utilization of microbial control agents is a proven method effective against mosquito immature of both Anophelines and Culicines. Commercial biolarvicide formulations of gram positive and

22 spore forming bacteria, Bacillus thuringiensis israelensis and Bacillus sphaericus are now being widely used across the globe in the vector control programmes. These strains have been well characterized both at the microbiological and molecular level. Based on these two Bacilli, there are several effective and well tested formulations commercially available including the wettable powder, slow-release granules, briquettes, tablets and emulsifiable concentrates. These formulations are often deployed as an integral components of the integrated vector management (IVM) strategy advocated by the World Health Organization and adopted by vector borne disease endemic countries.

2.3 BACILLI AS BIO-CONTROL AGENTS OF MOSQUITOES

In nature, a wide variety of organisms including viruses, protozoans, fungi and bacteria, effectively control mosquitoes [59]. Among the many bacteria that have been tested, strains of Bacillus thuringiensis (Bti) and Bacillus sphaericus (Bs) are most promising for vector control so far. Bacillus thuringiensis var. israelensis has an advantage of a broader host range. While Bacillus sphaericus has a narrow spectrum, its advantage is increased duration of larvicidal activity against specific mosquito species like Culex quinquefasciatus and recycling ability within mosquito cadavers [60]. There are options available for ‘stand-alone’ and combined formulations of these two Bacilli species and their strains for the vector control programmes.

2.3.1 Bacillus thuringiensis

Bacillus thuringiensis is a ubiquitous, sporulating aerobic bacterium which can be easily grown and cultured on routinely used media like nutrient agar. It can be isolated from a variety of sources [61]. On sporulation it produces two types of insecticidal crystal proteins or δ-endotoxins, Cry (for crystal) and Cyt (for cytolytic) proteins as seen in [Figure 5A], [Figure 5B] and further variations of each of these types. Cry proteins target lepidopteran insects, while few are toxic to dipteran or coleopteran insects. Cyt proteins show moderate toxicity to mosquito and black fly larvae occurring mostly in mosquitocidal subspecies e.g. B. thuringiensis subsp. israelensis [62].

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Fig. 5A- Diagrammatic representation of Bti spore crystal complex (Joleen Almeida©)

Fig. 5B- Diagrammatic representation of protein inclusions which are characteristic of Bacillus thuringiensis israelensis (Joleen Almeida©)

Cyt proteins have been studied less in comparison to Cry proteins. These proteins based primarily on studies of Cyt1Aa, are extremely important in the biology of mosquitocidal strains as they synergize mosquitocidal Cry proteins, such as Cry4Aa, Cry4Ba, and Cry11A, and delay the phenotypic expression of resistance as it would require multiple mutations at different loci. [63] The high degree of host specificity and the complexity of Bacillus thuringiensis mode of action, results from the interaction of the toxin within the complex environment of the insect’s midgut lumen and on the surface of the midgut epithelial cells. Although researchers discovered relatively early that the midgut was the primary site of δ-endotoxin activity as seen in (Fig. 6A), (Fig. 6B), the molecular mechanisms of Bt intoxication has continued to be the subject of intensive research. [64]

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Fig. 6A- Diagrammatic representation of mode of action of Bti toxin (Joleen Almeida©)

25

Fig. 6B - Cartoon showing death of mosquito larvae due to toxin action of biolarvicide (adapted from Dhindsa K., 2001)

Although Bti is proven to be effective against many mosquito species, operational application showed that it is more suitable against Aedes sp. such as Aedes aegypti and Ae. albopictus in comparison to other vector species [65]. More advantage for Aedes control may be due to their feeding behaviour, as most of the Bti toxins sediment to the base of the container during treatment where Aedes larvae frequently feed [66]. On the other hand, Anopheles balabacensis and Mansonia (Mansonioides) indiana were found comparatively less susceptible to the Bti (H-14) [67]. High susceptibility to B. thuringiensis H-14 was also shown by Cx. quinquefasciatus [68]. However, the latter species is more susceptible to B. sphaericus, being more effective in polluted water with high organic contents where Cx. quinquefasciatus prefers to breed as B. sphaericus has the ability to recycle in polluted water and persists longer than B. thuringiensis H-14 [69].

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2.3.2 Bacillus sphaericus

Bacillus sphaericus is a common soil bacterium with a few entomopathogenic strains. It is aerobic, rod-shaped and endospore forming. Kellen et al in 1965 reported the first discovery of a strain toxic to mosquito larvae [70]. The biolarvicidal activity of B. sphaericus is due to the presence of two unique binary proteins BinA (42 kDa) and BinB (51kDa). These binary proteins are cleaved by mosquito gut proteases, forming the active toxin by yielding peptides of 39 kDa and 43 kDa respectively. This associate and bind to the α-glucosidase receptor located on the midgut microvilli, resulting in lysis of midgut cells after ingestion [71], [72]. It is suspected that reported loss of toxicity i.e., resistance in target mosquito species to B. sphaericus may be due to the reduction or loss of interactions between BinA and BinB or BinB and its receptor [73]. In addition, another 100 kDa mosquitocidal protein appears to be synthesized but it is less toxic besides the production of some highly toxic proteins during the vegetative phase of growth [74].

2.3.3 Bacillus subtilis A strain of Bacillus subtilis producing a mosquitocidal toxin was reported to show significant larvicidal and pupicidal activity against three species of mosquitoes viz., Anopheles stephensi, Culex quinquefasciatus and Aedes aegypti. This strain was isolated from mangrove forests of Andaman and Nicobar Islands of India. It is the first Gram-positive bacterium reported for its high toxicity to mosquito pupae [75]. Its mosquitocidal activity is associated with a cyclic lipopeptide exotoxin identified as surfactin. It is highly active at both acidic and basic pH, temperature range of 25˚C - 42˚C, UV stable and non-toxic to mammals, suitable features for the development of a biolarvicide [76].

The overview of different bio-control agents, their strains and activity profile against target species, toxin genes and strain modifications with recombinant technology is shown in Table 2.

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Table 2: Overview of different bio-control agents, their strain, activity profile against target species, toxin genes and strain modifications with recombinant technology

Sr. Bacteria reported Strain Activity against Toxin gene Recombinant Reference No. mosquito species identified technology 1) Bacillus israelensis Ae. aegypti, Cx. Cry (crystal) 130 kDa toxin [77 - 79] thuringiensis (Bti) H-14 quinquefasciatus, and Cyt from Bti and VCRC An. stephensi, Ae. (cytolytic) introduced into B17 nigromaculis, An. proteins plasmid quadrimaculatus, pRK248 Cx. tarsalis expressed Caulobacter crescentus CB15. kurstaki (Btk) Ae. aegypti Cry protein NK [79] morrisoni NK Cry protein NK [80] jegathesan An. stephensi, Cry protein NK [81] (Btj) Cx. pipiens, (Cry19Aa1, Ae. aegypti Cry11Ba1) medellin Ae. aegypti, An. Cry protein NK [82] albimanus, Cx. (Cry11Bb1, quinquefasciatus Cyt1Ab1) higo Cx. pipiens Cry protein NK [83] (Cry 21Aa1) fukuokaensis Ae. aegypti Cry protein NK [83] (Cry 20Aa1) kyushuensis Ae. aegypti, An. Cry protein NK [83] stephensi, Cx. (Cyt2Aa1) pipiens tochigiensis Ae. aegypti, Cx. Cry protein NK [83] quinquefasciatus 2) Brevibacillus strains 921 Ae. aegypti, Toxic factors Enhanced [84 - 87] laterosporus and 615 likely to be larvicidal

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An. stephensi proteins of activity by bio- and Cx. pipiens. about 14 kDa encapsulation in Protozoa. 3) Bacillus neide H5a5b Culex sp., Binary (Bin) 1) Use of [88-91] sphaericus and VCRC Anopheles sp., toxins of 42 cyt1A B42 Aedes sp. (BinA) and promoters + 51(BinB) mRNA kDa+ MtX stabilizing (mosquitocid sequence to al) toxins synthesize high levels of Bs binary toxin in Bti strains. 2) Cry4Ba and cyt1Aa genes expressed in Bs2362 produced stable transformants 10 times more toxic to Aedes larvae than the host strain. 4) Clostridium malaysia An. sp., Ae. Toxic extract Toxin [92] bifermentans detritus, Ae. contains three encoding gene caspius, Ae. major cloned and aegypti, Cx. proteins of induced to be pipiens, blackfly 66, 18 and 16 expressed by larvae kDa transformed Bt, exhibited toxicity against mosquitoes.

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5) Bacillus alvei NK Culiseta NK NK [93] longiareolata 6) Bacillus brevis NK Culiseta NK NK [93] longiareolata 7) Bacillus circulans NK Cx. Toxicity due NK [94] quinquefasciatus, to spores An. gambiae 8) Pseudomonas migula strain An. stephensi, Cx. Mosquitocida NK [95 - 97] fluorescens VCRC B426 quinquefasciatus l exotoxin and Ae. aegypti (44kDa) 9) Bacillus subtilis subtilis An. stephensi, Cx. Exotoxin- NK [74] quinquefasciatus surfactin and Ae. aegypti 10) Bacillus strain VCRC An. stephensi, Cx. Production of NK [47] amyloliquefaciens B483 quinquefasciatus lipopeptide and Ae. aegypti (s) 11) Peanibacillus NK Ae. aegypti 80kDa NK [98] macerans parasporal protein 12) Chromobacterium chromobacte Ae. aegypti Colonize the NK [99] spp rium strain insect midgut Csp-P and displays entomopatho genic and anti-pathogen properties 13) Bacillus cereus VCRC B540 An. stephensi, NK NK [100-101] Aedes aegypti 14) Pseudomonas strain KUN2 Ae. aegypti Extracellular NK [102] aeruginosa toxins

*NK- not known, Bs- Bacillus sphaericus, Bt- Bacillus thuringiensis, Bti- Bacillus thuringiensis israelensis, An- Anopheles, Ae- Aedes, Cx- Culex, sp.-species

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2.4 BIOASSAYS, ISOLATION, CHARACTERIZATION AND IDENTIFICATION

Microbial isolates are constantly being screened and isolated from terrestrial and aquatic environments for mosquito control programmes [103]. The earlier method of isolating mosquito pathogenic Bacillus strains was cumbersome and time consuming, hence Dhindsa et al., 2002 developed a new soil screening method for the detection of mosquito pathogenic bacilli in the soil samples. This method involves the use of LB broth (buffered with Sodium acetate) and a heat shock step at 65°C [104]. Using this method, eight different Bacillus strains were successfully isolated, identified and evaluated for their larvicidal activity in Goa, India [104]. In a screening assay carried out by Radhika et al., 2011, ten bacilli were isolated from Tamil Nadu, India and tested for larvicidal activity against Ae. aegypti mosquito. Two microbial isolates (B. megaterium and Acinetobacter sp.) effectively caused 97% larval mortality at 48-hour incubation at bacterial concentrations of 4.1 ± 0.39 and 3.6 ± 0.71 mg/l [105]. In another study by Allwin et al., 2007 in India isolated native strains of B. thuringiensis from soil samples collected from different locations in Tamil Nadu, India and characterized them [106]. Soil samples collected from mangrove region of Vellar estuary in India yielded mosquitocidal bacteria Bacillus subtilis with significant activity against An. stephensi and Ae. aegypti [107]. Many other reports on the frequent occurrence of mosquito pathogenic bacterial isolates in the natural environment show high possibility of isolating novel strains [108].

2.5 RESISTANCE PHENOMENON AND OVERCOMING RESISTANCE

Since insecticide resistance can undermine efforts to control vector borne diseases, effective mosquito control can be successfully achieved only by overcoming insecticide resistance. Resistance being a complex phenomenon at genetic, evolutionary and ecological level is a serious threat to the success of microbial insecticide formulations used in public health settings [109].

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2.5.1 Strategies for management of resistance to biolarvicdes

Some measures to counter resistance include: (i) alternation or rotation of bacterial biolarvicidal toxins with other toxins, insecticides or biological control strategies (ii) less frequent biocide treatments (iii) utilization of slow–release, ultra-low volume (ULV) and thermal formulations which are active for longer durations (iv) use of source reduction methods and (v) constant resistance surveillance and monitoring. These principles when combined are essentially a blueprint for integrated pest management (IPM) which will successfully delay or prevent resistance development in vector populations [110]. Other methods of overcoming resistance have been listed below. a. Insecticide Mixtures

Studies have shown that by combining different classes of insecticides or their application by mosaic design can effectively overcome problem of resistance in the target insects. However, unless insecticides of different classes are combined and judiciously used, there is possibility of cross resistance if insecticides induce similar mechanism of resistance and have similar mode of action in target insect. If mixtures are used, there is inherent risk of resistance build up to multiple classes of insecticides making them useless or partly effective. But there is a drawback in this approach for their practical application mainly due to higher cost and practical difficulty as both compounds need to be present in equally high and persistent concentrations [111]. The use of two or more interventions has been advocated (e.g. IRS and LLINs) so that mosquitoes that survive contact with one (e.g. LLINs) are killed due to exposure to the second (e.g. IRS). In such a scenario, the use of biolarvicides where feasible, can also delay the onset of resistance and ease selection pressure of insecticide on target vectors.

b. ULV and thermal application

The dengue vector Aedes aegypti is a container breeder, hence use of Bti for its control is limited due to difficulty in its effective application. In this respect, ultra-low-volume cold fogging can be used effectively for larviciding purposes when the agent is applied correctly and under required conditions [112]. Seleena et al., 1996 reported that this technique was

32 highly effective in Aedes larval control using Bti H-14 and when used together with malathion it induced complete adult mortality [113]. In addition, the effectiveness of the thermal application of an aqueous suspension of Bti with and without pyrethroids using a thermal fogger has been reported without loss of its larvicidal activity [114], [115], [116].

c. Application of Ice granules containing endotoxins of microbial agents

A novel method for the aerial delivery of microbial mosquito control agents into vast aquatic sites in the form of ice granules was developed by Becker et al., 2003. The solutions containing formulations of Bti or Bs in powder form were transformed into ice pellets (named Icy Pearls) using a special ice-making machine and applied aerially. Successful field tests using Icy Pearls were conducted against larvae of Aedes vexans [117].

2.6 COMMERCIAL BIO-LARVICIDE FORMULATIONS AND THEIR FIELD EFFICACY

Two biolarvicide formulations—Bacticide® and VectoBac® containing viable endospore and delta endotoxin of Bti H-14 were evaluated in 2001 in Surat city, India against An. subpictus and Cx. quinquefasciatus. Both formulations were equally effective on larvae after second application [118]. Field testing and evaluation of the efficacy of bio-larvicide, Bactivec® SC (Bti H-14) was carried out in Bengaluru, India. It was found to be operationally feasible and easy to handle [119]. Kumar et al, 1995 and 1996 tested a formulation of Bactoculicide (Bti strain 164) in construction sites, abandoned overhead tanks and curing waters and a formulation of Spherix (B sphaericus H5a5b) in Goa, India respectively and found them highly effective [120], [121]. Bacillus sphaericus (Strain 101, Serotype H5a5b) was applied weekly in Panaji, Goa, India helped in malaria control and was identified as a useful bio control agent of An. stephensi [122]. Similarly, application of biolarvicide Bti strain 164 at 1 g/m2 surface area and introduction of larvivorous fish Aplocheilus blockii in major breeding habitats of An. stephensi was carried out in order to control malaria in Goa, India. This was found to successfully replace DDT and pyrethrum fogging [44].

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In addition, the efficacy of various formulations of Bti (Bactimos®, Teknar®, VectoBac®, Bactisand®, VectoPrime®, VectoMax®) and Bs (HIL-9® & HIL-10®, VectoLex®) in the form of tablets, granules, wettable powder, pellet, aqueous suspension, etc. were tested against mosquito vectors and found to be highly effective. Table 3 provides a list of the available commercial bio-larvicide formulations, their type, potency and field evaluation of these formulations.

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Table 3: Lists the available commercial bio-larvicide formulations, their type, potency and field evaluation of these formulations

Sr. Formulation Active Type Potency Field evaluation Reference No ingredient 1) HIL-9, Bacillus Dust NK V/s An. culicifacies [123] HIL- 10 sphaericus (doses- 0.05, 0.1 and 0.5 strain 1593 g/0.1 m2) 100% mortality in 3rd and 4th instar larvae. 2) Bactimos® Bacillus PT 3000 High mortality (96 - [124] thuringiensis ITU/mg 100%) in late larval israelensis against instars of Ae. albopictus, strain AM Aedes Cx. quinquefasciatus 65-52 aegypti from lab and field 24 larvae hours after application. 3) Bactisand® Bacillus FG 112 NK [125] thuringiensis ITU/mg israelensis against H-14 Aedes aegypti larvae 4) Bactoculicide® Bti Suspension 993 Culex, Aedes and [126] (strain 164) ITU/mg Anopheles larvae against breeding controlled (96 - Aedes 100%, for up to 5 weeks, aegypti dose - 0.5g/m2) in larvae industrial scrap in UP, India. 5) Spherix Bacillus WDG NK In lab evaluation in [127] sphaericus, Assam, India 90% H5a5b, mortality observed in Cx. strain B101 quinquefasciatus third instar larvae at 0.6 ppm.

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6) VectoBac® Bacillus WDG, 3000 1) In a study in Malaysia, [128-129] thuringiensis 12 AS, SC, ITU/mg VectoBac® G and israelensis G 1200 VectoBac® 12AS→ H-14 strain ITU/mg effective for 24 hrs. v/s AM 65-52 200 Ae. albopictus in ITU/mg discarded tires with > against 80% mortality. Aedes 2) VectoBac® WDG aegypti evaluated in the lab and larvae field in Bangalore, India v/s An. culicifacies and An. stephensi showed increased efficacy against An. stephensi. 7) Teknar® Bacillus SC 1200 Larvicidal efficacy v/s [130] thuringiensis ITU/mg Cx. quinquefasciatus was israelensis, against determined in lab and strain SA3A Aedes field in Pondicherry, aegypti India. In cesspits > 80% larvae reduction up to day 6 post-treatment and in unused wells > 80% reduction of pupae for 17 days post treatment was observed. 8) VectoLex® Bacillus FG 50 Efficacy against third sphaericus BSITU/mg instar larvae of Culex sp. [131] 2362, against Cx. and Ae. aegypti was Serotype quinquefasc studied in Queensland, H5a5b strain iatus larvae Australia. Both ABTS 1743 WDG 650 formulations were most BSITU/mg effective against Culex against Cx. sp., with the WDG 10-

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quinquefasc 100 times more effective iatus larvae than the FG on the basis of ITU/mosquito. 9) VectoMax® Bacillus FG 50 A trial v/s Ae. albopictus sphaericus BSITU/mg larvae in Spain took place [132] 2362, against at dosages of 10, 50, and Serotype Culex 577 kg/ha. Efficiency H5a5b, quinquefasc close to 100% for up to Strain ABTS iatus larvae 345 days post-treatment 1743 + was obtained. Bacillus WSP 50 Residual effectiveness of [133] thuringiensis BSITU/mg VectoMax® WSP when israelensis against applied to septic tanks Serotype H- Culex against 3rd and 4th stage 14 Strain quinquefasc larvae of Culex pipiens AM 65-52 iatus larvae was evaluated in a study in Turkey. > 96% control of larvae for 24 days was obtained.

10) VectoPrime® Bacillus FG 400 NK [134] thuringiensis ITU/mg israelensis strain AM 65-52 + (S)- methoprene

*FG = Fine Granule, G = Granule, SC = suspension concentrate, WDG = water-dispersible granule, WSP = water soluble pouch, SC = Suspension Concentrate, PT = Pellet, AS = aqueous suspension; NK = Not known; An- Anopheles; Ae- Aedes; Cx- Culex; sp.-species

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2.7 FUTURE PROSPECTS

Future prospects for the use of biolarvicide formulations against mosquito vectors will depend on low cost of production and development of cost-effective/competitive formulations. Cheaper formulations designed from the seeds of legumes, dried cow blood and mineral salts as well as the use of potato-based culture medium, bird feather waste and de-oiled rice bran waste as culture medium when assessed for growth and production of Bti toxins were shown to be more economical and effective against mosquito vectors [135], [136], [137]. This could help in media optimization for the economical production of Bacillus based insecticides [138]. In addition, enhanced activity of protein toxins by use of recombinant bacteria containing a mixture of endotoxins having different modes of action holds great promise. A few examples include newly discovered mosquitocidal proteins and peptides such as Mtx proteins and trypsin modulating oostatic factor which can be genetically engineered for development and use in vector control programs [139]. Research is also underway with respect to transgenic algae and cyanobacteria by expressing larvicidal endotoxins of Bacillus thuringiensis israelensisis and Bacillus sphaericus to allow the toxins to persist in the feeding zone for a longer duration as well as provide increased protection from sunlight (UV light). The most promising results were obtained with the expression of Cry toxins in the filamentous, nitrogen-fixing cyanobacteria Anabaena PCC 7120 [140]. A transgenic strain of this cyanobacteria was reported to protect the expressed δ- endotoxins of Bti from UV light inflicted damage. This organism has an added advantage as it has the ability to multiply in the breeding sites as well as serve as a food source to mosquito larvae [141]. Recently there has been a focus on the development of novel bio-larvicides and their applications. The use of entomopathogenic bacteria and fungi mainly Ascomycete’s fungi such as Metarhizium anisopliae and Beauveria bassiana, for control of both larval and adult stages of mosquito vectors such as Aedes [41]. The release of volatile chemicals into the air by the use of spatial repellents has been advocated. The additional advantage being their ability to induce modifications in insect behaviour and to reduce human-vector contact thereby reducing disease transmission [142]. Although mosquito traps have been used effectively as surveillance tools in order to capture vector mosquitoes for population and disease transmission studies, recently, lethal ovitraps

38 have been considered as a control strategy. These traps such as attractive baited lethal ovitrap (ALOT) are being developed on the principle of “attract and kill”. They have shown promising results in significant reduction of Aedes populations [143]. The use of attractive toxic sugar baits (ATSB) which work by attracting mosquitoes and having them feed on toxic sugar meals could also be a potential vector control tool. The future of vector control includes the use of Sterile Insect Technique (SIT) which has been successfully demonstrated against Ae. albopictus mosquitoes as a promising technique to control mosquito populations [144]. It has been recently combined with auto-dissemination, especially for the control of Aedes species, but this technique has not been used in large scale at present. Recently, a new control concept has been devised, named “boosted SIT” which might help in the area-wide eradication of mosquitoes [145]. In addition, the use of cytoplasmic incompatibility (CI) induced by the widespread bacterial endosymbiont Wolbachia can be an advantageous mosquito control method [146]. It shows great promise for mosquito control either alone or in combination with SIT [147]. Lately, mosquitoes modified with gene drive systems are being suggested as control tools [148]. The synergistic utilization and application of these control measures could have a major impact on the socio-economic health of populations particularly in developing countries. These methods are currently in the pipeline and when available could complement the integrated vector management programmes in which biolarvicides will play a critical role.

2.8 CONCLUSIONS

Mosquito borne diseases are a major public health concern. Effective vector control requires the deployment of a range of integrated interventions. It is of immense importance to focus on the development, evaluation and deployment of alternative vector control tools and strategies. However, for effective control and elimination of the mosquito vectors and vector borne diseases, these strategies will have to be locally adapted to account for vector biology and the intensity of disease transmission keeping in mind both human and financial resources. In addition, the discovery of a novel bacterium from nature is awaited which could be developed into an ideal biolarvicide having a broad spectrum of activity at very low concentrations without developing resistance in the target mosquito species.

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CHAPTER 3: MATERIALS AND METHODS

3.1. Sample Collection

Soil sample collection was carried out from the coastal region of Goa, India. The samples were collected in sterile condition from 3 sites viz., Ribandar (Salt pan), Miramar (beach), Curca (Salt pans and Mangrove soil) (Fig. 7). From each site, ~10 grams of soil devoid of organic debris was collected from the upper most layer of the habitat with the help of a sterile spatula into sterilized glass vials. The vials were sealed, labeled and brought to the laboratory. Soil sample vials were stored at room temperature till processed further. Maximum care was taken to avoid any contamination during sampling and processing.

Fig. 7: Sampling sites in Goa, India

3.2. Screening for Mosquito Pathogenic Bacilli In the earlier stages of the work (Ribandar sampling site), attempts were made to isolate mosquito pathogenic bacilli by plating the serial dilutions of the soil samples on nutrient agar plates (Appendix 1.1), colonies which had typical bacilli like morphologies were handpicked, purified and tested for pathogenicity. Three different methods were used.

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i. Method One:

Soil sample (1 g) was transferred in Nutrient broth (Appendix 1.2) and incubated on a shaker (100 rpm) for 48 hours after which 1 ml of the supernatant was screened for mosquitocidal activity as given below.

Method 1

1g of soil + 10ml NB

Shaker 1g(100 rpm, 48 hours)

1ml screened for mosquitocidal activity

ii. Method two:

The second method was direct inoculation of the soil in the Nutrient Yeast Extract Salt medium (NYSM) broth (Appendix 1.3). 1ml of this broth is used for testing the pathogenicity as given below.

Method 2

0.5g of the soil is weighed

Dispensed in 200ml of NYSM broth Shaker (200rpm, 48 hours)

1ml of the culture is used for toxicity testing

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iii. Method 3: Direct Isolation Method [ Dhindsa et al., 2002] [104]

This method of screening helps in directly isolating the mosquito pathogenic bacilli from soil.

0.5g soil sample

10 ml LB broth buffered with 0.25 M Sodium Acetate (pH 6.8) (Appendix 1.4) in 100 ml flask

Incubation on a shaker (200 rpm), 4 hours at 30˚C

1 ml aliquot heat shocked at 65˚C for 10 minutes in a pre-warmed 5ml glass tube

0.1 ml of sample removed and added to 1 ml of LB medium (Appendix 1.5), incubated for 24 hours at 30˚C

1ml of NYSM (sporulating medium) added and incubated for 2 days at 200rpm, 30˚C

0.1 ml of culture taken for preliminary toxicity

testing against mosquito vector species

(10 mosquito larvae in 10ml of sterile D/W)

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3.3. Isolation of Mosquito Pathogenic Bacilli

Isolation of the bacilli was carried out only from those soil samples which were found to be positive for mosquito-pathogenic bacilli following screening. Further only those samples (i.e., from Ribandar and Miramar) which produced more than > 50% mortality against 3rd instar larvae of the four mosquito test vectors, namely Anopheles stephensi, Culex quinquefasciatus, Aedes aegypti, and Aedes albopictus were further processed. These samples were processed by serial dilutions and plating followed by picking up of the morphologically distinct isolates (Protocol for Isolation of Mosquito Pathogenic Bacilli) [104]

The active soil sample identified via screening procedure

Each sample serially diluted and plated onto NA plates

Incubation at room temperature for 48 hours

Morphologically distinct colonies picked randomly

Purified by sub culturing on Nutrient Agar plates

Preliminary efficacy tests, microscopy, biochemical

characterization, etc.

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3.4. Maintenance of Mosquito Colony To test the mosquito-pathogenic activity of the isolates, the 3rd instar larvae of the 4 test vector species were obtained from the insectary of ICMR-National Institute of Malaria Research, Field Station, Goa. This table top insectary (Fig. 8) was maintained with the help of trained technicians, insect collectors and lab assistants. The mosquito colonies of the 4 test vector species (An. stephensi, Cx. quinquefasciatus, Ae. aegypti and Ae. albopictus) were maintained at ambient laboratory conditions i.e. temperature of 27 ± 2o C, relative humidity 70 ± 5% and 12 hours of day and night cycle. The methodology for mosquito collection from their breeding habitats and their rearing is briefly described here. Larval sampling: Mosquito larvae were collected from the breeding habitats of mosquitoes with the help of bowls of about 500 ml capacity. Larvae were picked up with the help of glass droppers having rubber bulbs and transported to the insectary for rearing and emergence of adults. The larvae were reared in the enamel or plastic trays of 5 x 10 x 3 inches dimension containing tap water. A pinch of larval food (Cerelac™ and finely ground fish meal in a 1:1 ratio) was provided to the larvae daily until the pupal stage. Adult emergence: The pupae formed were collected in plastic bowls containing tap water covered with a piece of nylon net having a small opening in the center to facilitate the removal of the adult mosquitoes with aspirator when found emerged. This opening was kept plugged with cotton to prevent the emerged adult mosquitoes from escaping. Such adults were identified and transferred to their respective cages which were labelled according to the species. The adult mosquitoes were identified based on morphological variations with the help of available keys. [150-157] Adult rearing: Following identification the adults were given feed of 10% glucose soaked in a cotton pad. The adult female mosquitoes were given a blood meal using a glass feeder in which blood was maintained at 37º C with the help of a circulating water bath. Plastic bowls containing 250ml tap water were kept inside the adult cages for oviposition by the gravid female mosquitoes. The eggs were transferred to the plastic trays where the larvae emerged. These larvae were either used in bioassays as they grew to 3rd instar stage or were reared further to adults as described above. In this fashion, a continuous cyclic colony was maintained in the insectary.

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Membrane feeding apparatus

Raisins and 10% Glucose pad

Collection of eggs Rearing of larvae and separating pupae Caging of adults

Fig 8: Flowchart of cyclic maintenance of mosquito colony

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3.5. Preliminary Toxicity Assay

The preliminary toxicity testing of the isolated bacterial colonies was carried out in order to select the isolates for main bioassays. For this, the individual colonies purified by sub culturing 2-3 times on NA plates, were grown on NYSM broth for 3-5 days till complete sporulation occurred. Each test was performed separately in sterile bioassay bowls containing 25 laboratory reared larvae of four different species Anopheles stephensi, Culex quinquefasciatus, Aedes aegypti and Aedes albopictus in 250ml of sterilized distilled water. Sample (1 ml) from the whole culture grown on NYSM broth (sporulating medium) was used for screening the larvicidal activity and concurrent controls containing 1ml un- inoculated NYSM broth was maintained as the control. The mortality was scored by counting the number of dead larvae after 24 hours and 48 hours of treatment and the percentage mortality was calculated and recorded. The percentage mortality was calculated using the formula:

Number of dead larvae % Mortality = X 100 Total number of larvae tested

If the % mortality was > 5% in the control larvae, then the corrected mortalities were determined by Abbot’s formula [158].

(% mortality in the experiment) − (% mortality in the control) % Mortality = X 100 100 − (% mortality in the control)

The isolates that showed high larvicidal activity (> 50%) against the 3rd instar larvae of Anopheles, Culex and Aedes spp. were chosen for further investigations.

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3.2.5 Storage of the isolates The working isolates were code named BGUMS14, BGUMS16, BGUMS101 from the Ribandar salt pan sampling site and C1A, C2A, C2C, B1C from the Miramar beach sampling site. They were maintained on NA plates and slants which were kept in refrigerator at 4° C and sub cultured periodically. A pure sample of each isolate was kept safe at -20° C in 20% (v/v) glycerol [159]. For this the cultures were first grown on NYSM for 3 - 5 days for complete sporulation and then an equal amount of sterilized stock of glycerol (40% v/v) was added.

3.6. Morphological and Biochemical Characterization of the Isolates

Colonial, morphological and biochemical characteristics of the isolate were studied as per the tests listed in Bergey’s Manual of Systematic Bacteriology Vol 3, Bacteria: Firmicutes [160].

3.6.1. Colony characteristics: The isolates were grown on NA plates for 12 - 16 hours and their colony morphologies were observed under a compound microscope. The characteristic features including the size, shape, color, margin, elevation, surface texture, opacity and motility was recorded.

3.6.2 Gram and Endospore staining

i. Gram staining:

A thin smear of the bacterial culture was made on a glass slide and air dried and heat fixed which was followed by covering it with crystal violet for 30 seconds and washed with distilled water. It was then covered with iodine solution for 30 seconds and washed with 95% ethyl alcohol followed by washing with distilled water. The slides were then drained and counterstained with safranine for 30 seconds. The slides were again washed with distilled water, blotted, air dried and observed under oil immersion.

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ii. Endospore staining:

Endospore staining was performed with a 48 - 72 hours culture grown on a Nutrient Agar plate. The spore shapes and sizes were noted. Staining was done according to Schaeffer- Fulton spore staining technique (HiMedia Schaeffer and Fulton’s spore stain kit K006).

3.6.3. Scanning Electron Microscopy (SEM) The bacterial cultures were scanned after fixation, using the Zeiss EVO 18 SEM model at the instrumentation facility of Goa University. Samples were fixed with 2% glutaraldehyde overnight followed by dehydration using increasing concentrations of acetone (30%, 50%, 70%, 90%, and 100%). This was followed by air drying overnight and sputter coating with gold to observe the external morphology of bacterial cells.

3.6.4. Biochemical tests The biochemical tests for bacilli identification were carried out as per the tests listed in Bergey’s Manual of Systematic Bacteriology Vol 3, Bacteria: Firmicutes [160]. The morphological and biochemical characteristics of the new isolates were recorded and compared with the two reference strains for their basic identification.

1. Nitrate test: The medium (Appendix 1.6) was distributed in test tubes and the culture was inoculated and incubated for 2 - 7 days at growth temperature. For recording the results 1ml of the test reagent Griess Illosvay reagent (Appendix 2.2) was added to the test cultures and the control. The color change to red was noted.

2. Catalase test: Bacterial colonies were taken on a slide with the help of a nichrome loop and on this a drop of H2O2 was added. Effervescence caused by the liberation of free oxygen as gas bubbles was noted which indicated the presence of catalase enzyme in the culture.

3. Indole test: The sterilized broth (Appendix 1.7) in the test tubes was inoculated and incubated at 37° C for 2 - 7 days after which Kovacs reagent (Appendix 2.3) was added, shaken and allowed to stand. If a deep red color developed due to the presence of indole that separates out in the alcohol layer, it was recorded as a positive test.

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4. Oxidase test: The oxidase reaction was carried out by touching and spreading a well isolated colony on the oxidase disc. The reaction was observed within 5 - 10 seconds at 25°- 30° C. A change later than 10 seconds or no change at all was considered as a negative reaction.

5. Sugar fermentation (glucose): The media (Appendix 1.8) was dispersed in each test tube and was autoclaved and then cooled following which 3ml of the 20% stock of each sugar (sterilized separately) and few drops of the indicator (phenol red) were added to them. These were inoculated with the test cultures and incubated for 3 - 5 days. The change in the color to red was noted for results.

6. MR-VP test: The test tubes containing broth (Appendix 1.8) were inoculated and incubated. For recording the results a few drops of the indicator O’Meara’s reagent (Appendix 2.1) was added and the color change was observed. The color change to red was recorded as a positive test.

7. Gelatin liquefaction: A heavy inoculum of an 18-to-24-hour old test bacteria was stab-inoculated into tubes containing nutrient gelatin (Appendix 1.9). The inoculated tubes and an un-inoculated control tube were incubated at the test bacterium’s optimal growth temperature, for up to 1 week, and checked every day for gelatin liquefaction.

8. Starch hydrolysis: The poured dried plates of the medium (Appendix 1.10) were inoculated by streaking once across the surface and incubated at 27° C overnight. For testing, the plates were flooded with 5 - 10 ml Gram’s iodine and clear zones formed due to starch hydrolysis were noted.

9. Degradation of tyrosine: Culture was spot inoculated onto dry, sterile Tyrosine agar (Appendix 1.11) plate. Plate was incubated inverted at room temperature for 24 hours. Plate was checked for zone of clearance around colony indicating production of tyrosinase enzyme.

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10. Hydrolysis of Caesin: Culture was spot inoculated onto dry, sterile Caesin agar (Appendix 1.12) plate. Plate was incubated inverted at room temperature for 24 hours. Plate was checked for zone of clearance around colony indicating production of Caesinase enzyme.

11. Hydrolysis of Tween compounds: Poured dried plates of the medium containing tween (Appendix 1.13) were inoculated by streaking once across the surface and were incubated for 24 hours. The presence of any clear zone surrounding the bacterial growth due to the hydrolysis was noted.

12. Hydrolysis of Arginine: Tubes containing medium (Appendix 1.14) were stab inoculated in duplicates with each culture and then one each of the two tubes was covered with liquid paraffin. The color change to red due to the hydrolysis of Arginine, if any was noted.

13. Hugh leifson Test: Two tubes of Hugh Leifsons basal medium (Appendix 1.15) was inoculated with the test organism using a sterilized nichrome loop by stabbing half way to the bottom of the tube. One tube of each pair was covered with 1 cm layer of sterile mineral oil and incubated at 35° C for 48 hours.

14. Urease test: Slants containing the urease basal medium were prepared and inoculated with the culture. The tubes were observed for color change to pink due to the production of urease enzyme.

15. Optimization of Growth Conditions: i) Growth and toxicity in different media: A pure colony of each of the eight bacterial isolates to be tested for mosquito larvicidal activity was grown in 100 ml conical flasks containing 20 ml of Nutrient yeast sporulating (NYSM) medium, Nutrient broth (NB), Media D (MD) (Appendix 1.16) and 1/4th strength Nutrient broth (1/4th NB) (Appendix 1.17). Incubation was at 28 ± 2o C on a rotary shaker at 200rpm for 72 - 96 hours. The toxicity was assessed against the 3rd instar larvae of the test vectors and the percentage mortality was recorded following 24 hours of exposure. Appropriate controls were maintained.

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ii) Temperature: 10ml of LB Broth (HiMedia) (10 ml) was prepared in 50ml conical flasks inoculated with each culture and incubated on the shaker for 24 - 48hrs. One set of the cultures were incubated at 50° C and the other at 65° C. The bacilli growth in these two sets of culture was observed. iii) Growth and toxicity at different time periods: Bacterial isolates were grown in NYSM for 24hrs, 48hrs, 72hrs, 96hrs, 120hrs and 144hrs at a temperature of 27 ± 2° C, on shaker at 200rpm. Bioassays were performed after each time period against healthy 3rd instar larvae of the test vectors and the percentage mortality was recorded following 24 hours of exposure. Appropriate controls were maintained. iv) Growth and toxicity in different concentrations of NaCl: Each culture was inoculated into 10ml of NYSM containing different concentrations of NaCl (Appendix 1.18) and incubated on a rotary shaker at room temperature. The toxicity was assessed against the 3rd instar larvae of the test vectors and the percentage mortality was recorded following 24 hours of exposure. Appropriate controls were maintained.

16. Antibiotic sensitivity: The isolates were spread on Nutrient Agar plates and the antibiotic discs Streptomycin (10mg/disc), Kanamycin (30mg/disc), Erythromycin (15mg/disc) were aseptically placed onto the surface of the agar with the help of a sterilized spatula and gently tapped in place. Plates were incubated inverted at 37º C overnight. Diameters of the respective antibiotic sensitivity zones were measured and recorded.

3.6.5. Bio surfactant production by the method described by Morikawa et al., 1993 The bacterial isolates were grown separately in 50ml NYSM broth in a conical flask and incubated on a rotary shaker (200rpm) for 96 hours at 28 ± 2o C. The culture was harvested by centrifugation at 8000rpm for 20 minutes at 4o C and the culture supernatant obtained was tested for bio-surfactant activity. Seven ml of distilled water was dispensed in a sterile petri dish following which 8µl of sterile mineral oil was placed on its surface. Five µl of culture supernatant was gently placed on to the center of the thin membrane of oil formed. Clear halo was visible under light and the area of the circle was measured [161]. The same test was performed with un-inoculated plain culture medium.

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3.7. Crude protein extraction and determination of concentration by nanodrop technique The bacterial samples were prepared and crude protein extracted by the method of Murphy and Stevens., 1992 with slight modifications as given below [162, 163]. Protein concentration in mg/ml was determined by Nanodrop technique using the Thermoscientific Nanodrop 2000c spectrophotometer at Goa University.

10 ml culture grown in NYSM

Centrifuged at 8500rpm for 15 minutes

Supernatant is discarded and pellet resuspended in 1ml Lysis solution (vortexed) (Appendix 2.5.1)

Add 50mg Lysozyme, vortex and incubated for 10 minutes

Centrifuged at 8500rpm for 15 minutes

Washed with Lysis solution (Appendix 2.5.1)

Add 500µl Extraction buffer (Appendix 2.5.2)

Add 500µl Dissociation buffer or sample buffer (Appendix 2.5.3)

Kept in boiling water for 5 minutes. 50µl can be loaded for SDS PAGE analysis in mini gel wells

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3.8. DNA extraction and PCR amplification

3.8.1. DNA extraction The bacterial genomic DNA was extracted from 24 hours old culture grown in Nutrient broth using the phenol-chloroform method described by Sambrook et al., 2006. The extraction is performed as described in following protocol [164].

Bacterial isolates are grown in 10ml Nutrient broth overnight

In 1.5ml Eppendorf tubes take 500µl of culture broth

and add 200µl of nuclease free water

Wash by centrifugation (5000rpm, 3 minutes)

Discard the supernatant and resuspended the pellet

in 464µl of TE buffer, add 20µl of lysozyme

Slightly vortex the tubes and incubate at 37o C for 1.5 hours

o Add 30µl of SDS (10%) and incubate at 60 C for 20

minutes and for 5 minutes in room temperature Slightly vortex the tubes.

Add equal volume of phenol: chloroform

Centrifugation at 12000rpm for 10 minutes

Transfer the aqueous layer into a fresh tube and

repeat the phenol: chloroform step again

53

Centrifugation at 12000rpm for 10 minutes

Transfer the aqueous layer into a fresh tube and add equal

volume of chloroform: isoamyl alcohol (24:1)

Centrifugation at 12000rpm for 10 minutes

th Transfer the aqueous layer into a fresh tube and add 1/10 total volume of sodium acetate and 0.6 of the total volume of chilled propanol

Centrifugation at 12000rpm for 10 minutes

Discard the supernatant and add 1ml of 70% chilled ethanol, slight vortex

Centrifugation at 12000rpm for 10 minutes

Discard the supernatant and air dry the tube

Suspend in 50µl TE buffer

Genomic DNA extraction protocol by using the phenol-chloroform method

54

3.8.2. PCR AMPLIFICATION The amplification of the 16S rRNA gene, was carried out using the universal primers (S-D- Bact-0011-a-5-17: 5`-GTTTGATCCTGGCTCAG-3`) and (S-*-Univ 1392-b-A-15: 5`- ACGGGCGGTGTGTNC-3`) [165]. PCR reaction mixture consisted of 15μl of PCR Master

Mix (0.05U/μl taq DNA polymerase, reaction buffer, 4mM MgCl2, and 4×1.25 ml nuclease free water), 1 μl of primers respectively. The extracted bacterial genomic DNA template (1.5 µl) of each sample was added in sterile PCR tubes. The final volume was made up to 30 μl by using nuclease free water. The samples were amplified in a thermal cycler (Bioer XP Cycler). The PCR conditions followed were the initial denaturation for 5 mins at 94°C, this was followed by 30 cycles of denaturation at 94° C for 1 min duration, annealing and extension at 55° C and 72° C respectively for 2 mins, final extension for 5 mins at 72° C. The amplified PCR products were purified using Qiagen Q1A quick PCR purification kit followed by sequencing at Eurofins India Pvt. Ltd., Bangalore. The sequences obtained were edited using the Bioedit sequence alignment editor version 7.0.4.1 software and compared with sequences of other closely related species retrieved from the GenBank database http://www.ncbi.nlm.nih.gov/BLAST and identified based on sequence homology.

3.8.3. AGAROSE GEL ANALYSIS: Pouring and setting the gel- The gel casting apparatus was assembled and 1% agarose gel (prepared in lX TBE buffer) was poured and allowed to set along with a comb. Gel loading- The set gel was placed in the tank containing TBE buffer. To check for amplification and verify the size of the PCR product 8μl of PCR amplified products was run along with the 10µl of a 500bp DNA ladder. Separation- The apparatus was connected to the power pack and electrophoresis was carried out at 100V. The current was switched off when the dye reached the other end of the gel. The gel was viewed using a gel documentation system (Quantum Vilber Lourmat system).

3.8.4. Phylogenetic Analysis: Phylogenetic analysis was carried out using the neighbour-joining method [166]. ClustalX version 2.0. was used to generate multiple sequence alignment between closely related species. Phylogenetic tree was constructed using neighbour-joining (NJ) method. Tree was obtained with 100 seeds and 1000 bootstraps. Final tree was rooted and drawn using MEGA4.

55

3.9. Large Scale Production of Active Isolates 3.9.1. Mass Production of culture

For large scale production of culture each bacterial isolate was sub-cultured on Nutrient agar plates and incubated for 24 hours at 27° C. Loopful of a single colony was taken and inoculated in 100ml of NYSM broth followed by incubation in a rotary shaker at 200rpm for 24 hours at 27° C (pilot scale). 1000ml of NYSM was dispensed into five 500 ml flasks with 200ml of media in each. 1 ml of the pilot culture was now inoculated in each of the flasks with NYSM broth, incubated for 72 - 96 hours in rotary shaker at 200rpm. The media was now harvested by centrifugation at 8000rpm for 20 min. The cell pellets were pooled together and lyophilized.

3.9.2. Lyophilization

Lyophilization of the culture was carried out in Lyophilizer (Scanvac) instrument at Goa University. For this the cultures were grown on NYSM for 3 - 5 days till complete sporulation occurred and centrifuged at 8000rpm for 20 min. The pellet was washed twice with distilled water, resuspended in the minimum quantity of water and frozen at -80°C. The lyophilizer unit was switched on and the temperature lowered to -110˚C. The tube was tightly screwed to the lyophilizer unit. Vacuum was then applied for 4 - 6 hours till complete drying of the sample was obtained. The water is thus removed by sublimation under vacuum. The viability and pathogenicity of the lyophilized samples was tested. The lyophilized samples were stored in sealed glass vials at 4˚ C until use.

56

Culture grown in NYSM Rotary shaker (100rpm), Centrifugation (8000 rpm, 96hrs, 27±2°C 20mins)

Bioassays Dilutions prepared from lyophilized Cell pellet lyophilized powder

Fig 9: Scaling – up the bacterial cultures

57

3.10. Harvesting of Culture Supernatant and Crude Mosquitocidal Toxin (CMT) i) Culture supernatant The culture supernatant (CS) was obtained by centrifugation of the culture broth (loopful of bacteria culture inoculated in 100ml NYSM and incubated for 96 hours at 200rpm) at 8000 rpm for 20 minutes. This CS was tested against 3rd instar larvae and pupae of the test vector species and the percentage mortality was recorded. ii) Crude mosquitocidal toxin The cell free broth obtained above was used for the isolation of the mosquitocidal toxin by acid precipitation with 6 N HCl. The precipitate containing the mosquitocidal toxin was collected by centrifugation and resuspended in water, adjusted to pH 7.0 and subsequently lyophilized [76]. This material was designated as crude mosquitocidal toxin (CMT). The CMT obtained following 96 hours of growth was bio-assayed against the lab reared 3rd instar larvae and pupae of different vector mosquito’s viz., Anopheles stephensi, Culex quinquefasciatus, Aedes aegypti and Ae. albopictus.

58

3.11. Toxin Studies Toxin genes were detected by using the following toxin gene specific primers- Cry2 (UN), Cry4 (UN), Cyt1 (UN), Cyt2 (UN), BinA, BinB, Mtx, Gyrase, and Surfactin. The PCR conditions were standardized and the product visualized on a 1% agarose gel using a Gel documentation system.

Table 4: List of primers used for amplification of toxin genes

Sr. No. Gene Primer sequence Expected References amplicon size (bp) 1 Cry 5ˈGAGTTTAATCGACAAGTAGATAATTT 3ˈF 526 [167] 2(UN) 5ˈGGAAAAGAGAATATAAAAATGGCCAG 3ˈR

2 Cry4 5ˈGCATATGATGTAGCGAAACAAGCC 3ˈF 439 [167] (UN) 5ˈGCGTGACATACCCATTTCCAGGTCC 3ˈR 3 Cyt1 5ˈCCTCAATCAACAGCAAGGGTTATT 3ˈF 480 [167] (UN) 5ˈTGCAAACAGGACATTGTATGTGTAATT3ˈR 4 Cyt2 5ˈATTACAAATTGCAAATGGTATTCC 3 ˈF 356 [167] (UN) 5ˈTTTCAACATCCACAGTAATTTCAAATGC 3 ˈF

5 BINA 5ˈGTACATTCGCGTTATGG 3ˈ F 720 [168]

5ˈGTATCATAGGTGAACC 3ˈ R 6 BINB 5ˈCACGGAATGGTTATGGTT -3ˈ F 1054 [168] 5ˈAGGTGCATTAGGATACGA -3ˈ R 7 Mtx gene 5ˈAATGAAAAGGACCAAATTACTTTTTTAT 3ˈF 800-900 [169] 5ˈTTATTTAAAAGAAATTTCTTTAACATCTATT A 3ˈR 8 Gyrase 5ˈCAGTCAGGAAATGCGTACGTCCTT3ˈF 1070 [76] 5ˈCAAGGTAATGCTCCAGGCATTGCT3R

9 Surfactin 5ˈ ATGAAGATTTACGGAATTTA 3ˈF 675 [170] 5ˈ TTATAAAAGCTCTTCGTACG 3ˈF

59

3.12. Main Bioassays The main bioassays of the active isolates were carried out by the WHO procedures [171]. The bioassays of the bacterial isolates were carried out against laboratory reared 3rd instar larvae of An. stephensi, Cx. quinquefasciatus, Ae. aegypti and Ae. albopictus mosquitoes. Stock dilutions in parts per million (ppm) were prepared by dissolving the weighed lyophilized powder in appropriate amount of sterile distilled water. A series of doses from 1ppm - 200ppm based on the results obtained from preliminary screening were prepared and tested. Sterile plastic bowls (500ml capacity) were filled with 250ml of sterile distilled water. Healthy 25 larvae were transferred to each bowl. Four replicates were maintained for each dose. Same number of replicates for the negative controls were also maintained under similar conditions simultaneously, each consisting of 25 larvae in 250ml distilled water + same dose of un-inoculated NYSM broth). All the bioassay cups were covered with nylon mesh and experiments were conducted at room temperature (28 ± 2º C), and at 80% relative humidity. The mortality of the larvae was recorded after 24 and 48 hours of exposure by counting the dead larvae. The corrected mortalities were determined using Abbott’s formula. The experiment was rejected if the mortality was more than 20 % in the negative control.

Fig 10: Experimental set up for main bioassays for testing mosquito pathogenic activity

3.12.1 Calculation of LC50 and LC90 values: Efficacy of the isolates against An. stephensi, Aedes aegypti, Aedes albopictus and Culex rd quinquefasciatus 3 instar larvae in terms of LC50 was studied by analysing dose mortality responses of individual strains by Probit analysis using SPSS version 16 software [172].

60

3.13. Effect of sub-lethal dose of Bacilli/toxin on the survival and

fecundity of F1 generation of mosquito species (modified from Zuharah et al., 2016) [173]

rd. Fully fed females Treatment of 3 Adult F1 females → • No. of eggs laid/No. of transferred to paper instar larvae with sub- given a blood meal eggs hatched cup individually lethal (LC ) dose • Survival/longevity of 50 adults and development period of the immatures • Egg hatch rate, pupation percentage, adult emergence rate

Fig 11: Pictorial representation of the protocol to study effect of sub lethal dose on the test vector species

The sub lethal effect of Sporosarcina sp. strain BGUMS101 were studied at the adult F1 generation stage of Anopheles stephensi, Aedes aegypti and Culex quinquefasciatus mosquito which had been exposed to the microbial agent at the larval stage in comparison to the control untreated group. Biological studies included fecundity (number of eggs produced) at the parental F1 stage, life span and survival rate of treated populations, development period (number of days required at every life stage), egg hatchability, pupation percentage and adult emergence rate. 25 late-third instar larvae of laboratory cultured An. stephensi, Ae. aegypti and Cx. quinquefasciatus respectively were treated with Sporosarcina sp. strain BGUMS101 at LC50 value = 3.244ppm against An. stephensi larvae, 1.014ppm against Ae. aegypti larvae and 0.225ppm against 3rd instar Cx. quinquefasciatus larvae for 24 hours in bioassay bowls containing 250ml of sterile distilled water. The experiment was carried out using 6 replicates for test and 6 replicates for control containing untreated larvae under similar conditions for comparison. The surviving larvae (after 24 hours treatment) were washed twice with sterile distilled water and transferred to a clean container with for further observation and pupation. The pupae were taken in bowls with 250ml water and

61 placed in cloth cages till the emergence of adults. Both males and females were transferred separately into oviposition cages using a manual aspirator and allowed to mate in cloth cages of 1 cu ft. dimension. Following 3 - 5 days post emergence the adult females (F1 generation) were given a blood meal using artificial glass feeders and (n = 10) fully fed females were transferred to a paper cup individually. Sucrose was provided using a cotton pad. Subsequent studies on life traits such as fecundity, life span and survival rate of treated populations, development period, egg hatchability, pupation percentage and adult emergence rate were conducted on the next generation

• Fecundity For fecundity study, the number of eggs laid by each blood fed female were counted daily until no further eggs were laid. The number of eggs was counted using a dissecting microscope at the magnification of 40 X. After counting, the filter paper with the eggs was submerged into a tray with 1000ml distilled water to study and record the egg/ offspring hatchability. Eggs that hatched and turned into first instar larvae were counted for egg hatchability (fertility) studies.

• Longevity and Survival The eggs collected for the fecundity study, were used to study the development period from offspring to adult, the egg hatch rate, pupal percentage, adult emergence rate, longevity and survival of the adults. For this purpose, the number of larvae that hatched was recorded daily and were maintained through larval instars development (L1 - L4), pupal formation and adult emergence (F1 generation). The larvae were transferred to another culture tray after emergence to the next life stage. The larval food consisted of a 1:1 mixture of Cerelac™ and fish food. The emerged pupae were recorded daily, taken in bowls with 250ml water and placed in cloth cages till the emergence of adults. The survival and longevity of the F1 generation was counted in days till no adults survived. Control batches of mosquitoes without any treatment were also set up and the results recorded.

62

• Data analysis The following formulas were used:

∑ number of eggs hatched in to 1st instar larvae Egg hatch rate = X 100 ∑ number of eggs laid by fully fed F1 females (n = 10)

∑ number of pupae formed from 4th instar larvae Pupation percentage = X 100 ∑ number of 4th instar larvae

∑ number of emerged adults Adult emergence rate = X 100 ∑ number of pupae

Data was statistically analyzed by the t-test and ^Chi square test using the Microsoft Excel 2010 computer programme. In addition, the Mann-Whitney Rank sum test and Log rank test was also used for fecundity and life span studies.

63

CHAPTER 4: RESULTS

4.1. Sampling, Isolation and Screening of soil samples for mosquito- pathogenic bacilli

Three sites (Ribandar, Miramar and Curca) located along the coastal region of Goa were selected for screening for the presence of mosquito-pathogenic bacilli. At these sites, soil samples were collected from three types of regions viz., salt pans, beach and mangroves and brought to the laboratory to isolate the mosquito larvicidal bacterial strains as described in Chapter 3. Preliminary screening was carried out against the 3rd instar larvae of the mosquito test vector species. Subsequently, the isolates showing significant (> 50% mortality) were bio assayed at different doses to determine their LC50 and LC90 value

i) From the Ribandar sampling site (Fig. 12), a total of eight morphologically distinct bacterial strains were obtained and screened for mosquito larvicidal activity. The bacterial isolates were provisionally code named as BGUMS14, BGUMS16, BGUMS101, BGUMS93, BGUMS94, BGUMS06, BGUMS473-C and BGUMS837. Preliminary screening with 1 ml of culture suspension showed that three bacterial isolates possessed mosquito larvicidal properties (BGUMS14, BGUMS16 and BGUMS101).

Fig. 12: Ribandar salt pan sampling site (15° 30′ 8.1″ N and 073° 51′ 19.6″ E)

64 ii) To screen for the presence of mosquito pathogenic Bacilli from the Miramar beach sampling site (Fig. 13), a total of nine soil samples from three different zones i.e., tidal (Sample A1, B1, C1), intertidal (Sample A2, B2, C2) and sea shore (Sample A3, B3, C3) were collected and further processed. Among the three methods used for screening of the soil samples, the method proposed by Dhindsa et al., 2002 showed the best results.

Fig. 13: Miramar beach sampling site (15.4827° N, 73.8074° E)

The soil samples processed using Method # 1 showed mosquito pathogenic activity between (0 - 10%) against the 3rd instar larvae of the test vector species (Fig 14). Sample B1 and C1 collected from the tidal zone showed 10% mortality against Anopheles stephensi and Aedes aegypti respectively. Soil samples A2 and C2 collected from the intertidal zone showed 10% mortality against Culex quinquefasciatus and Aedes aegypti respectively. Soil sample B3 collected from the sea shore showed 10% mortality against Anopheles stephensi larvae. No mortality was observed in other soil samples (Fig. 14).

65

100

80 Anopheles stephensi 60 Aedes aegypti Culex quinquefasciatus

40 Mortality(%) 20

0 A1 B1 C1 A2 B2 C2 A3 B3 C3

Tidal Inter- Tidal Sea shore

Fig. 14: Screening of soil samples by Method 1 using Nutrient broth (NB)

These soil samples when processed by Method # 2 showed mosquito pathogenic activity between (0-20%) only against the 3rd instar Culex quinquefasciatus larvae (Fig. 15). Sample C1 collected from the tidal zone showed 10% mortality against Culex quinquefasciatus larvae. Soil sample A3 and C3 collected from the sea shore showed 20% mortality against Culex quinquefasciatus. No mortality was observed in other soil samples.

100

80 Anopheles stephensi 60 Aedes aegypti Culex quinquefasciatus

40 Mortality(%) 20

0 A1 B1 C1 A2 B2 C2 A3 B3 C3

Tidal Inter- Tidal Sea shore

Fig. 15: Screening of soil samples by Method 2 using Nutrient Yeast

Sporulating Medium (NYSM)

66

However, when the soil samples were processed by Method # 3 (i.e., Dhindsa et al., 2002) moderate to high mosquito pathogenic activity (10 - 100%) was observed against the 3rd instar larvae of the test vector species (Fig. 16). Sample B1 collected from the tidal zone showed 100% mortality against all the three test vector species, whereas, sample C1 obtained from the same zone showed 100% and 40% mortality against 3rd instar Culex quinquefasciatus and Anopheles stephensi larvae respectively. Soil sample A2 and C2 obtained from the intertidal zone showed 30% and 100% mortality against Culex quinquefasciatus larvae respectively. Also sample C2 from the same zone showed 10% mortality against Anopheles stephensi larvae. No mortality was observed in other soil sample.

100

80 Anopheles stephensi Aedes aegypti 60 Culex 40 quinquefasciatus

Mortality(%) 20

0 A1 B1 C1 A2 B2 C2 A3 B3 C3

Tidal Inter- Tidal Sea shore

Fig. 16: Screening of soil sample using Method 3 (Dhindsa et al., 2002)

The 3 soil samples (B1, C1, C2) which showed significant larvicidal activity were considered to be ideal for isolation of mosquito pathogenic Bacilli. From these soil samples nine morphologically distinct bacterial isolates were obtained by serial dilution and plating and code named as B1A, B1B, B1C, C1A, C2A, C2B, C2C, C2D, and C2E. Preliminary screening showed that 4 isolates i.e. C1A, C2A, C2C and BIC showed moderate to high toxicity (60 - 100%) against 3rd instar larvae of the test vector species. iii) From the Curca Salt pans and Mangrove sampling site (Fig. 17) four soil samples were obtained and coded as M1, M2, SP1 and SP2 respectively.

67

Fig. 17: Curca Mangrove and Salt pan sampling site (15.4584° N, 73.8728° E)

The soil samples processed using Method # 1, once again, did not show mosquito pathogenic activity against the 3rd instar larvae of the test vector species.

The soil samples processed by Method 2 showed mosquito pathogenic activity between (0 - 30%) against the 3rd instar larvae of the test vector species (Fig. 18). Sample M1 collected from the mangrove soil showed 10% mortality against Anopheles stephensi larvae, and 20% mortality against Aedes aegypti and Culex quinquefasciatus larvae. Sample M2 and SP2 showed 20% and 30% mortality against Aedes aegypti and Culex quinquefasciatus respectively. Sample SP2 collected from the salt pan site showed no mortality against the test vector species.

68

Anopheles stephensi 100 Aedes aegypti 80 Culex quinquefasciatus

60

40 Mortality(%) 20 0 M1 M2 SP1 SP2

Mangrove Salt pan Fig. 18: Screening of soil samples by Method # 2 using Nutrient Yeast Sporulating Medium (NYSM) and testing against mosquito larvae

Similarly, the soil samples processed by Method # 3 (i.e., Dhindsa et al., 2002) showed moderate to high mosquito pathogenic activity (20 - 80%) against the 3rd instar larvae of the 3 test vector species (Fig 19). Sample M1 and M2 showed 20% and 30% mortality against Aedes aegypti larvae respectively. Against Culex quinquefasciatus 3rd instar larvae sample M1 and M2 showed 30% and 20% mortality respectively. Sample SP1 and SP2 showed 50% and 30% mortality against Aedes aegypti larvae respectively. Sample SP2 showed 30% mortality against both Aedes aegypti and Culex quinquefasciatus larvae. Sample SP2 showed highest mortality of 80% against Anopheles stephensi 3rd instar larvae.

69

100 Anopheles stephensi 80 Aedes aegypti Culex quinquefasciatus 60

40 Mortality(%) 20

0 M1 M2 SP1 SP2

Mangrove Salt pan Fig 19: Screening of soil samples by Method # 3 (Dhindsa et al., 2002) and testing against mosquito larvae

From these soil samples six morphologically distinct bacterial isolates were obtained (M21, M22, SP11, SP12, SP21, and SP22). However, since during preliminary screening against the 3rd instar larvae of the 4 test vector species (i.e. Anopheles stephensi, Aedes aegypti, Aedes albopictus and Culex quinquefasciatus), extremely low percentage mortality (< 50%) was observed these samples were not processed further.

4.2. Maintenance of Mosquito Colony

The colonies of Anopheles stephensi, Culex quinquefasciatus, Aedes aegypti and Aedes albopictus mosquitoes were successfully maintained at the insectary of ICMR- National Institute of Malaria Research as described in Chapter 3. The healthy larvae were used for bioassay experiments as they grew to the 3rd instar larvae and pupal stage.

4.3. Preliminary toxicity testing of bacterial isolates The potential soil samples from the 3 sampling sites were processed for the isolation of mosquito pathogenic bacilli. These were subjected to serial dilution and plating as shown in the protocol for isolation (Chapter 3) for the selective isolation of bacilli. The morphologically different colonies were picked up from the Nutrient Agar plates. They were observed for their morphology, gram stained and purified on Nutrient Agar plates. These purified isolates were grown on NYSM for 3 - 5 days and individually tested for larvicidal activity against 3rd instar larvae of the four test vector species i.e., Anopheles stephensi, Culex quinquefasciatus, Aedes aegypti and Aedes albopictus to separate pathogenic strains from the non-pathogenic ones.

70

i) The results of preliminary activity of the bacterial isolates obtained from Ribandar assessed using four different media are presented in Fig. 20 A, B & C. Out of the 8 morphologically distinct bacterial isolates obtained, three promising isolates (BGUMS14, BGUMS16, BGUMS101) when grown in NYSM medium for 96 hours showed pronounced larvicidal activity after 24 hours of treatment against the above mentioned test vector species. The levels of mosquito larvicidal activity expressed in the form of percentage mortality by counting the number of dead larvae greatly varied with BGUMS14 showing the highest percentage mortality of 80% against An. stephensi larvae, BGUMS16 showing the highest percentage mortality of 65% against An. stephensi as well as Ae. aegypti larvae and BGUMS101 showed the highest percentage mortality of 64% against Cx. quinquefasciatus larvae when grown in NYSM medium. The rest of the isolates showed a percentage mortality of 50% or less in all the media tested. Since Nutrient yeast sporulating medium (NYSM) achieved the highest toxicity for all bacterial isolates as compared to the other tested media i.e., Nutrient broth (NB) medium, Media D (MD) medium and 1/4th NB medium it was used for all further studies.

20A

100 MD media 1/4th NB media 80 NYSM 60 NB

40 Mortality(%)

20

0 BGUMS14 BGUMS16 BGUMS101 BGUMS93 BGUMS94 BGUMS06 BGUMS473-c BGUMS837 Isolate

71

20B

100 MD media 1/4th NB media 80 NYSM NB 60

40 Mortality (%)Mortality 20

0 BGUMS14 BGUMS16 BGUMS101 BGUMS93 BGUMS94 BGUMS06 BGUMS473-c BGUMS837 Isolate

20C:

100 MD media

80 1/4th NB media NYSM 60 NB

40 Mortality(%)

20

0 BGUMS14 BGUMS16 BGUMS101 BGUMS93 BGUMS94 BGUMS06 BGUMS473-c BGUMS837 Isolate

Fig. 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

72

In order to determine whether the mosquito pathogenic activity of the promising bacterial isolates is affected by the duration of incubation in NYSM or not, bioassays were performed after growing three promising bacterial isolates in NYSM for 24hrs, 48hrs, 72hrs, 96hrs, 120hrs and 144hrs following which bioassays were carried out. The highest mosquito pathogenic activity for the 3 isolates against the 3rd instar larvae of the test vectors was following 96 hours of incubation in NYSM. A steep increase in mosquito-pathogenic activity was observed after 24 hours of incubation in NYSM with the activity reaching a peak at 96 hours of incubation in NYSM after which a sharp decline in activity was observed in the case of all the 3 isolates (Fig. 21, 22, 23). Hence the period of 96 hours incubation in NYSM was considered most optimum duration for incubation and was subsequently followed for all further bioassay experiments.

100 Isolate 14 Isolate 16 80 Isolate 101

60

40 Mortality(%)

20

0 24 48 72 96 120 144 Time (in hrs)

Fig. 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

73

Isolate 14 100 Isolate 16 Isolate 101 80

60

40 Mortality(%)

20

0 24 48 72 96 120 144 Time(in hrs)

Fig. 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

100 Isolate 14

Isolate 16 80 Isolate 101

60

40 Mortality(%)

20

0 24 48 72 96 120 144 Time (in hrs)

Fig. 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

74

Since the mosquito pathogenic isolates were obtained from a saline environment, preliminary screening was carried out by supplementing the standard NYSM medium (containing 0.5% salt concentration) with salt concentrations ranging from 1%-7% followed by the dose-mortality bio-assays. This was carried out in order to determine the optimum salt concentration for the growth and toxicity of the bacterial isolates. The results for the promising isolates have been represented below indicating that the isolates show best activity against mosquito larvae in standard NYSM medium with 0.5% salt concentration hence no additional salt supplementation is necessary (Fig. 24, 25, 26).

100 Cx quinquefasciatus An stephensi 80 Aedes aegypti

60

40 Mortality(%)

20

0 0.5% NaCl 1% NaCl 3% NaCl 7% NaCl NaCl Concentrations

Fig 24: Percentage mortality of the vector species with Isolate BGUMS14 at varying salt concentrations

75

100 Cx quinquefasciatus 80 An stephensi Aedes aegypti 60

40 Mortality(%)

20

0 0.5% NaCl 1% NaCl 3% NaCl 7% NaCl NaCl Concentrations

Fig. 25: Percentage mortality of the vector species with Isolate BGUMS16 at varying salt concentrations

100 Cx quinquefasciatus 80 An stephensi Aedes aegypti 60

40 Mortality(%)

20

0 0.5% NaCl 1% NaCl 3% NaCl 7% NaCl NaCl Concentrations

Fig. 26: Percentage mortality of the vector species with Isolate BGUMS101 at varying salt concentrations

76 ii) Among the 9 Miramar isolates (B1A, B1B, B1C, C1A, C2A, C2B, C2C, C2D, and C2E) against 3rd instar Anopheles stephensi larvae, isolate B1C showed 90% and 100% activity on 24hrs and 48hrs of exposure respectively. On 48 hours of exposure, the order of activity observed with isolate C2C (40% activity) >Isolate C2E (30% activity) > C2A (20% activity) while Isolate C2D and Isolate CIA showed 10% mortality (Fig. 27 and 28). Against 3rd instar Aedes aegypti larvae, isolate B1C showed 100% activity on 24 hours of exposure. On 48 hours of exposure, the order of activity observed with isolate C2E was (30% activity) > Isolate C2C and isolate C2D (20% activity) > C2A (10% activity). Isolate C1A showed no activity. Against 3rd instar Culex quinquefasciatus larvae, Isolate B1C, Isolate C2A, Isolate C2C and Isolate C2E showed 100% activity on 24 hours of exposure. While Isolate C2D and Isolate C1A showed 90% and 20% activity respectively. An increase in activity to 60% was observed on 48 hours of treatment with Isolate C1A (Fig. 27 and 28).

Anopheles stephensi Culex quinquefasciatus Aedes aegypti Aedes albopictus

100

80

60

40 Mortality%

20

0 B1A B1B B1C C1A C2A C2B C2C C2D C2E Isolates

rd. Fig. 27: Preliminary screening results following 24 hours of treatment of 3 instar larvae of the 4 test vector species with the Isolates

77

Anopheles stephensi Culex quinquefasciatus Aedes aegypti Aedes albopictus

100

80

60

40 Mortality%

20

0 B1A B1B B1C C1A C2A C2B C2C C2D C2E Isolates

rd. Fig. 28: Preliminary screening results following 48 hours of treatment of 3 instar larvae of the 4 test vector species with the Isolates

iii) Since the isolates obtained from the Curca site showed low percentage mortality against the 3rd instar larvae of the test vector species these samples were not processed further (Fig. 29, 30, 31, 32).

100

80 24hrs 48hrs

60

40

Mortality(%)

20

0 M21 M22 SP1 1 SP1 2 SP2 1 SP2 2

Fig. 29: Preliminary screening of the isolates against 3rd instar larvae of Anopheles stephensi

78

100

80 24hrs 48hrs 60

40 Mortality(%) 20

0 M21 M22 SP1 1 SP1 2 SP2 1 SP2 2

Fig. 30: Preliminary screening of the isolates against 3rd instar larvae of Culex quinquefasciatus

100

80 24hrs 48hrs 60

40 Mortality(%) 20

0 M21 M22 SP1 1 SP1 2 SP2 1 SP2 2

Fig. 31: Preliminary screening of the isolates against 3rd instar larvae of Aedes aegypti

79

100

80 24 hours 48 hours 60

40

Mortality(%) 20

0 M21 M22 SP1 1 SP1 2 SP2 1 SP2 2

Fig. 32: Preliminary screening of the isolates against 3rd instar larvae of Aedes albopictus

Only those isolates that exhibited 50% mortality with the above-mentioned mosquito vector species were retained for further study. Using this criterion 7 working isolates were identified for main bioassays viz., BGUMS14, BGUMS16, BGUMS101 from Ribandar isolation site and C1A, C2A, C2C and BIC from Miramar beach site.

4.4. Morphological and biochemical characterization of new isolates Microscopic examination following Gram staining and endospore staining showed that all isolates were Gram positive, rod shaped and endospore positive. Biochemical tests were performed and compared with commercial strains of Bacillus thuringiensis israelensis H-14 and Bacillus sphaericus Neide 2362. The morphological and biochemical characteristics of these isolates are presented in Table 5 and 6. The scanning electron microscopy (SEM) images also revealed similar morphological features (Fig. 33). Based on Gordon (1973) and Berkeley (1984) simplified key for Bacillus species and Bergeys Manual of Systemic Bacteriology Vol 3, Bacteria: Firmicutes (Sneath et al., 1986), the isolates were found to belong to Bacillus spp. (Table 5 and 6) [160, 174, 175].

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(i) Colony characteristics

Isolate BGUMS14, BGUMS16 and B1C showed irregular, undulate and flat colony characteristics, while Isolates BGUMS101, BGUMS93, C2A, C2C and the Bti commercial strain showed round, entire and raised characteristics. BGUMS94 showed the same characteristics, however elevation was convex. Isolate BGUMS06, C1A, C2D and C2E showed irregular, undulate and raised characteristics. Both Isolate BGUMS473-C and BGUMS837 showed filamentous and flat colony characteristics. The commercial Bs strain showed round, entire and flat characteristics.

(ii) Gram staining and endospore staining

The gram staining of the bacterial isolates showed purple stained and gram-positive rods of Bacillus sp. The size of the rods varied in the different isolates.

(iii) SEM imaging

All the isolates were endospore producers. The detailed microscopic examination of the larvicidal bacilli was observed by scanning electron microscopy (SEM) at the instrumentation facility at Goa University and the photographs were taken. It was observed that vegetative cells of all the active isolates were rod shaped though their sizes were slightly different in the different isolates. These rods appeared swollen due to the presence of sporangia in some of the isolates. The cells of the isolate BGUMS14 appeared small as compared to other isolates. The isolates BGUMS16, BGUMS101, C2A, C2C and BIC showed rods of intermediate sizes, while C1A showed long rods. The racket shaped swollen sporangia containing the central rounded endospores in isolate C1A, while central endospores in isolate BGUMS101 were also observed.

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Scanning Electronic Micrograph of Scanning Electronic Micrograph of BGUMS16 BGUMS14 at 10 KX magnification (small at 10 KX magnification (Rods=2.005 µm) rods=1.326 µm)

Scanning Electronic Micrograph of Scanning Electronic Micrograph of C1A at 10 BGUMS101 at 10 KX magnification KX magnification (long rods=6.041 µm) (Rods=2.959 µm)

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Scanning Electronic Micrograph of C2A at 10 Scanning Electronic Micrograph of C2C at 10 KX magnification (Rods= 2.775 µm) KX magnification (Rods= 2.617 µm)

Scanning Electronic Micrograph of B1C at 10 KX magnification (Rods= 3.120 µm)

Fig. 33: Scanning Electronic Micrographs of the active bacterial isolates

83

(iv) A. Biochemical characterization

i) The 8 isolates from Ribandar sampling site showed a positive catalase, starch hydrolysis and oxidase test indicating the presence of catalase, amylase and oxidase enzymes respectively. They were negative for indole test and Hugh leifson test. Isolate BGUMS14, BGUMS16 and BGUMS101 had the ability to liquefy gelatin indicating the presence of gelatinase enzyme. Isolate BGUMS14, BGUMS16, BGUMS93, BGUMS94, BGUMS06, BGUMS473-C and BGUMS837 tested positive for hydrolysis of Caesin, V-P test and tween test indicating the production of Caesinase enzyme, acetoin production in the medium and hydrolysis of tween respectively. These isolates also lacked the ability to degrade tyrosine while Isolate BGUMS101 tested positive. Isolate BGUMS06 and BGUMS94 showed the ability to perform mixed acid fermentations as indicated by the positive methyl red test. Isolates BGUMS93, BGUMS06, BGUMS473-C and BGUMS837 showed the ability to ferment sugar (glucose). Isolates BGUMS14, BGUMS101, BGUMS93, BGUMS473-C, BGUMS837, BGUMS06 showed the ability to reduce nitrate by producing the enzyme nitrate reductase. Isolate BGUMS93, BGUMS473-C, BGUMS837 were found to be positive for citrate, isolates BGUMS94 and BGUMS16 were however, found to be weakly positive and isolate BGUMS101, BGUMS14 and BGUMS06 were found to be negative for citrate utilization indicating the inability to utilize citrate as the sole carbon source. Isolates BGUMS473-C, BGUMS837 and BGUMS06 tested positive for arginine dihydrolase production. Isolate BGUMS101, BGUMS93 and BGUMS06 showed a positive urease test. Except isolate BGUMS101 all the isolates showed growth at a temperature of 50° C while none of the isolates grew at a temperature of 65° C. All the isolates were motile except the Isolates BGUMS101 and BGUMS473-C. All the Isolates had the ability to grow in a medium containing 2% and 5% NaCl. None of the isolates had the capacity to grow in the medium containing 7% NaCl.

84

on

red test red

yl

Isolate Colony oncharacteristics platesNA (Form, margin, elevation) Staining Gram stainingEndospore positiEndospore Catalase Oxidase test Indoletest Hugh test leifson 65°Growth at C Starch hydrolysis Caesin hydrolysis 50°Growth at C Motility test Vouges Proskauer test test Tween reductionNitrate test utilizationCitrate test Sugar (Glucose) fermentation test Meth test Urease Tyrosine test Arginine test Gelatin liquefaction Tentative identification BGUMS Irregular, + + Terminal + W - - - + + + + + + + ------+ Bacillus spp. 14 undulate, flat or sub terminal BGUMS Irregular, + + Central + + - - - + + + + + + - W - - - - - + Bacillus spp. 16 undulate, flat BGUMS Round, + + Central + + - - - + - - - - - + - - - + + - + Bacillus spp. 101 entire, raised BGUMS Round, + + Central + + - - - + + + + + + + + + - + - - - Bacillus spp. 93 entire, raised BGUMS Round, + + Central + W - - - + + + + + + - W - + - - - - Bacillus spp. 94 entire, convex BGUMS Irregular, + + Central + + - - - + + + + + + + - + + + - + - Bacillus spp. 06 undulate, raised BGUMS Filamentous, + + Sub + + - - - + + + - + + + + + - - - + - Bacillus spp. 473-C filamentous, terminal flat BGUMS Filamentous, + + Sub + + - - - + + + + + + + + + - - - + - Bacillus spp. 837 filamentous, terminal flat Bti* Round, + + Sub + + - - - + + + + + - + + + + - - + + Bacillus entire, raised terminal thuringiensis Bs* Round, + + Terminal + + - - - - V + + ------+ Bacillus entire, flat or sub sphaericus terminal

Table 5: Morphological and Biochemical characteristics of the Ribandar isolates

*Bti- Bacillus thuringiensis israelensis H-14* Bs- Bacillus sphaericus Neide 2362 V- variable W- weakly positive

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ii) All the isolates from Miramar sampling site were catalase positive indicating the

presence of catalase enzyme which has the ability to breakdown H2O2 and release oxygen. They also showed positive reaction for oxidase indicating the ability to produce oxidase enzyme. All the isolates were negative for Indole, VP, Hugh leifson and Gelatin (absence of gelatinase enzyme). All the isolates showed positive results for MR indicating the ability to perform mixed acid fermentation, Sugar fermentation indicating the ability to ferment glucose, Tyrosine indicating the presence of tyrosinase enzyme, Arginine indicating production of arginine dihydrolase except B1C. B1C, C1A, C2E showed positive test results for nitrate by producing the enzyme nitrate reductase while others tested negative. Starch results were positive for B1C, C2E indicating the presence of amylase enzyme and for others it was recorded negative. Starch results of C1A was variable. Except for B1C and C1A all Isolates had the ability to hydrolyse Caesin due to the presence of Caesinase enzyme. All the Isolates had the ability to grow in a medium containing 2% and 5% NaCl (except C1A). None of the isolates had the capacity to grow in medium containing 7% NaCl. None of the isolates could withstand higher temperatures like 50° C and 65° C.

86

Table 6: Morphological and Biochemical characteristics of the Miramar isolate

margin, margin,

st

Isolate Colony oncharacteristics platesNA (Form, Staining Gram elevation) stainingEndospore positionEndospore Catalase Oxidase test Indoletest Hugh test leifson 65°CGrowth at Starch hydrolysis Caesin hydrolysis 50°CGrowth at Motility test Vouges Proskauer test test Tween reductionNitrate te utilizationCitrate test Sugar (Glucose) fermentation test Methyl test red test Urease Tyrosine test Arginine test Gelatin liquefaction Tentative identification C1A Irregular, + + Sub + + - - -- V - - + - - + - - - + + - - Bacillus spp. undulate, terminal raised C2A Round, + + Sub - - - - - + - + ------Bacillus spp. entire, terminal + + + raised C2C Round, + + Sub + + - - - - + - + - + ------Bacillus spp. entire, terminal raised C2D Irregular, + + Sub + + - - - - + -- + - + ------Bacillus spp. undulate, terminal raised C2E Irregular, + + Sub + + - - - + + + - + + ------Bacillus spp. undulate, terminal raised BIC Irregular, + + Sub + + - - - + - - + - + + + + + - - + - Bacillus spp. undulate, terminal flat Bti* Round, + + Sub + + - - - + + + + + - + + + + - - + + Bacillus entire, terminal thuringiensis raised Bs* Round, + + Sub + + - - - - V + + ------+ Bacillus entire, flat terminal sphaericus

*Bti- Bacillus thuringiensis israelensis H-14* Bs- Bacillus sphaericus Neide 2362 V- variable W- weakly positive

87

B. Antibiotic sensitivity tests

The pathogenic strains of Bacillus sphaericus are generally known to exhibit natural resistance to Streptomycin and Chloramphenicol [176]. The study bacterial isolates were tested for antibiotic sensitivity against streptomycin (10mg per disc), kanamycin (30mg per disc) and erythromycin (15mg per disc) and the zone of clearance radius was measured in centimetres (Table 7). All isolates were found to be sensitive to Streptomycin except isolate C2A, C2C, C2D and C2E which were found to be resistant. All isolates showed sensitivity to Kanamycin except C1A. All isolates were however, sensitive to erythromycin.

Table 7: Antibiotic sensitivity tests

Isolate Streptomycin Kanamycin Erythromycin (10mg/disc) (30mg/disc) (15mg/disc)

Sensitivity Zone Sensitivity Zone Sensitivity Zone radius radius radius (cms) (cms) (cms)

BGUMS14 S 2.2 S 2.3 S 4

BGUMS16 S 2.8 S 2 S 3.1

BGUMS101 S 2 S 3 S 3

BGUMS93 S 2.4 S 2.5 S 3

BGUMS94 S 2.8 S 2.3 S 2.7

BGUMS06 S 2 S 1.6 S 3

BGUMS473-C S 2.2 S 2.6 S 3.8

BGUMS837 S 1.8 S 2.3 S 2.6

C1A S 2 R - S 3

C2A R - S 2.3 S 2.5

C2C R - S 2.8 S 3.5

C2D R - S 2 S 1.8

C2E R - S 3 S 3

B1C S 2.5 S 2.2 S 2.9

*S- Sensitive *R-Resistant

88

(v) Bio-surfactant production

Isolate BGUMS14, B1C and Bti commercial strain did not produce bio-surfactant as indicated by the absence of a clear halo in the oil film. However Isolate BGUMS16, BGUMS101, BGUMS93, BGUMS94, BGUMS473-C, BGUMS837 and BGUMS06 produced a clear halo in the oil film (Fig. 34). The clearance zone produced was measured in centimeters. Maximum bio-surfactant production was observed in case of Isolate BGUMS473-c as indicated by the clearance zone of 4 cms (Table 8).

Fig. 34: Production of a bio- surfactant by Isolate Oil film BGUMS101

Clearance Zone

89

Table 8: Bio-surfactant production

Sr. No. Isolate No. Bio-surfactant activity Clearance zone in centimeters (cms) 1 BGUMS14 -ve 0 2 BGUMS16 +ve 2.6 3 BGUMS101 +ve 0.5 4 BGUMS93 +ve 0.8 5 BGUMS94 +ve 1 6 BGUMS06 +ve 2.1 7 BGUMS473-c +ve 4 8 BGUMS837 +ve 1 9 C1A +ve 2.1 10 C2A +ve 0.7 11 C2C +ve 1.2 12 C2D +ve 0.5 13 C2E +ve 3 14 B1C -ve 0 15 Bti* -ve 0 16 Bs* +ve 2.9

*Bti – Bacillus thuringiensis israelensis *Bs- Bacillus sphaericus

90

4.5. Crude protein extraction and determination of concentration by Nano drop technique

The bacterial samples were prepared and crude protein extracted by the method of Murphy and Stevens, 1992 with slight modifications as described in Chapter 3 [162]. Protein concentration in mg/ml was determined by Nano drop technique (Table 9).

Table 9: Determination of protein concentrations (mg/ml) using Nanodrop technique

Sr. No Isolate code Protein conc. mg/ml

1 BGUMS 14 101.560

2 BGUMS 16 110.092

3 BGUMS 101 108.970

4 BGUMS 93 100.870

5 BGUMS 94 99.768

6 BGUMS 06 101.439

7 BGUMS 473-C 100.609

8 BGUMS 837 104.876

9 Isolate C1A 102.435

10 Isolate C2A 102.908

11 Isolate C2C 113.737

12 Isolate C2D 98.634

13 Isolate BIC 94.125

14 Bti* 109.833

15 Bs* 105.800

*Bti – Bacillus thuringiensis israelensis *Bs- Bacillus sphaericus 4.6. i) Molecular identification of the new isolates Molecular characterization of these bacterial isolates by PCR amplification of the 16S rRNA gene yielded amplicons of the size 1500 bps (Fig. 35 and 36). Following PCR amplification,

91 the samples were sent for sequencing. The nucleotide sequences were blasted against the NCBI database using BLASTN, Isolate BGUMS14, Isolate BGUMS16 and Isolate BGUMS101 showed sequence similarity with Bacillus aerius (100%), Bacillus safensis (99%) and Sporosarcina soli (99%) species respectively. These sequences were submitted to NCBI GenBank database with NCBI accession no’s MN533914, MN443618 and MN443634 respectively.

1 2 3 4 5 6 7 8 9 10

1500 bp

1000 bp

500 bp

Wells from Left to Right- 1-500bp ladder 2-Isolate 14 3-Isolate 16 4-Isolate 101 5-Isolate 93 6-Isolate 94 7-Isolate 06 8-Isolate 837 9-Isolate 473-C 10-NTC

Fig. 35: Gel image of PCR amplification using 16S rRNA primers

Further, Isolate C1A showed 99.93% sequence similarity with Aneurinibacillus sp, while Isolate C2A and C2C showed high similarity (99.2% and 100% respectively) with Lysinibacillus sp. Isolate B1C showed 100% sequence similarity with Bacillus thuringiensis strain. All sequences have been submitted to NCBI GenBank database with accession no’s MN595035, MN606113, MN606137 and MN606138 respectively.

92

11 2 2 3 3 4 4 5 6 5 7 6 8 79 10 8 9 10

1500 bp 1000 bp

500 bp

Well from left to right-

1-500bp ladder 2-C1A 3-B1C 4-C2A 5-C2C 6-C2D 7-C2E 8-+ve control 9-+ve control 10-NTC

Fig. 36: Gel image of PCR amplification using 16S rRNA primers

4.6. ii) Phylogenetic analysis

The phylogenetic tree construction was based on the comparison of the 16S rRNA sequences generated in this study with sequences of species belonging to Bacillus genus, and other closely related organisms. The strain BGUMS14 formed a coherent branch with Bacillus sp. group including Bacillus aerius, Bacillus pumilus and other closely related species. While the strain BGUMS101 formed a branch with Sporosarcina group including Sporosarcina ginsengisoli, Sporosarcina soli and closely related species. Strain BGUMS16 aligned with Bacillus safensis group with Bacillus australimaris, Bacillus pumilus and other related Bacillus sp. forming outgroups (Fig. 37).

Similarly, the isolate C1A formed a coherent branch with Aneurinibacillus migulanus group. While the isolates C2A and C2C formed a branch with Lysinibacillus group. Isolate B1C aligned with Bacillus thuringiensis group (Fig. 38).

93

Fig. 37: Phylogenetic tree showing the phylogenetic relationships among isolate BGUMS14, BGUMS16, BGUMS101 and their close relatives inferred from 16S rRNA gene sequences from NCBI GenBank.

94

Fig. 38: Phylogenetic tree showing the phylogenetic relationships among isolate BIC, C1A, C2A, C2C and their close relatives inferred from 16S rRNA gene sequences from NCBI GenBank.

95

4.7. Analysis of Culture supernatant and Crude Mosquitocidal Toxin

(CMT) for larvicdal and pupicidal activity

A) Culture Supernatant i) Larvicidal activity The culture supernatant obtained by centrifugation of the bacterial culture and described in chapter 3, was tested against the 3rd instar larvae of Anopheles stephensi, Culex quinquefasciatus and Aedes aegypti. The percentage mortality was recorded and found to be isolate Sporosarcina sp. strain BGUMS101 > Bacillus sp. strain BGUMS14 > Bacillus safensis strain BGUMS16 > Bacillus sp. strain BGUMS473-C > Bacillus sp. strain BGUMS06 > Bacillus sp. strain BGUMS94 > Bacillus sp. strain BGUMS93 > Bacillus sp. strain BGUMS837 against Anopheles stephensi larvae following 24 hours of exposure (Fig. 39). Against Culex quinquefasciatus larvae Bacillus sp. strain BGUMS14 > Sporosarcina sp. strain BGUMS101 > Bacillus safensis strain BGUMS16 > Bacillus sp. strain BGUMS94 > Bacillus sp. strain BGUMS93 > Bacillus sp. strain BGUMS837 > Bacillus sp. strain BGUMS06 > Bacillus sp. strain BGUMS473-C on 24hrs of exposure (Fig. 40). Least activity was observed against Aedes aegypti larvae, Sporosarcina sp. strain BGUMS101 showed 16% ± 1 , Bacillus sp. strain BGUMS837 showed 12% ± 1, Bacillus sp. strain BGUMS14, Bacillus safensis strain 16, Bacillus sp. strain BGUMS93, Bacillus sp. strain BGUMS94, Bacillus sp. strain BGUMS06 all showed 8% mortality and Bacillus sp. strain BGUMS473-C showed 4% ± 0.707 mortality following 24 hours of exposure (Fig. 41). The percentage mortality following 48 hours of exposure was also recorded and found to be between 20% - 76% against Anopheles stephensi larvae, between 76% - 100% against Culex quinquefasciatus larvae and between 8% - 24% against Aedes aegypti larvae. Since larval mortality was observed the CS was processed further for isolation of the Crude mosquitocidal toxin (CMT) in order to perform bioassays. The CS of the isolates obtained from the Miramar sampling site did not show any larvicidal activity hence were not processed further.

96

100 Anopheles larvae - 24hrs 80 Anopheles larvae- 48hrs 60

40

20 Mortality(%)

0 Bacillus sp. Bacillus Sporosarcina Bacillus sp. Bacillus sp. Bacillus sp. Bacillus sp. Bacillus sp. strain safensis strain sp. strain strain strain strain strain strain BGUMS14 BGUMS16 BGUMS101 BGUMS93 BGUMS94 BGUMS06 BGUMS473-C BGUMS837 Isolate Fig. 39: Preliminary screening of culture supernatant of Bacillus sp. against 3rd instar Anopheles stephensi larvae

100 Culex larvae 24hrs Culex larvae 48hrs 80

60

40 Mortality(%) 20

0 Bacillus sp. Bacillus Sporosarcina Bacillus sp. Bacillus sp. Bacillus sp. Bacillus sp. Bacillus sp. strain safensis strain sp. strain strain strain strain strain strain BGUMS14 BGUMS16 BGUMS101 BGUMS93 BGUMS94 BGUMS06 BGUMS473-C BGUMS837 Isolate

Fig. 40: Preliminary screening of culture supernatant of Bacillus sp. against 3rd instar

Culex quinquefasciatus larvae

97

100 Aedes aegypti larvae- 24 hrs

80 Aedes aegypti larvae- 48hrs

60

40 Mortality(%) 20

0 Bacillus sp. Bacillus Sporosarcina Bacillus sp. Bacillus sp. Bacillus sp. Bacillus sp. Bacillus sp. strain safensis strain sp. strain strain strain strain strain strain BGUMS14 BGUMS16 BGUMS101 BGUMS93 BGUMS94 BGUMS06 BGUMS473-C BGUMS837 Isolate Fig. 41: Preliminary screening of the culture supernatant of Bacillus sp. against 3rd

instar Aedes aegypti larvae

ii) Pupicidal activity

The isolates showed variable pupicidal activity. Sporosarcina sp. strain BGUMS101 showed best pupicidal activity with a percentage mortality of 48% ± 1.414 against Anopheles stephensi pupae, 40% ± 2.828 against Culex quinqufasciatus pupae and 32% against Aedes aegypti pupae. The remaining isolates showed low pupicidal activity (Fig. 42, 43, 44).

98

100

80

60

40 Mortality(%) 20

0 Bacillus sp. Bacillus Sporosarcina Bacillus sp. Bacillus sp. Bacillus sp. Bacillus sp. Bacillus sp. strain safensis strain sp. strain strain strain strain strain strain BGUMS14 BGUMS16 BGUMS101 BGUMS93 BGUMS94 BGUMS06 BGUMS473-C BGUMS837 Isolate

Fig. 42: Preliminary screening of the culture supernatant of Bacillus sp. against

Anopheles stephensi pupae

100

80

60

40 Mortality(%) 20

0 Bacillus sp. Bacillus Sporosarcina Bacillus sp. Bacillus sp. Bacillus sp. Bacillus sp. Bacillus sp. strain safensis strain sp. strain strain strain strain strain strain BGUMS14 BGUMS16 BGUMS101 BGUMS93 BGUMS94 BGUMS06 BGUMS473-C BGUMS837 Isolate

Fig. 43: Preliminary screening of culture supernatant of Bacillus sp. against Culex

quinquefasciatus pupae

99

100

80

60

40 Mortality (%)Mortality 20

0 Bacillus sp. Bacillus Sporosarcina Bacillus sp. Bacillus sp. Bacillus sp. Bacillus sp. Bacillus sp. BGUMS14 safensis strain sp. strain BGUMS93 BGUMS94 BGUMS06 BGUMS473-C BGUMS837 BGUMS16 BGUMS101 Isolate

Fig. 44: Preliminary screening of culture supernatant of Bacillus sp. against Aedes

aegypti pupae

B) Crude Mosquitocidal Toxin (CMT)

i) Larvicidal activity

The crude mosquitocidal toxin (CMT) of the bacterial isolates obtained by acid precipitaion of the culture supernatant was subjected to bioassays against the 3rd instar larvae of Anopheles stephensi, Culex. quinquefasciatus, Aedes aegypti and Aedes albopictus (Table 10). The CMT of Sporosarcina sp. strain BGUMS101 did not show significant mosquito larvicidal activity as compared to the commercial strains, being most toxic against Cx. quinquefasciatus (41 ± 0.5 % mortality) on 48 hours of treatment. Similarly, the CMT of Bacillus safensis strain BGUMS16 was most toxic to Cx. quinquefasciatus larvae (46 ± 3 % mortality) on 48 hours treatment with the highest dose of 20 ppm. The larvae of Ae. aegypti were found to be most susceptible to the CMT of Bacillus sp. strain BGUMS14 with 36 ± 1.414 % mortality. The CMT of the commercial strains also evaluated. Bti was highly toxic to Cx. quinquefasciatus and Ae. aegypti with 100% mortality while Bs showed a similar activity profile with 89 ± 0.5 % and 91 ± 1.707 % mortality against Cx. quinquefasciatus and Ae. aegypti respectively on 48 hours of treatment with the highest dose of 20 ppm. The CMT of the remaining active bacterial isolates did not show any activity hence were excluded (Table 10).

100

Table 10: Larvicidal activity (% mortality) of Crude mosquitocidal toxin against 3rd instar larvae of Culex quinquefasciatus, Anopheles stephensi, Aedes aegypti and Aedes albopictus.

Mortality (%) against mosquito species

Anopheles stephensi Culex quinquefasciatus Aedes aegypti Aedes albopictus Isolate Code Dose 24hrs 48hrs 24hrs 48hrs 24hrs 48hrs 24hrs 48hrs (ppm)

1 4 ± 1 6 ± 0.5 11 ± 0.957 14 ± 1 7 ± 0.433 10 ± 0.5 2 ± 0.577 6 ± 0.577 Sporosarcina 5 6 ± 1.290 9 ± 0.829 15 ± 0.957 20 ± 0.816 11 ± 0.433 15 ± 0.433 3 ± 0.957 7 ± 1.258 sp. strain BGUMS101 10 8 ± 0.816 13 ± 0.433 21 ± 0.957 29 ± 0.957 14 ± 1.118 19 ± 0.433 6 ± 1.290 14 ± 1.732

20 21 ± 0.957 25 ± 0.829 31 ± 1.707 41 ± 0.5 18 ± 1.118 28 ± 0.707 9 ± 1.5 17 ± 1.5

1 11 ± 0.957 12 ± 1.414 16 ± 2.160 21 ± 2.061 10 ± 0.577 13 ± 0.5 4 ± 0.816 9 ± 0.5 Bacillus 5 13 ± 1.258 14 ± 1 23 ± 1.258 38 ± 2.380 14 ± 0.577 17 ± 0.957 7 ± 0.5 17 ± 1.258 safensis strain 10 14 ± 1.290 16 ± 0.816 32 ± 3.162 45 ± 2.629 15 ± 1.707 17 ± 1.892 11 ± 1.258 36 ± 2.449

BGUMS16 20 16 ± 0.816 18 ± 0.577 35 ± 2.217 46 ± 3 23 ± 0.957 26 ± 1.914 14 ± 1.290 44 ± 1.154

101

Bacillus sp. 1 0 2 ± 0.577 0 5 ± 0.5 8 ± 1.414 10 ± 1.290 9 ± 0.957 11 ± 0.5 strain 5 5 ± 0.5 10 ± 0.577 2 ± 0.577 9 ± 0.5 10 ± 1.732 12 ± 1.414 10 ± 1.290 12 ± 0.816 BGUMS 14 10 6 ± 1.290 10 ± 1 7 ± 1.258 10 ± 0.577 20 ± 0.816 25 ±0.957 13 ± 0.5 13 ± 0.957

20 8 ± 1.414 13 ± 1.258 11 ± 0.5 11 ± 0.5 27 ± 1.5 36 ± 1.414 16 ± 1.414 17 ± 1.5

1 21 ± 2.753 25 ± 2.753 96 ± 0.816 97 ± 0.957 94 ± 1.290 95 ± 0.957 30 ± 2.516 39 ± 2.217 Bti commercial strain 5 37 ± 0.957 43 ± 0.957 98 ± 0.577 99 ± 0.5 100 100 65 ± 2.217 72 ± 1.825

10 43 ± 1.707 46 ± 2.645 100 100 100 100 73 ± 2.362 78 ± 1.290

20 54 ± 1.914 61 ± 3.774 100 100 100 100 81 ± 1.707 89 ± 1.707

1 10 ± 1.290 13 ± 0.957 29 ± 1.707 45 ± 4.112 34 ± 1.290 43 ± 2.061 32 ± 2.160 36 ± 2.160 Bs commercial 5 13 ± 0.957 17 ± 0.5 40 ± 0.816 55 ± 0.957 73 ± 5.909 83 ± 4.716 39 ± 2.5 47 ± 1.258 strain

10 19 ± 1.258 23 ± 0.957 51 ± 1.707 62 ± 0.577 83 ± 2.753 86 ± 1.914 43 ± 3.304 54 ± 3.109

20 31 ± 1.707 45 ± 1.28 84 ± 0.816 89 ± 0.5 88 ± 2.160 91 ± 1.707 61 ± 74 ±3.109 1.892

*All values are in percentage ± standard error

102

ii) Pupicidal activity

The Crude mosquitocidal toxin (CMT) obtained following acid precipitation was tested against the pupae of the test vector species and the percentage mortality recorded following 24 hours of exposure was recorded. Sporosarcina sp. strain BGUMS101 showed best pupicidal activity with a percentage mortality of 56.66% ± 4.509 against Anopheles stephensi pupae, 51.66% ± 1.527 against Culex quinquefasciatus pupae, 5% and against Aedes aegypti pupae. The remaining isolates showed extremely low pupicidal activity (Fig. 45 - 47) .

100

80

60

40 Mortality(%) 20

0 Bacillus sp. Bacillus Sporosarcina Bacillus sp. Bacillus sp. Bacillus sp. Bacillus sp. Bacillus sp. strain safensis strain sp. strain strain strain strain strain strain BGUMS14 BGUMS16 BGUMS101 BGUMS93 BGUMS94 BGUMS06 BGUMS473-C BGUMS837 Isolate

Fig. 45: Effect of the Crude mosquitocidal toxin (CMT) of Bacillus sp. against

Anopheles stephensi pupae

103

100

80

60

40 Mortality(%) 20

0 Bacillus sp. Bacillus Sporosarcina Bacillus sp. Bacillus sp. Bacillus sp. Bacillus sp. Bacillus sp. strain safensis strain sp. strain strain strain strain strain strain BGUMS14 BGUMS16 BGUMS101 BGUMS93 BGUMS94 BGUMS06 BGUMS473-C BGUMS837 Isolate

Fig. 46: Effect of the Crude mosquitocidal toxin (CMT) of Bacillus sp. against Culex

quinquefasciatus pupae

100

80

60

Mortality(%) 40

20

0 Bacillus sp. Bacillus Sporosarcina Bacillus sp. Bacillus sp. Bacillus sp. Bacillus sp. Bacillus sp. strain safenis strain sp. strain strain strain strain strain strain BGUMS14 BGUMS16 BGUMS101 BGUMS93 BGUMS94 BGUMS06 BGUMS473-C BGUMS837 Isolate

Fig. 47: Effect of the Crude mosquitocidal toxin (CMT) of Bacillus sp. against Aedes

aegypti pupae

104

iii) The dead pupae were observed under 40X magnification of a stereo microscope. It was observed that in Anopheles stephensi and Culex quinquefasciatus the death of the pupae was not only by direct action of the crude mosquitocidal toxin however in certain cases the fully formed adult mosquito was unable to emerge from the pupal casing hence seem to be causing emergence inhibition. This mode of action was not observed in case of Aedes aegypti pupae (Fig. 48 - 50).

105

Fig Mosquito Live Pupae Dead pupae Emerged and dead No. species

48. Anopheles stephensi

49. Culex quinquefasciatus

50. Aedes spp. -

Fig. 48 - 50: Showing the effect of crude mosquitocidal toxin (CMT) of Isolate Sporosarcina sp. strain BGUMS101 against the pupae of Anopheles stephensi, Culex quinquefasciatus and Aedes spp.

106

4.8. Toxin Studies

The primers used for detection of toxin genes have been listed in the previous chapter. Isolate BGUMS 101 was negative for all the toxin genes tested indicating the presence of a novel toxic component responsible for its larvicidal activity. The Isolate CIA was found to possess BinA, BinB and MtX toxin gene. Commercial strains of Bacillus thuringiensis israelensis H-14 and Bacillus sphaericus Neide 2362 were used as controls (Fig 53 - 61, Table 11). Cry2 (UN) has been reported to be specific to lepidopterans hence no amplification in mosquito pathogenic bacterial strains was detected [177].

Table 11: Toxin gene detection by PCR based methods

Sr. Gene Cry Cry4 Cyt1 Cyt2 BINA BINB Mtx Gyrase Surfactin No. 2(UN) (UN) (UN) (UN) gene

1 Sporosarcina sp. ------strain BGUMS101 2 Bacillus sp. strain ------BGUMS14 3 Bacillus safensis. ------strain BGUMS16 4 Bacillus sp. strain ------BGUMS 93 5 Bacillus sp. strain ------BGUMS 94 6 Bacillus sp. strain ------BGUMS 06 7 Bacillus sp. strain ------+ - BGUMS 473-C 8 Bacillus sp. strain ------+ - BGUMS 837 9 Aneurinibacillus sp. - - - - + + + - - strain C1A 10 Lysinibacillus sp. - - - - + + + - - strain C2A 11 Lysinibacillus sp. - - - - + + + - - strain C2C 12 Lysinibacillus sp. - - - - + + + - - strain C2D 13 Bacillus sp. strain - + + + - - - - - BIC 14 Lysinibacillus sp. - - - - + + + - - strain C2E 15 Bti* - + + + - - - - -

16 Bs* - - - - + + + - -

*Bti – Bacillus thuringiensis israelensis H-14 *Bs- Bacillus sphaericus Neide 2362

107

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

439 bp

Wells from Left to Right Wells from Left to Right

1- 500bp ladder 2- Bacillus sp. strain BGUMS14 3- Bacillus safensis 1- 100bp ladder 2- Bacillus sp. strain BGUMS14 3- Bacillus safensis strain BGUMS16 4- Sporosarcina sp. strain BGUMS101 5- Bacillus sp. strain BGUMS16 4- Sporosarcina sp. strain BGUMS101 5- Bacillus strain BGUMS94 6- Bacillus sp. strain BGUMS93 7- Bacillus sp. strain sp. strain BGUMS94 6- Bacillus sp. strain BGUMS93 7- Bacillus sp. BGUMS06 8- Bacillus sp. strain BGUMS473-C 9- Bacillus sp. strain strain BGUMS06 8- Bacillus sp. strain BGUMS473-C 9- Bacillus sp. BGUMS837 strain BGUMS837 10- Aneurinibacillus sp. strain C1A 10-Aneurinibacillus sp. strain C1A 11- Lysinibacillus sp. strain C2A 11- Lysinibacillus sp. strain C2A 12- Lysinibacillus sp. strain C2C 12- Lysinibacillus sp. strain C2C 13- Lysinibacillus sp. strain C2D 13- Lysinibacillus sp. strain C2D 14- Bacillus sp. strain B1C 14- Lysinibacillus sp. strain C2E 15- Bacillus sp. strain B1C 16- -ve 15- +ve control (Bti) 16- Lysinibacillus sp. strain C2E 17- -ve control control (Bs)

Fig. 51: Detection of Cry2(UN) gene Fig. 52: Detection of Cry4 (UN) gene

108

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

480 bp 356 bp

Wells from Left to Right Wells from Left to Right

1- 100bp ladder 2- Bacillus sp. strain BGUMS14 3- Bacillus safensis 100bp ladder 2- Bacillus sp. strain BGUMS14 3- Bacillus safensis strain BGUMS16 4- Sporosarcina sp. strain BGUMS101 5- Bacillus sp. strain BGUMS16 4- Sporosarcina sp. strain BGUMS101 5- Bacillus strain BGUMS94 6- Bacillus sp. strain BGUMS93 7- Bacillus sp. strain sp. strain BGUMS94 6- Bacillus sp. strain BGUMS93 7- Bacillus sp. BGUMS06 8- Bacillus sp. strain BGUMS473-C 9- Bacillus sp. strain strain BGUMS06 8- Bacillus sp. strain BGUMS473-C 9- Bacillus sp. BGUMS837 10- Aneurinibacillus sp. strain C1A 11- Lysinibacillus sp. strain BGUMS837 10- Aneurinibacillus sp. strain C1A strain C2A 12- Lysinibacillus sp. strain C2C 13- Lysinibacillus sp. 11- Lysinibacillus sp. strain C2A 12- Lysinibacillus sp. strain C2C strain C2D 14- Bacillus sp. strain BIC 15- Bacillus sp. strain B1C 16- 13- Lysinibacillus sp. strain C2D 14- Bacillus sp. strain BIC 15- Bti Lysinibacillus sp. strain C2E 17-NTC 18- Bti +ve control 19- Bacillus +ve control 16- Lysinibacillus sp. strain C2E 17- Bacillus sphaericus sphaericus –ve control -ve control

Fig. 53: Detection of Cry1 (UN) gene Fig. 54: Detection of Cyt 2 (UN) gene

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

1054 bp

720 bp

Wells from Left to Right Wells from Left to Right

1- 500bp ladder 2- Bacillus sp. strain BGUMS14 3- Bacillus safensis 1- 500bp ladder 2- Bacillus sp. strain BGUMS14 3- Bacillus safensis strain BGUMS16 4- Sporosarcina sp. strain BGUMS101 5- Bacillus strain BGUMS16 4- Sporosarcina sp. strain BGUMS101 5- Bacillus sp. sp. strain BGUMS94 6- Bacillus sp. strain BGUMS93 7- Bacillus sp. strain BGUMS94 6- Bacillus sp. strain BGUMS93 7- Bacillus sp. strain strain BGUMS06 8- Bacillus sp. strain BGUMS473-C 9- Bacillus BGUMS06 8- Bacillus sp. strain BGUMS473-C 9- Bacillus sp. strain sp. strain BGUMS837 10- Lysinibacillus sp. strain C2C 11- Bacillus BGUMS837 10- Aneurinibacillus sp. strain C1A 11- Lysinibacillus sp. sp. strain BIC 12- Lysinibacillus sp. strain C2A 13- Aneurinibacillus strain C2A 12- Lysinibacillus sp. strain C2C 13- Lysinibacillus sp. sp. strain C1A 14- ISOLATE C2D 15- Bs –ve control strain C2D 14- Bacillus sp. strain BIC 15- Lysinibacillus sp. strain 16- Lysinibacillus sp. strain C2E C2E 16- Bs Negative control

Fig. 55: Detection of BinA gene Fig. 56: Detection of BinB gene

109

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

600 – 800 bp

Wells from Left to Right

1- 500bp ladder 2- Bacillus sp. strain BGUMS14 3- Bacillus safensis strain BGUMS16 4- Sporosarcina sp. strain BGUMS101 5- Bacillus sp. strain BGUMS94 6- Bacillus sp. strain BGUMS93 7- Bacillus sp. strain BGUMS06 8- Bacillus sp. strain BGUMS473-C 9- Bacillus sp. strain BGUMS837 10- Aneurinibacillus sp. strain C1A 11- Lysinibacillus sp. strain C2A 12- Lysinibacillus sp. strain C2C 13- Lysinibacillus sp. strain C2D 14- Lysinibacillus sp. strain C2E 15- +ve control 16- Bacillus sp. strain BIC 17- Negative control

Fig. 57: Detection of Mtx toxin gene

1 2 3 4 5 6 7 8 9 10 11 12 1 2 3 4 5 6 7 8 9 10 11 12

1075 1075 bp bp

Wells from Left to Right Wells from Left to Right

1- 100bp ladder 2- Bacillus sp. strain BGUMS14 3- Bacillus safensis 1- 500bp ladder 2- Aneurinibacillus sp. strain C1A 3- Lysinibacillus strain BGUMS16 4- Sporosarcina sp. strain BGUMS101 5- Bacillus sp. sp. strain C2A 4- Lysinibacillus sp. strain C2C 5- Lysinibacillus sp. strain BGUMS93 6- Bacillus sp. strain BGUMS94 7- Bacillus sp. strain strain C2D 6- Bacillus sp. strain BIC 7- Lysinibacillus sp. strain C2E BGUMS06 8- Bacillus sp. strain BGUMS473-C 9- Bacillus sp. strain 8- Bs 9- Bti 10- Negative control 11- Isolate KS1 +ve control BGUMS837 10- KS1 +ve control 11- KS2 +ve control 12- Negative 12- Isolate KS2 +ve control control

Fig. 58: Detection of Gyrase toxin gene

110

1 2 3 4 5 6 7 8 9 10 11 12 1 2 3 4 5 6 7 8 9 10 11 12

675 bp

675 bp

Wells from Left to Right Wells from Left to Right

1- 100bp ladder 2- Bacillus sp. strain BGUMS14 3- Bacillus safensis 1- 500bp ladder 2- Aneurinibacillus sp. strain C1A 3- Lysinibacillus

strain BGUMS16 4- Sporosarcina sp. strain BGUMS101 5- Bacillus sp. sp. strain C2A 4- Lysinibacillus sp. strain C2C 5- Lysinibacillus sp.

strain BGUMS93 6- Bacillus sp. strain BGUMS94 7- Bacillus sp. strain strain C2D 6- Bacillus sp. strain BIC 7- Lysinibacillus sp. strain C2E

BGUMS473-C 8- Bacillus sp. strain BGUMS837 9- BGUMS06 10- KS1 8- Bs 9- Bti 10- Negative control 11- Isolate KS1 +ve control

+ve control 11- KS2 +ve control 12- Negative control 12- Isolate KS2 +ve control

Fig. 59: Detection of Surfactin toxin gene

4.9. i) Main Bioassay tests

rd The LC50 and LC90 values of the three most active isolates against laboratory reared 3 instar larvae of four test mosquito species were calculated by carrying out the main bioassay as described in chapter 3. Sporosarcina sp. strain BGUMS101 showed a broad spectrum of mosquito larvicidal activity across the four test vector species at lowest doses. The mortality increased with increase in the dose and time period of exposure. The larvae of Aedes albopictus was the most susceptible to Sporosarcina sp. strain BGUMS101 (LC50 = 0.18 ppm) followed by Culex quinquefasciatus (LC50 = 0.22 ppm) and Aedes aegypti (LC50 =

1.01 ppm). Anopheles stephensi larvae was the least susceptible (LC50 = 3.22 ppm). When treated with Bacillus safensis strain BGUMS16, Aedes species showed highest mortality

(LC50 = 34.55 and 64.05 ppm against Aedes albopictus and Aedes aegypti respectively) followed by Anopheles stephensi (LC50 = 96.45 ppm). The larvae of Culex quinquefasciatus was least susceptible (LC50 = 132.04 ppm). A similar pattern of susceptibility was observed on treatment with Bacillus sp. strain BGUMS14. Aedes species showed highest susceptibility (LC50 = 82.02 and 129.07 ppm against Aedes albopictus and Aedes aegypti

111 respectively) followed by Anopheles stephensi and Culex quinquefasciatus (LC50 = 130.8 ppm and 131.98 ppm respectively) on 24 hours of treatment. The LC50 and LC90 values of commercial strains Bacillus thuringiensis israelensis (Bti) and Bacillus sphaericus Neide (Bs) were also recorded and compared (Table 12 and 13). All the toxic isolates showed similar pattern with the mortality increasing with increase in the dose (ppm). Isolate Anuerinibacillus sp. strain C1A showed the highest toxicity against the 3rd instar larvae of the 3 test vector species with Culex quinquefasciatus being the most susceptible (LC50 =

0.08 ppm) followed by Aedes aegypti (LC50 = 13.12 ppm) and Anopheles stephensi (LC50 = 39.42 ppm).

112

Table 12: Larvicidal activity in parts per million (ppm) of newly isolated Bacillus sp. against 3rd instar larvae of Anopheles stephensi, Culex quinquefasciatus, Aedes aegypti and Aedes albopictus.

Mosquito species Isolate Code Anopheles stephensi Culex quinquefasciatus Aedes aegypti Aedes albopictus 24hrs 48hrs 24hrs 48hrs 24hrs 48hrs 24hrs 48hrs

Sporosarcina 3.22 1.62 0.22 0.10 1.01 0.66 0.18 0.13 sp. strain (1.47-7.02) (0.73-3.57) (0.12- 0.41) (0.04-0.22) (0.62-1.64) (0.36-1.20) (0.11-0.29) (0.07-0.22) BGUMS101 LC50 46.78 32.40 3.60 1.52 5.61 6.09 0.67 0.58 LC90 (21.46- (14.70- (1.94-6.69) (0.70-3.27) (3.46-9.09) (3.35- (0.42-1.08) (0.33-1.00) 101.98) 71.39) 11.05)

Bacillus 96.45 62.76 132.04 99.29 64.04 42.65 34.55 16.76 safensis (59.71- (37.46- (79.76- (56.94- (43.34- (27.13- (9.47- (5.50-51.04) strain BGUMS16 155.79) 105.14) 218.59) 173.13) 94.64) 67.06) 125.98) LC50

835.17 615.82 1266.94 1224.30 335.39 271.14 923.80 314.25 LC90 (517.05- (367.60- (765.31- (702.15- (227.30- (172.46- (253.33- (103.19- 957.01) 1349.00) 1031.65) 2097.38) 2134.75) 496.35) 426.27) 1368.74)

113

Bacillus sp. 130.81 62.46 131.97 84.95 129.07 66.80 82.02 51.43 strain (75.91- (42.68- (86.36- (58.60- (73.71- (63.40- (31.27- (21.76-121.51) BGUMS 14 225.41) 91.42) 201.67) 123.15) 225.98) 102.82) 215.12)

LC50 704.67 288.71 852.68 286.98 852.66 294.02(215 809.41 441.12

(490.99- (211.22- (401.45- (211.03- (571.60- .083- (308.62- (186.70- 1042.21) LC90 1011.34) 394.62) 825.13) 390.27) 1271.73) 401.93) 1122.85)

0.34 0.21 0.0002 0.00009 0.00104 0.00045 0.00252 0.00015 Bti commercial (0.19-0.61) (0.09-0.46) (0.0001- (0.00002- (0.00021- (0.00009- (0.00057- (0.0002-0.00129) strain 0.0021) 0.00031) 0.00508) 0.0022) 0.01121) LC50 1.83 0.63 0.02 0.003 0.09 0.020 0.271 0.048

LC90 (1.02-3.25) (0.38- 1.02) (0.002- (0.001- (0.02-0.48) (0.0042- (0.06-1.20) (0.005-0.421) 0.31) 0.012) 0.101)

Bs commercial 1.86 0.88 0.00006 0.00002 0.12 0.05 0.23 0.02 strain (0.72-4.82) (0.31-2.45) (0.00001- (0.00001- (0.04- (0.01- (0.09- (0.008- 0.091)

LC50 0.00029) 0.0001) 0.35) 0.16) 0.59)

20.23 18.25 0.007 0.0049 2.411 0.995 3.594 0.380 LC90 (7.83- (7.45-44.68) (0.001- (0.00074- (0.872- (0.319- (1.439- (0.110-1.313) 52.26) 0.034) 0.03267) 6.663) 3.100) 8.977)

*All values are in parts per million (ppm)

114

Table 13: Larvicidal activity in parts per million (ppm) of newly isolated Bacillus sp. against 3rd instar larvae of Anopheles stephensi,

Culex quinquefasciatus, Aedes aegypti and Aedes albopictus.

Mosquito species Isolate Code Anopheles stephensi Culex quinquefasciatus Aedes aegypti Aedes albopictus 24hrs 48hrs 24hrs 48hrs 24hrs 48hrs 24hrs 48hrs

Aneurinibacillus 39.42 30.68 0.08 0.032 13.18 3.65 23.58 20.593 sp. strain C1A (17.38- (12.36- (0.038- (0.012- (7.81- (2.01- (12.54-50.08) (9.579-

LC50 89.36) 80.88) 0.169) 0.078) 22.26) 6.63) 44.273)

303.70 261.28 0.87 0.45 61.15 23.22 636.723 190.259 LC90 (133.96- (184.36- (0.423- (0.174- (36.21- (12.78- (151.245- (88.497- 688.49) 461.46) 1.854) 1.183) 103.24) 42.17) 1156.542) 409.034)

Lysinibacillus sp. 95.74 38.28 0.30 0.16 13.18 3.65 16.500 12.729 strain C2A (39.43 - (9.85- (0.174- (0.102- (7.81- (2.01- (9.563- (7.689-

LC50 232.42) 148.68) 0.502) 0.300) 22.26) 6.63) 28.468) 21.073)

408.76 299.17 1.32 0.62 399.95 96.31 89.769 60.629 LC90 (280.28- (149.44- (0.77-2.24) (0.34- (194.7- (52.42- (52.030- (36.622- 820.07) 445.80) 1.00) 821.27) 176.93) 154.883) 100.373)

115

Lysinibacillus sp. 54.22 29.35 0.25 0.15 13.18 3.65 41.848 24.907 strain C2C (17.39- (12.361- (0.13-0.45) (0.08- (7.81- (2.01- (28.614- (16.401-

LC50 89.37) 80.89) 0.28) 22.26) 6.63) 61.203) 37.824)

323.78 144.33 1.37 0.70 399.95 96.31 124.802 92.703 LC90 (133.96- (54.81- (0.75-2.51) (0.37- (194.7- (52.42- (85.334- (61.044- 688.49) 358.68) 1.33) 821.27) 176.93) 182.523) 140.780)

2.011 0.37 0.162 0.13 0.14 0.13 0.189 0.161 Bacillus sp. (0.83-4.85) (0.18-0.75) (0.11-0.23) (0.091- (0.10- (0.09- (0.131-0.272) (0.112-0.233) strain B1C 0.197) 0.19) 0.17) LC50

50.2 3.99 0.528 0.461 0.384 0.315 0.660 0.530

LC90 (20.79- (1.96-8.11) (0.36-0.76) (0.31- (0.28- (0.23- (0.458- (0.368-0.763)

121.17) 0.68) 0.52) 0.43) 0.951)

0.34 0.21 0.0002 0.00009 0.00104 0.00045 0.00252 0.00015 Bti commercial (0.19-0.61) (0.09-0.46) (0.0001- (0.00002- (0.00021- (0.00009 (0.00057- (0.0002- strain 0.0021) 0.00031) 0.00508) -0.0022) 0.01121) 0.00129) LC50 1.83 0.63 0.02 0.003 0.09 0.020 0.271 0.048

(1.02-3.25) (0.38- (0.002- (0.001- (0.02- (0.0042- (0.06-1.20) (0.005-0.421) LC90 1.02) 0.31) 0.012) 0.48) 0.101)

116

Bs commercial 1.86 0.88 0.00006 0.00002 0.12 0.05 0.23 0.02 strain (0.72-4.82) (0.31-2.45) (0.00001- (0.00001- (0.04- (0.01- (0.09- 0.59) (0.008- 0.091)

LC50 0.00029) 0.0001) 0.35) 0.16)

20.23 18.25 0.007 0.0049 2.411 0.995 3.594 0.380 LC90 (7.83- (7.45- (0.001- (0.00074- (0.872- (0.319- (1.439- (0.110-1.313) 52.26) 44.68) 0.034) 0.03267) 6.663) 3.100) 8.977)

*All values are in parts per million (ppm)

117 ii) Mode of action of the larvicidal bacteria

Mode of action of the larvicidal bacilli was studied against An. stephensi, Cx. quinquefasciatus, Ae. aegypti and Aedes albopictus during the main bioassays by observing the internal and external changes in the larvae. The dead larvae were observed under 40X magnification of a stereo microscope (Fig. 60 - 63).

On microscopic observation, the gut wall of the dead larvae were seen lysed mainly in the mid- gut region. In the case of An. stephensi on certain occasions, the active larvae were seen consuming the sluggish and dead larvae. No such activity (cannibalistic behaviour) was observed in the case of Cx. quinquefasciatus and Ae. aegypti larvae.

Hence, it was concluded that the mode of action of the bacterial isolates was due to lysis of the midgut of the larvae resulting in septicemia and death of the larvae.

118

Fig Mosquito Untreated Treated with Sporosarcina Treated with Aneurinibacillus No. Species sp. strain BGUMS101 sp. strain C1A 60. Anopheles stephensi

61. Culex quinquefasciatus

62. Aedes aegypti

63. Aedes albopictus

Fig. 60 - 63: Depicting the death of larvae of the test species due to midgut lysis when exposed to mosquito-pathogenic bacilli producing endotoxins

119

4.10. Effect of sub-lethal dose of the bacilli/toxin

The sub lethal effect of Sporosarcina sp. strain BGUMS101 which showed a broad spectrum of mosquito larvicidal activity was studied at the adult F1 generation stage of Anopheles stephensi, Aedes aegypti and Culex quinquefasciatus mosquito which had been exposed to the microbial agent at the larval stage in comparison to the control untreated group. Biological studies included fecundity (number of eggs produced) at the F1 generation, life span and survival rate of treated populations, development period (number of days required at every life stage), egg hatchability, pupation percentage and adult emergence rate. The results were recorded.

Anopheles stephensi:

Table 14: A comparison of Egg Hatch, Pupation and Adult emergence Rates in Test and Control

CONTROL TEST P-value^ n % n % Eggs 677 - 421 - - Larvae 610 90.10* 368 87.41* 0.1644 Pupae 255 41.80** 226 61.41** <0.001 Adults 242 94.90$ 193 85.39$ 0.0004 ^Chi-square test

The larval formation rate was lower in the test than control group, but the difference was not statistically significant (P-value = 0.164, using chi-square test). The pupation rate was higher in test than control group, it was also statistically significant (P-value < 0.001, using chi-square test). In addition, adult emergence was lower in test group than control group, with a statistically significant difference between them (P-value = 0.0004, using chi-square test) as seen in Table 14.

120

Supplementary table 14A indicating pupation rate and adult emergence rate from egg and from 1st instar larvae of Anopheles stephensi respectively

Control Test P-value* n % n %

From egg

Pupation rate 255 37.7 226 53.7 <0.001

Adult emergence rate 242 35.7 193 45.8 0.001

From 1st instar Larvae

Pupation rate 255 41.8 226 61.4 <0.001

Adult emergence rate 242 39.7 193 52.5 <0.001

^Chi-square test

Table 15: A comparison of Fecundity (oviposition) of Anopheles stephensi F1 females in test and control groups

Gonotrophic Control Test P- cycle n Median Min – Max n Median Min – Max value*

1 10 57.5 0 – 98 10 55.5 0 – 75 0.639

2 9 33 0 – 104 8 15 0 – 68 0.588

3 7 18 0 – 38 4 15 10 – 18 0.704

4 4 0 0 – 73 4 0 0 – 0 0.317

Total 30 20 0 – 104 26 13 0 – 75 0.485

*Mann-Whitney rank sum test

Median egg laid was 13 and 20 for test and control group respectively, but the difference was not statistically significant (P-value = 0.485, using Mann-Whitney rank sum test). In the same manner, each of the gonotrophic cycle did not show significant difference between the test and control group (Table 15).

121

50 CONTROL No. of eggs laid 45 40 CONTROL No. of eggs hatched 35 female TEST No. of eggs laid 30 TEST No. of eggs hatched 25 20 15 10

Anopheles stephensi 5

per per 0

Mean number Meannumber eggsof laid/hatched 1 2 3 4 Gonotrophic cycle

Fig. 64: Mean number of eggs laid and hatched per treated and control Anopheles stephensi female during each gonotrophic cycle

Table 16: Hatchability of eggs produced by F1 Anopheles stephensi females during different gonotrophic cycles

Gonotrophic Control Test P-

cycle n Median Min – Max n Median Min – Max value*

1 10 51 0 – 90 10 28 0 – 70 0.434

2 9 28 0 – 98 8 9 0 – 61 0.589

3 7 11 0 – 31 4 7.5 4 – 15 0.704

4 4 0 0 – 62 4 0 0 – 0 0.317

Total 30 15.5 0 – 98 26 5.5 0 – 70 0.345

*Mann Whitney rank sum test

The median egg hatchability was 5.5 and 15.5 for test and control group respectively, but the difference was not statistically significant (P-value = 0.345, using Mann-Whitney rank sum

122 test). In the same manner, each of the gonotrophic cycle did not show significant difference between the test and control group (Fig. 64, Table 16).

Supplementary table 16A of overall egg hatchability in Control vs. Test population of Anopheles stephensi

Group Egg hatchability P- Total Yes No value*

Control 871 (89.6%) 101 (10.4%) 972 <0.001 Test 511 (76.2%) 160 (23.8%) 671

Total 1382 (84.1%) 261 (15.9%) 1643

^Chi-square test

Supplementary table 16B taking number of females of Anopheles stephensi in control and test into account

Egg Hatchability (%) Group P-value# n Mean Min – Max

Control 18 84.82 (12.33) 50.00 – 97.14 0.052 Test 15 70.87 (26.00) 8.57 – 94.34

Total 33 78.48 (20.64) 8.57 – 97.14

#t-test

The mean egg hatchability was 70.87 and 84.82 for test and control respectively. Hence egg hatchability was more in control group than test group, the difference was statistically significant among them (P-value = 0.052, t-test) as seen in Table 16A and B.

123

Table 17A: Life span of the F1 generation Anopheles stephensi test female mosquitoes

Test Female Cum. Survival Day Number Mortality SE 95% CI Mortality probability 1 148 34 34 0.7703 0.0346 0.6937 - 0.8300 2 114 17 51 0.6554 0.0391 0.5729 - 0.7258 3 97 25 76 0.4865 0.0411 0.4039 - 0.5640 4 72 5 81 0.4527 0.0409 0.3712 - 0.5306 5 67 7 88 0.4054 0.0404 0.3261 - 0.4832 6 60 21 109 0.2635 0.0362 0.1955 - 0.3363 7 39 9 118 0.2027 0.033 0.1423 - 0.2708 8 30 1 119 0.1959 0.0326 0.1365 - 0.2634 9 29 2 121 0.1824 0.0317 0.1251 - 0.2485 10 27 5 126 0.1486 0.0292 0.0970 - 0.2107 11 22 2 128 0.1351 0.0281 0.0860 - 0.1954 12 20 3 131 0.1149 0.0262 0.0699 - 0.1721 13 17 2 133 0.1014 0.0248 0.0594 - 0.1562 14 15 5 138 0.0676 0.0206 0.0346 - 0.1156 16 10 1 139 0.0608 0.0196 0.0299 - 0.1072 17 9 1 140 0.0541 0.0186 0.0253 - 0.0987 19 8 1 141 0.0473 0.0174 0.0209 - 0.0900 23 7 3 144 0.027 0.0133 0.0089 - 0.0631 25 4 1 145 0.0203 0.0116 0.0055 - 0.0537 26 3 1 146 0.0135 0.0095 0.0027 - 0.0439 29 2 1 147 0.0068 0.0067 0.0006 - 0.0340 33 1 1 148 0 - - SE: Standard error, CI: Confidence interval (Lower and Upper limit)

124

Table 17B: Life span of the F1 generation Anopheles stephensi Test male mosquitoes

Test Male Cum. Survival Day Number Mortality SE 95% CI Mortality probability 1 45 0 0 1.0000 - - 2 45 14 14 0.6889 0.0690 0.5320 - 0.8025 3 31 3 17 0.6222 0.0723 0.4646 - 0.7455 5 28 1 18 0.6000 0.0730 0.4427 - 0.7260 6 27 4 22 0.5111 0.0745 0.3579 - 0.6450 8 23 1 23 0.4889 0.0745 0.3374 - 0.6241 9 22 2 25 0.4444 0.0741 0.2972 - 0.5816 10 20 3 28 0.3778 0.0723 0.2391 - 0.5157 11 17 1 29 0.3556 0.0714 0.2203 - 0.4932 12 16 1 30 0.3333 0.0703 0.2018 - 0.4704 13 15 1 31 0.3111 0.0690 0.1837 - 0.4473 15 14 4 35 0.2222 0.0620 0.1150 - 0.3514 16 10 2 37 0.1778 0.0570 0.0833 - 0.3010 17 8 1 38 0.1556 0.0540 0.0684 - 0.2751 18 7 2 40 0.1111 0.0468 0.0407 - 0.2213 19 5 2 42 0.0667 0.0372 0.0173 - 0.1639 20 3 1 43 0.0444 0.0307 0.0081 - 0.1333 22 2 2 45 0 . - SE: Standard error, CI: Confidence interval (Lower and Upper limit)

125

Table 17C: Life span of the F1 generation Anopheles stephensi control female mosquitoes

Control Female Cum. Survival Day Number Mortality SE 95% CI Mortality probability 1 122 9 9 0.9262 0.0237 0.8630 - 0.9609 2 113 25 34 0.7213 0.0406 0.6326 - 0.7921 3 88 7 41 0.6639 0.0428 0.5727 - 0.7401 4 81 11 52 0.5738 0.0448 0.4811 - 0.6558 5 70 14 66 0.459 0.0451 0.3689 - 0.5444 6 56 11 77 0.3689 0.0437 0.2840 - 0.4537 7 45 16 93 0.2377 0.0385 0.1666 - 0.3161 9 29 1 94 0.2295 0.0381 0.1595 - 0.3072 10 28 3 97 0.2049 0.0365 0.1386 - 0.2804 11 25 1 98 0.1967 0.036 0.1317 - 0.2714 12 24 3 101 0.1721 0.0342 0.1114 - 0.2441 14 21 1 102 0.1639 0.0335 0.1047 - 0.2348 16 20 1 103 0.1557 0.0328 0.0981 - 0.2256 17 19 1 104 0.1475 0.0321 0.0915 - 0.2163 18 18 3 107 0.123 0.0297 0.0722 - 0.1879 19 15 2 109 0.1066 0.0279 0.0598 - 0.1686 20 13 1 110 0.0984 0.027 0.0537 - 0.1589 21 12 2 112 0.082 0.0248 0.0419 - 0.1391 22 10 2 114 0.0656 0.0224 0.0307 - 0.1188 23 8 4 118 0.0328 0.0161 0.0108 - 0.0759 25 4 1 119 0.0246 0.014 0.0067 - 0.0645 26 3 1 120 0.0164 0.0115 0.0032 - 0.0527 27 2 1 121 0.0082 0.0082 0.0007 - 0.0408 29 1 1 122 0 - - SE: Standard error, CI: Confidence interval (Lower and Upper limit)

126

Table 17D: Life span of the F1 generation Anopheles stephensi control male mosquitoes

Control Male Cum. Survival Day Number Mortality SE 95% CI Mortality probability 1 101 13 13 0.8713 0.0333 0.7887 - 0.9231 2 88 12 25 0.7525 0.0429 0.6561 - 0.8254 3 76 26 51 0.4950 0.0497 0.3944 - 0.5878 5 50 5 56 0.4455 0.0495 0.3471 - 0.5392 6 45 6 62 0.3861 0.0484 0.2917 - 0.4796 7 39 12 74 0.2673 0.0440 0.1854 - 0.3560 8 27 2 76 0.2475 0.0429 0.1684 - 0.3347 9 25 2 78 0.2277 0.0417 0.1517 - 0.3133 10 23 2 80 0.2079 0.0404 0.1352 - 0.2915 12 21 1 81 0.1980 0.0397 0.1270 - 0.2806 13 20 4 85 0.1584 0.0363 0.0952 - 0.2361 14 16 1 86 0.1485 0.0354 0.0875 - 0.2247 15 15 7 93 0.0792 0.0269 0.0370 - 0.1422 16 8 1 94 0.0693 0.0253 0.0305 - 0.1297 17 7 4 98 0.0297 0.0169 0.0080 - 0.0772 19 3 3 101 0.0000 - - SE: Standard error, CI: Confidence interval (Lower and Upper limit)

127

Kaplan-Meier survival estimates

1.00

0.75

0.50

Survival probability 0.25

0.00 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60 Days

Test Female Control Female

Fig. 65: Kaplan-Meier survival curves of the test and control F1 generation Anopheles stephensi females

Kaplan-Meier survival estimates

1.00

0.75

0.50

Survival probability 0.25

0.00 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60 Days

Test Male Control Male

Fig. 66: Kaplan-Meier survival curves of the test and control F1 generation Anopheles stephensi males

128

Supplementary table 18A depicting a comparison of mean survival time of test and control females and males of Anopheles stephensi

Median survival days Group P-value* Test Control

Female 3 5 0.0606 Male 8 3 0.0099

*Log-rank test

The median survival days of females for test and control group was 3 and 5 respectively, and the survival curve were not statistically significant between groups (P-value = 0.0606, using Log-rank test) as seen in Table 17 A, B, C, D, Fig. 65 and 66. Median survival days of male for test and control group were 8 and 3 respectively, and the survival curves were statistically significant between the groups (P-value = 0.0099, using Log-rank test) as seen in Table 18.

129

700 Emergence DAY 10 - DAY 16 = 7 DAYS 400 Emergence DAY 9 - DAY 17 = 9 DAYS Pupae DAY 9 - DAY 15 = 7 DAYS Pupae DAY 8 - DAY 18 = 11 DAYS 600 350 L1-L4 DAY 1 - DAY 12 = 12 DAYS L1-L4 DAY 1 - DAY 18 = 18 days

500 300

250 400 200

Number 300 Number 150 200 100

100 50

0 0 1 2 3 4 5 6 7 8 9 10111213141516171819 1 2 3 4 5 6 7 8 9 10111213141516171819 DAYS DAYS

Fig. 67A and B: Larval development (L1-L4), pupal formation and adult emergence in the control and treated population of Anopheles stephensi mosquitoes

The developmental period is increased in the test population of Anopheles stephensi as compared to the control population of Anopheles stephensi mosquito. In the test population larval development: L1 - L4 = 18 days, pupal development and adult emergence = 11 days and in the control population the larval development: L1 - L4 = 12 days, pupal development and adult emergence = 7 days. (Fig. 67A and B).

130

Aedes aegypti:

Table 19: A comparison of Egg Hatch, Pupation and Adult emergence Rates in Test and Control

CONTROL TEST P-value^ n % n % Eggs 603 - 308 - - Larvae 585 97.01 293 95.12 0.150 Pupae 410 70.08 244 83.27 <0.001 Adults 398 97.07 201 82.37 <0.001 ^Chi-square test

The larval formation rate was lower in test than control group, but the difference was not statistically significant (P-value = 0.150, using chi-square test). However, pupation rate was higher in test than control group, it was also statistically significant (P-value < 0.001, using chi- square test). In addition, adult emergence was lower in test group than control group, it was also statistically significant (P-value < 0.001, using chi-square test) as seen in Table 19.

Supplementary Table 19A indicating Pupation rate and adult emergence rate from egg and from 1st instar larvae of Aedes aegypti respectively

Control Test P-value* n % n %

From egg

Pupation rate 410 68.0 244 79.2 <0.001

Adult emergence rate 398 66.0 201 65.3 0.823

From 1st instar Larvae

Pupation rate 410 70.1 244 83.3 <0.001

Adult emergence rate 398 68.0 201 68.6 0.865

^Chi-square test

131

Table 20: A comparison of Fecundity (oviposition) of Aedes aegypti F1 females in test and control groups

Gonotrophic Control Test P-

cycle n Median Min – Max n Median Min – Max value*

1 10 68.5 0 – 77 10 24 0 – 75 0.360

2 10 53 0 – 68 8 47.5 30 – 55 0.180

3 9 55 0 – 80 8 58 0 – 71 0.698

4 7 31 20 – 41 6 16.5 0 – 38 0.100

5 6 15 0 – 33 4 5 0 – 11 0.330

6 5 42 0 – 48 4 18 8 – 28 0.142

7 5 32 21 – 61 3 31 27 – 34 0.655

8 5 42 0 – 61 3 25 21 – 31 0.653

9 4 48 0 – 55 2 28 25 – 31 0.348

10 3 0 0 – 0 1 0 0 – 0 -

Total 64 41 0 – 80 49 30 0 – 75 0.222

*Mann Whitney rank sum test

The median egg laid was 30 and 41 for test and control group respectively, but the difference was not statistically significant (P-value = 0.222, using Mann-Whitney rank sum test). In the same manner, each of the gonotrophic cycle was also not significantly different between the test and control group as seen in Table 20.

132

60 CONTROL No. of eggs laid CONTROL No. of eggs hatched 50 TEST No. of eggs laid

40 TEST No. of eggs hatched female 30

20

Aedes aegypti 10 per per 0

Mean number Meannumber eggsof laid/hatched 1 2 3 4 5 6 7 8 9 10 Gonotrophic cycle

Fig. 68: Mean number of eggs laid and hatched per treated and control Aedes aegypti female during each gonotrophic cycle.

133

Table 21: Hatchability of eggs produced by F1 Aedes aegypti females during different gonotrophic cycles

Gonotrophic Control Test P-

cycle n Median Min – Max n Median Min – Max value*

1 10 63.5 0 – 71 10 21 0 – 71 0.208

2 10 51 0 – 67 8 40.5 20 – 51 0.130

3 9 55 0 – 79 8 55.5 0 – 69 0.735

4 7 25 19 – 40 6 12.5 0 – 35 0.133

5 6 13 0 – 31 4 2.5 0 – 8 0.279

6 5 40 0 – 45 4 16.5 5 – 21 0.142

7 5 30 20 – 58 3 27 25 – 30 0.368

8 5 40 0 – 59 3 20 18 – 30 0.653

9 4 46 0 – 54 2 22.5 20 – 25 0.355

10 3 0 0 – 0 1 0 0 – 0 -

Total 64 39.5 0 – 79 49 25 0 – 71 0.145

*Mann Whitney rank sum test

The median egg hatchability was 25 and 39.5 for test and control group respectively, but the difference was not statistically significant (P-value = 0.145, using Mann-Whitney rank sum test) as seen Fig. 68, Table 21. In the same manner, each of the gonotrophic cycle was also not significant between the test and control group.

134

Supplementary Table 21A of overall egg hatchability in Control vs. Test population of Aedes aegypti

Group Egg Hatchability Total P-value* Yes No

Control 2207 (94.6%) 126 (5.4%) 2333 <0.001 Test 1325 (88.4%) 174 (11.6%) 1499

Total 3532 (92.2%) 300 (7.8%) 3832

^Chi-square test

Supplementary Table 21B taking number of females of Aedes aegypti in control and test into account

Egg Hatchability (%) Group P-value# n Mean Min – Max

Control 49 93.75 (4.73) 77.42 – 100.00 <0.001 Test 41 84.99 (12.28) 50.00 – 100.00

Total 90 89.76 (9.95) 50.00 – 100.00

#t-test

The mean egg hatchability was 85.00 and 93.75 for test and control respectively. Hence egg hatchability was more in control group than test group, and it was a statistically significant difference between the groups (P-value < 0.001, t-test) as seen in Table 21A or B.

135

Table 22A: Life span of the F1 generation Aedes aegypti Test female mosquitoes

Test Female Cum. Survival Day Number Mortality SE 95% CI Mortality probability 1 106 4 4 0.9623 0.0185 0.9026 - 0.9857 2 102 5 9 0.9151 0.0271 0.8432 - 0.9549 3 97 2 11 0.8962 0.0296 0.8205 - 0.9411 4 95 2 13 0.8774 0.0319 0.7982 - 0.9269 5 93 1 14 0.8679 0.0329 0.7872 - 0.9196 6 92 1 15 0.8585 0.0339 0.7763 - 0.9122 7 91 2 17 0.8396 0.0356 0.7548 - 0.8971 8 89 3 20 0.8113 0.0380 0.7230 - 0.8739 9 86 1 21 0.8019 0.0387 0.7125 - 0.8660 10 85 6 27 0.7453 0.0423 0.6510 - 0.8176 11 79 9 36 0.6604 0.0460 0.5618 - 0.7418 12 70 3 39 0.6321 0.0468 0.5328 - 0.7159 13 67 1 40 0.6226 0.0471 0.5232 - 0.7071 15 66 6 46 0.5660 0.0481 0.4664 - 0.6540 16 60 2 48 0.5472 0.0483 0.4477 - 0.6361 17 58 5 53 0.5000 0.0486 0.4016 - 0.5906 18 53 3 56 0.4717 0.0485 0.3744 - 0.5629 19 50 6 62 0.4151 0.0479 0.3208 - 0.5066 20 44 10 72 0.3208 0.0453 0.2344 - 0.4102 22 34 1 73 0.3113 0.0450 0.2260 - 0.4003 24 33 3 76 0.2830 0.0438 0.2009 - 0.3705 25 30 3 79 0.2547 0.0423 0.1764 - 0.3403 26 27 2 81 0.2358 0.0412 0.1602 - 0.3200 27 25 1 82 0.2264 0.0406 0.1522 - 0.3097 29 24 5 87 0.1792 0.0373 0.1132 - 0.2576 30 19 2 89 0.1604 0.0356 0.0981 - 0.2363 31 17 2 91 0.1415 0.0339 0.0833 - 0.2147 32 15 1 92 0.1321 0.0329 0.0761 - 0.2038 34 14 1 93 0.1226 0.0319 0.0689 - 0.1927 35 13 1 94 0.1132 0.0308 0.0619 - 0.1816 38 12 5 99 0.0660 0.0241 0.0291 - 0.1239 39 7 1 100 0.0566 0.0224 0.0232 - 0.1118 40 6 1 101 0.0472 0.0206 0.0176 - 0.0995 41 5 2 103 0.0283 0.0161 0.0076 - 0.0738 46 3 1 104 0.0189 0.0132 0.0037 - 0.0602 57 2 1 105 0.0094 0.0094 0.0008 - 0.0465 58 1 1 106 0 - - SE: Standard error, CI: Confidence interval (Lower and Upper limit)

136

Table 22B: Life span of the F1 generation Aedes aegypti Test male mosquitoes

Test Male Cum. Survival Day Number Mortality SE 95% CI Mortality probability 1 90 3 3 0.9667 0.0189 0.9002 - 0.9891 2 87 5 8 0.9111 0.0300 0.8301 - 0.9545 3 82 3 11 0.8778 0.0345 0.7902 - 0.9304 4 79 4 15 0.8333 0.0393 0.7389 - 0.8960 5 75 2 17 0.8111 0.0413 0.7139 - 0.8781 6 73 2 19 0.7889 0.0430 0.6892 - 0.8598 7 71 4 23 0.7444 0.0460 0.6410 - 0.8222 8 67 7 30 0.6667 0.0497 0.5592 - 0.7537 9 60 6 36 0.6000 0.0516 0.4913 - 0.6927 10 54 3 39 0.5667 0.0522 0.4581 - 0.6615 11 51 3 42 0.5333 0.0526 0.4253 - 0.6299 12 48 3 45 0.5000 0.0527 0.3930 - 0.5978 13 45 4 49 0.4556 0.0525 0.3507 - 0.5544 15 41 3 52 0.4222 0.0521 0.3194 - 0.5213 16 38 2 54 0.4000 0.0516 0.2989 - 0.4990 17 36 4 58 0.3556 0.0505 0.2584 - 0.4538 18 32 2 60 0.3333 0.0497 0.2385 - 0.4308 19 30 1 61 0.3222 0.0493 0.2287 - 0.4193 20 29 7 68 0.2444 0.0453 0.1615 - 0.3367 21 22 6 74 0.1778 0.0403 0.1071 - 0.2630 24 16 1 75 0.1667 0.0393 0.0984 - 0.2504 25 15 5 80 0.1111 0.0331 0.0569 - 0.1856 26 10 3 83 0.0778 0.0282 0.0342 - 0.1447 27 7 4 87 0.0333 0.0189 0.0089 - 0.0861 29 3 1 88 0.0222 0.0155 0.0043 - 0.0702 30 2 1 89 0.0111 0.0110 0.0010 - 0.0540 38 1 1 90 0.0000 - - SE: Standard error, CI: Confidence interval (Lower and Upper limit)

137

Table 22C: Life span of the F1 generation Aedes aegypti control female mosquitoes.

Control Female Cum. Survival Day Number Mortality SE 95% CI Mortality probability 1 213 12 12 0.9437 0.0158 0.9029 - 0.9676 2 201 13 25 0.8826 0.0221 0.8313 - 0.9191 3 188 16 41 0.8075 0.0270 0.7479 - 0.8544 4 172 2 43 0.7981 0.0275 0.7377 - 0.8461 5 170 5 48 0.7746 0.0286 0.7124 - 0.8251 6 165 5 53 0.7512 0.0296 0.6874 - 0.8038 7 160 14 67 0.6854 0.0318 0.6184 - 0.7432 8 146 3 70 0.6714 0.0322 0.6039 - 0.7300 9 143 3 73 0.6573 0.0325 0.5893 - 0.7167 10 140 14 87 0.5915 0.0337 0.5224 - 0.6541 11 126 7 94 0.5587 0.0340 0.4894 - 0.6224 12 119 4 98 0.5399 0.0342 0.4706 - 0.6041 13 115 3 101 0.5258 0.0342 0.4566 - 0.5903 14 112 7 108 0.4930 0.0343 0.4242 - 0.5580 15 105 11 119 0.4413 0.0340 0.3738 - 0.5066 16 94 4 123 0.4225 0.0338 0.3557 - 0.4877 17 90 5 128 0.3991 0.0336 0.3332 - 0.4640 18 85 5 133 0.3756 0.0332 0.3108 - 0.4402 19 80 5 138 0.3521 0.0327 0.2886 - 0.4162 20 75 6 144 0.3239 0.0321 0.2621 - 0.3871 21 69 5 149 0.3005 0.0314 0.2403 - 0.3628 22 64 6 155 0.2723 0.0305 0.2144 - 0.3333 24 58 8 163 0.2347 0.0290 0.1803 - 0.2935 25 50 5 168 0.2113 0.0280 0.1593 - 0.2683 26 45 13 181 0.1502 0.0245 0.1061 - 0.2016 27 32 3 184 0.1362 0.0235 0.0942 - 0.1858 29 29 8 192 0.0986 0.0204 0.0633 - 0.1431 30 21 1 193 0.0939 0.0200 0.0595 - 0.1376 33 20 1 194 0.0892 0.0195 0.0558 - 0.1322 35 19 1 195 0.0845 0.0191 0.0521 - 0.1267 38 18 7 202 0.0516 0.0152 0.0274 - 0.0871 39 11 1 203 0.0469 0.0145 0.0240 - 0.0813 40 10 1 204 0.0423 0.0138 0.0208 - 0.0754 41 9 4 208 0.0235 0.0104 0.0089 - 0.0509 47 5 1 209 0.0188 0.0093 0.0063 - 0.0444 54 4 1 210 0.0141 0.0081 0.0039 - 0.0378 55 3 1 211 0.0094 0.0066 0.0019 - 0.0310 56 2 2 213 0.0000 - - SE: Standard error, CI: Confidence interval (Lower and Upper limit)

138

Table 22D: Life span of the F1 generation Aedes aegypti control male mosquitoes

Control Male Cum. Survival Day Number Mortality SE 95% CI Mortality probability 1 171 15 15 0.9123 0.0216 0.8587 - 0.9462 2 156 12 27 0.8421 0.0279 0.7783 - 0.8889 3 144 12 39 0.7719 0.0321 0.7014 - 0.8278 4 132 2 41 0.7602 0.0326 0.6889 - 0.8174 5 130 1 42 0.7544 0.0329 0.6827 - 0.8121 6 129 1 43 0.7485 0.0332 0.6764 - 0.8069 7 128 16 59 0.6550 0.0364 0.5786 - 0.7209 8 112 11 70 0.5906 0.0376 0.5131 - 0.6600 9 101 4 74 0.5673 0.0379 0.4896 - 0.6376 10 97 7 81 0.5263 0.0382 0.4489 - 0.5979 11 90 19 100 0.4152 0.0377 0.3409 - 0.4878 12 71 2 102 0.4035 0.0375 0.3298 - 0.4759 13 69 9 111 0.3509 0.0365 0.2802 - 0.4223 14 60 7 118 0.3099 0.0354 0.2422 - 0.3799 15 53 14 132 0.2281 0.0321 0.1684 - 0.2933 16 39 2 134 0.2164 0.0315 0.1581 - 0.2807 17 37 2 136 0.2047 0.0309 0.1479 - 0.2680 18 35 5 141 0.1754 0.0291 0.1227 - 0.2360 19 30 1 142 0.1696 0.0287 0.1178 - 0.2295 20 29 1 143 0.1637 0.0283 0.1128 - 0.2230 21 28 4 147 0.1404 0.0266 0.0933 - 0.1968 22 24 3 150 0.1228 0.0251 0.0790 - 0.1768 24 21 3 153 0.1053 0.0235 0.0650 - 0.1566 25 18 3 156 0.0877 0.0216 0.0514 - 0.1360 26 15 5 161 0.0585 0.0179 0.0299 - 0.1005 27 10 5 166 0.0292 0.0129 0.0110 - 0.0629 29 5 2 168 0.0175 0.0100 0.0048 - 0.0467 30 3 1 169 0.0117 0.0082 0.0023 - 0.0382 34 2 1 170 0.0058 0.0058 0.0005 - 0.0297 38 1 1 171 0.0000 - - SE: Standard error, CI: Confidence interval (Lower and Upper limit)

139

Kaplan-Meier survival estimates

1.00

0.75

0.50

Survival probability

0.25 0.00 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60 Days

Test Female Control Female

Fig. 69: Kaplan-Meier survival curves of the test and control F1 generation Aedes aegypti females

Kaplan-Meier survival estimates

1.00

0.75

0.50

Survival probability

0.25 0.00 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60 Days

Test Male Control Male

Fig. 70: Kaplan-Meier survival curves of the test and control F1 generation Aedes aegypti males

140

Supplementary Table 23: A comparison of mean survival time of test and control females and males

Median survival days Group P-value* Test Control

Female 17 14 0.0701

Male 12 11 0.0743

*Log-rank test

The median survival days of females for test and control group were 17 and 14 respectively, and the survival curve were also not statistically significant between groups (P-value = 0.070, using Log-rank test) as seen in Table 22A, B, C, D, Fig. 69 and 70. Median survival days of males for test and control group were 12 and 11 respectively, and the survival curve were not statistically significant between groups (P-value = 0.074, using Log-rank test) in Table 23.

Emergence DAY8 - DAY15 = 8 DAYS 350 Emergence DAY8 - DAY15 = 8 DAYS 700 Pupae DAY7 - DAY14 = 8 DAYS Pupae DAY7 - DAY14 = 8 DAYS L1-L4 DAY1 - DAY14 = 14 DAYS 300 600 L1-L4 DAY1 - DAY14 = 14 DAYS 250 500 200 400

Number 150

Number 300

200 100

100 50

0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 DAYS DAYS Fig. 71A and B: F1 Larval development (L1-L4), pupal formation and adult emergence in the control and treated Aedes aegypti mosquitoes

The larval (Day1- Day 14) and pupal development (Day 7 - Day 14) and adult emergence (Day 8 - Day 15) was of the same duration in treated population v/s control population i.e. 14 days from larval stages L1 - L4, pupal development and adult emergence = 8 days. (Fig. 71A and B).

141

Culex quinquefasciatus:

Table 24: A comparison of Egg Hatch, Pupation and Adult emergence Rates in Test and Control

CONTROL TEST P-value^ n % n % Eggs 338 - 196 - - Larvae 324 95.86 187 95.41 0.805 Pupae 228 70.37 176 94.12 <0.001 Adults 202 88.60 125 71.02 <0.001 ^Chi-square test

Larval rate was nearly equal between test and control group, and it was also not statistically significant between groups (P-value = 0.805, using chi-square test). Pupation rate was higher in test than control group, it was also statistically significant (P-value < 0.001, using chi-square test). Adult emergence was lower in test group than control group, it was statistically significant (P-value < 0.001, using chi-square test) as seen in Table 24.

Supplementary Table 24A indicating Pupation rate and adult emergence rate from egg and from 1st instar larvae of Culex quinquefasciatus respectively

Control Test P-value* n % n %

From egg

Pupation rate 228 67.5 176 89.8 <0.001

Adult emergence rate 202 59.8 125 63.8 0.359

From 1st instar Larvae

Pupation rate 228 70.4 176 94.1 <0.001

Adult emergence rate 202 62.4 125 66.8 0.307

^Chi-square test

142

Table 25: A comparison of Fecundity (oviposition) of Culex quinquefasciatus F1 females in test and control groups

Gonotrophic Control Test P-value* cycle n Median Min – Max n Median Min – Max

1 10 48 0 – 80 10 22 0 – 70 0.274

2 10 0 0 – 118 8 0 0 – 102 0.613

3 10 53.5 0 – 89 7 24 0 – 112 0.762

4 7 0 0 – 105 5 0 0 – 100 0.749

5 5 0 0 – 134 5 0 0 – 125 0.368

6 5 0 0 – 104 4 0 0 – 60 0.467

7 2 0 0 – 0 4 0 0 – 0 -

8 2 0 0 – 0 1 0 0 – 0 -

Total 51 0 0 – 134 44 0 0 – 125 0.183

*Mann Whitney rank sum test

The median egg laid was 0 for both test and control group, but the difference was not statistically significant (P-value = 0.183, using Mann-Whitney rank sum test). In the same manner, each of the gonotrophic cycle was also not statistically significant between the test and control group as seen in Table 25.

143

CONTROL No. of eggs laid 60 CONTROL No. of eggs hatched

50 TEST No. of eggs laid female 40 TEST No. of eggs hatched

30

20

10 Culex Culex quinquefasciatus

Meanno. eggsof laid/hatched per 0 1 2 3 4 5 6 7 8 Gonotrophic cycle

Fig. 72: Mean number of eggs hatched per treated and control Culex quinquefasciatus female during each gonotrophic cycle

144

Table 26: Hatchability of eggs produced by F1 Culex quinquefasciatus females during different gonotrophic cycles

Gonotrophic Control Test P-value* cycle n Median Min – Max n Median Min – Max

1 10 45.5 0 – 78 10 19.5 0 – 68 0.241

2 10 0 0 – 114 8 0 0 – 95 0.613

3 10 50 0 – 85 7 21 0 – 100 0.761

4 7 0 0 – 101 5 0 0 – 97 0.749

5 5 0 0 – 130 5 0 0 – 119 0.368

6 5 0 0 – 100 4 0 0 – 58 0.467

7 2 0 0 – 0 4 0 0 – 0 -

8 2 0 0 – 0 1 0 0 – 0 -

Total 51 0 0 – 130 44 0 0 – 119 0.159

*Mann Whitney rank sum test

The median egg hatchability was 0 for both test and control group, but the difference was not statistically significant (P-value = 0.159, using Mann-Whitney rank sum test). In the same manner, each of the gonotrophic cycle was also not statistically difference between the test and control group as seen in Fig. 72, Table 26.

145

Supplementary Table 26A of overall egg hatchability in Control vs. Test population of Culex quinquefasciatus

Group Egg hatchability P- Total Yes No value*

Control 1776 (95.8%) 77 (4.2%) 1853 <0.001 Test 992 (92.5%) 80 (7.5%) 1072

Total 2768 (94.6%) 157 (5.4%) 2925

^Chi-square test

Supplementary Table 26B taking number of females of Culex quinquefasciatus in control and test into account

Egg Hatchability (%) P- Group # n Mean Min – Max value

Control 23 95.70 (1.66) 92.59 – 98.79 0.0003 Test 15 92.18 (3.68) 87.50 – 97.14

Total 38 94.31 (3.13) 87.50 – 98.79

#t-test

The mean egg hatchability was 92.18 and 95.70 for test and control respectively. So egg hatchability was more in control group than test group, the difference was statistically significant among them (P-value = 0.0003, t-test) as seen in Table 26A and B.

146

Table 27A: Life span of the F1 generation Culex quinquefasciatus Test female mosquitoes

Test Female Cum. Survival Day Number Mortality SE 95% CI Mortality probability 1 63 2 2 0.9683 0.0221 0.8790 - 0.9920 2 61 7 9 0.8571 0.0441 0.7434 - 0.9230 3 54 4 13 0.7937 0.0510 0.6713 - 0.8746 4 50 11 24 0.6190 0.0612 0.4876 - 0.7260 5 39 4 28 0.5556 0.0626 0.4249 - 0.6679 6 35 4 32 0.4921 0.0630 0.3642 - 0.6078 7 31 11 43 0.3175 0.0586 0.2074 - 0.4331 14 20 1 44 0.3016 0.0578 0.1940 - 0.4164 15 19 1 45 0.2857 0.0569 0.1807 - 0.3996 16 18 3 48 0.2381 0.0537 0.1419 - 0.3482 18 15 2 50 0.2063 0.0510 0.1171 - 0.3131 19 13 1 51 0.1905 0.0495 0.1050 - 0.2953 21 12 1 52 0.1746 0.0478 0.0931 - 0.2772 23 11 1 53 0.1587 0.0460 0.0815 - 0.2589 35 10 2 55 0.1270 0.0419 0.0594 - 0.2214 36 8 2 57 0.0952 0.0370 0.0388 - 0.1825 37 6 1 58 0.0794 0.0341 0.0293 - 0.1623 57 5 1 59 0.0635 0.0307 0.0205 - 0.1416 59 4 1 60 0.0476 0.0268 0.0126 - 0.1202 60 3 1 61 0.0317 0.0221 0.0060 - 0.0979 62 2 1 62 0.0159 0.0157 0.0013 - 0.0749 64 1 1 63 0.0000 - - SE: Standard error, CI: Confidence interval (Lower and Upper limit)

147

Table 27B: Life span of the F1 generation Culex quinquefasciatus Test male mosquitoes

Test Male

Cum. Survival Day Number Mortality SE 95% CI Mortality probability

1 48 1 1 0.9792 0.0206 0.8612 - 0.9970 2 47 1 2 0.9583 0.0288 0.8435 - 0.9894

3 46 14 16 0.6667 0.0680 0.5148 - 0.7807 4 32 2 18 0.6250 0.0699 0.4728 - 0.7446

5 30 1 19 0.6042 0.0706 0.4521 - 0.7262 9 29 1 20 0.5833 0.0712 0.4317 - 0.7076

12 28 4 24 0.5000 0.0722 0.3526 - 0.6307 13 24 13 37 0.2292 0.0607 0.1230 - 0.3549

14 11 1 38 0.2083 0.0586 0.1076 - 0.3317 15 10 1 39 0.1875 0.0563 0.0926 - 0.3080

16 9 3 42 0.1250 0.0477 0.0508 - 0.2344 17 6 1 43 0.1042 0.0441 0.0382 - 0.2086

18 5 1 44 0.0833 0.0399 0.0267 - 0.1821 19 4 1 45 0.0625 0.0349 0.0163 - 0.1545

21 3 1 46 0.0417 0.0288 0.0077 - 0.1257 24 2 1 47 0.0208 0.0206 0.0017 - 0.0958

29 1 1 48 0.0000 - -

SE: Standard error, CI: Confidence interval (Lower and Upper limit)

148

Table 27C: Life span of the F1 generation Culex quinquefasciatus control female mosquitoes

Control Female Cum. Survival Day Number Mortality SE 95% CI Mortality probability 1 101 3 3 0.9703 0.0169 0.9107 - 0.9903 2 98 3 6 0.9406 0.0235 0.8725 - 0.9729 3 95 15 21 0.7921 0.0404 0.6992 - 0.8591 4 80 8 29 0.7129 0.0450 0.6139 - 0.7907 6 72 10 39 0.6139 0.0484 0.5117 - 0.7009 7 62 9 48 0.5248 0.0497 0.4232 - 0.6166 8 53 7 55 0.4554 0.0496 0.3565 - 0.5490 9 46 8 63 0.3762 0.0482 0.2826 - 0.4695 10 38 3 66 0.3465 0.0474 0.2555 - 0.4391 11 35 4 70 0.3069 0.0459 0.2201 - 0.3979 12 31 5 75 0.2574 0.0435 0.1769 - 0.3454 13 26 1 76 0.2475 0.0429 0.1684 - 0.3347 14 25 2 78 0.2277 0.0417 0.1517 - 0.3133 15 23 2 80 0.2079 0.0404 0.1352 - 0.2915 16 21 1 81 0.1980 0.0397 0.1270 - 0.2806 18 20 1 82 0.1881 0.0389 0.1189 - 0.2696 23 19 1 83 0.1782 0.0381 0.1109 - 0.2585 27 18 1 84 0.1683 0.0372 0.1030 - 0.2473 28 17 1 85 0.1584 0.0363 0.0952 - 0.2361 29 16 1 86 0.1485 0.0354 0.0875 - 0.2247 32 15 1 87 0.1386 0.0344 0.0799 - 0.2133 36 14 1 88 0.1287 0.0333 0.0724 - 0.2018 38 13 1 89 0.1188 0.0322 0.0650 - 0.1901 39 12 1 90 0.1089 0.0310 0.0578 - 0.1783 40 11 1 91 0.0990 0.0297 0.0507 - 0.1664 45 10 1 92 0.0891 0.0283 0.0438 - 0.1544 50 9 1 93 0.0792 0.0269 0.0370 - 0.1422 51 8 1 94 0.0693 0.0253 0.0305 - 0.1297 53 7 1 95 0.0594 0.0235 0.0243 - 0.1171 54 6 2 97 0.0396 0.0194 0.0129 - 0.0909 58 4 1 98 0.0297 0.0169 0.0080 - 0.0772 59 3 1 99 0.0198 0.0139 0.0038 - 0.0630 61 2 1 100 0.0099 0.0099 0.0009 - 0.0486 64 1 1 101 0.0000 - - SE: Standard error, CI: Confidence interval (Lower and Upper limit

149

Table 27D: Life span of the F1 generation Culex quinquefasciatus control male mosquitoes

Control Male Cum. Survival Day Number Mortality SE 95% CI Mortality probability 1 92 5 5 0.9457 0.0236 0.8744-0.9770 2 87 2 7 0.9239 0.0276 0.8470-0.9630 3 85 15 22 0.7609 0.0445 0.6599-0.8355 4 70 1 23 0.7500 0.0451 0.6482-0.8262 5 69 7 30 0.6739 0.0489 0.5679-0.7593 6 62 4 34 0.6304 0.0503 0.5233-0.7199 7 58 7 41 0.5543 0.0518 0.4472-0.6489 8 51 10 51 0.4457 0.0518 0.3425-0.5436 9 41 6 57 0.3804 0.0506 0.2820-0.4781 10 35 5 62 0.3261 0.0489 0.2331-0.4222 11 30 9 71 0.2283 0.0438 0.1488-0.3181 12 21 1 72 0.2174 0.0430 0.1398-0.3062 13 20 11 83 0.0978 0.0310 0.0480-0.1686 14 9 1 84 0.0870 0.0294 0.0406-0.1553 15 8 1 85 0.0761 0.0276 0.0335-0.1417 16 7 1 86 0.0652 0.0257 0.0267-0.1279 18 6 1 87 0.0543 0.0236 0.0202-0.1138 23 5 1 88 0.0435 0.0213 0.0142-0.0993 24 4 1 89 0.0326 0.0185 0.0088-0.0843 25 3 1 90 0.0217 0.0152 0.0042-0.0688 29 2 1 91 0.0109 0.0108 0.0010-0.0530 40 1 1 92 0.0000 - - SE: Standard error, CI: Confidence interval (Lower and Upper limit)

150

Kaplan-Meier survival estimates

1.00

0.75

0.50

Survival probability 0.25

0.00 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60 Days Test Female Control Female

Fig. 73: Kaplan-Meier survival curves of the test and control F1 generation Culex quinquefasciatus females

Kaplan-Meier survival estimates 1.00

0.75

0.50

Survival probability

0.25

0.00 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60 Days

Test Male Control Male

Fig. 74: Kaplan-Meier survival curves of the test and control F1 generation Aedes aegypti males

151

Supplementary Table 28: A comparison of mean survival time of test and control females and males

Median survival days Group P-value* Test Control

Female 6 8 0.754

Male 12 8 0.054

*Log-rank test

Median survival day of females for test and control group were 6 and 8 respectively, and the survival curve was also not statistically significant between groups (P-value = 0.754, using Log- rank test) in Table 27A, B, C, D and Fig. 73 and 74. Median survival day of males for test and control group were 12 and 8 respectively, and the survival curve was statistically significant between them (P-value = 0.054, using Log-rank test) as seen in Table 28.

152

Emergence DAY 9- DAY 16 = 8 DAYS Emergence DAY 10 - DAY 17 = 8 DAYS Pupae DAY 8 - DAY 15 = 8 DAYS Pupae DAY 8 - DAY 16 = 9 DAYS 350 200 L1-L4 DAY 1- DAY 15 = 15 DAYS L1-L4 DAY1 - DAY 15 = 15 DAYS 180 300 160 250 140 120 200 100

Number 150 Number 80

100 60 40 50 20 0 0 1 2 3 4 5 6 7 8 9 10 11 12 1314 15 1617 18 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 DAYS DAYS

Fig. 75A and B: F1 Larval development (L1-L4), Pupal formation and adult emergence in the control and treated Culex quinquefasciatus mosquitoes

The larval development (Day 1 - Day 15) was of the same duration in treated population v/s control population i.e., 15 days. Pupal development (Day 8 - Day 15) and adult emergence (Day 9 - Day 16) was of 8 days in the control population while pupal development (Day 8 - Day 16) was of 9 days and adult emergence (Day 10 - Day 17) was of 8 days respectively in the treated population (Fig. 75A and B).

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CHAPTER 5: DISCUSSION

Mosquitoes represent a significant threat to human health due to their ability to vector disease causing pathogens which afflict millions of people worldwide [178]. They are responsible for infecting more than 700 million people every year in more than 80 countries, approximately 80% of the world’s population is at risk of acquiring infections of mosquito borne-diseases [179]. Several species belonging to genera Anopheles, Aedes and Culex are vectors of diseases such as malaria, dengue fever, dengue haemorrhagic fever, Japanese encephalitis, chikungunya, Zika, filariasis, etc. [180]. Malaria is the largest single component of the disease burden and epidemic malaria, in particular, remains a major public health concern in the low-income tropical countries [181]. Vector control is an essential requirement in the control/elimination of these diseases that are transmitted by different species of mosquitoes [182]. In past few years, the emergence of resistance to insecticides and chemical agents have been increasing rapidly, hence there have been persistent efforts to develop new insecticidal agents from natural environments. In this regard, soil is believed to be an excellent habitat for the microbes which have the ability to produce endotoxins and secondary metabolites inimical to mosquitoes and which can be applied in bio control activities at mass scale [105]. For example, Bacillus sp., Streptomyces sp., Penicillium sp., and Trichoderma sp. are being developed as effective microbial control agents which can act as successful alternatives to chemical compounds [183]. Marine ecosystems such as salt pans, beaches and mangroves are considered as extreme environments and which are known to be inhabited by organisms that thrive in high salinities, temperatures and withstand severe solar radiations [184]. This renewed our interest in exploring soil samples from these regions for the isolation of previously unreported strains of mosquito pathogenic bacteria. In the present study, in this quest, we isolated, identified and evaluated three novel bacterial strains from salt pans which showed pronounced larvicidal activity against the 3rd instar larvae of An. stephensi, Cx. quinquefasciatus, Ae. aegypti and Ae. albopictus. In addition, in a recent study by Suryadi et al., 2016 the beach area of Lombok Island, Indonesia yielded four toxic isolates of B. sphaericus. These isolates showed mild toxicity when tested against the larvae of three mosquito species (Culex quinquefasciatus, Anopheles aconitus and Aedes aegypti) [185].

154

In another study, Poopathi et al., 2014 reported the isolation of Bacillus cereus strain VCRC-B520, a novel mosquitocidal bacterium from marine soil collected from east coastal areas at Pondicherry (India). The LC50 and LC90 values for C. quinquefasciatus were 0.30 and 2.21 mg/L, respectively [186]. Hence, soil samples were collected from a coastal region of Goa (i.e., Miramar beach) to isolate mosquito-pathogenic bacilli. From this sampling location three different zones i.e., tidal, intertidal and seashore were explored. However, mosquito pathogenic isolates were obtained only from two zones i.e., tidal and intertidal. No mosquito pathogenic bacteria could be detected in the soil sample collected from the sea-shore zone. This is supported by Manonmani et al., 1987 who reported that Bacillus spores are known to settle rapidly in watery areas [187]. Previously, Nabar et al., 2015 have opined that the extreme natural environment conditions have been consistently generating microbial species which contributed to the control of diseases and their transmission [188]. A Bacillus subtilis subsp. subtilis strain was isolated by Geetha et al in 2007 from mangrove forests of Andaman and Nicobar Islands of India showing thereby that novel bacilli from saline environment possess mosquito larvicidal and pupicidal activity [54, 76]. In another similar study soil samples were collected from 100 different sites in and around Devakottai (Tamil Nadu) for the isolation of B. sphaericus. Susceptibility of Cx. quinquefasciatus to B. sphaericus was analyzed through bioassays against 1st, 2nd, 3rd and 4th instar larvae of lab reared Cx. quinquefasciatus [60].

The method for isolation of mosquito-pathogenic bacilli proposed by Travers et al., 1987 [189] has long been considered as a standard. It uses heat treatment and acetate selection which permits selective isolation of bacilli. As this process is cumbersome, time consuming and involves the wastage of media by plating of all the soil samples which may include both mosquito pathogenic and non-pathogenic Bacilli strains, an alternative was badly needed. It was the protocol devised by Dhindsa et al., 2002 [104] for the screening of soil samples for the presence of mosquito pathogenic bacilli prior to isolation which resolved the issue. When this protocol was used in the present study, it proved successful. This method involves the use of LB broth (buffered with Sodium acetate) and a heat shock step at 65° C which eliminates the non-spore formers hence allowing for direct pre-screening of the soil containing Bacillus strains prior to isolation. Using this method in a previous study, eight Bacillus strains were successfully

155 isolated from the upper crust of soil in mosquito breeding habitats in Goa and evaluated for their larvicidal activity [104]. Applying this novel technique for isolation of mosquito-pathogenic bacilli of Dhindsa et al., 2002 in the present study, three out of nine soil samples from Miramar beach were found to contain Bacillus strains pathogenic to Anopheles stephensi, Culex quinquefasciatus, Aedes aegypti and Aedes albopictus larvae which are highly prevalent vectors of mosquito borne diseases in India and elsewhere.

In totality, the isolation of 7 promising strains of mosquito pathogenic isolates from 4 soil samples indicate that soils of Goa are rich source of these strains. In addition, the novel isolates identified in the present study were found to be different from the ones reported earlier in Goa. This study presents the first report of larvicidal bacteria isolated from a marine environment of Goa. Bacilli culture media composition is of utmost importance in the growth and the translation of larvicidal endotoxins. Previously, Rashad et al in 2012 reported that the variation in the composition of the media in their contents of carbon and nitrogen sources as well as minerals affect both yield and toxicity of biocides [190]. During preliminary screening of bacilli in the present study, a steady increase in the mosquito larvicidal activity was observed when the isolates were grown in Nutrient Yeast Sporulating Medium (NYSM) for 96 hours as compared to other tested media. The yield dropped beyond 96 hours. This result is supported by a study conducted by Foda et al., 2003 in which it was concluded that NYSM medium supported the highest growth, sporulation and toxicity of mosquito pathogenic bacteria due to the presence of Mn2+ , Ca2+ and Mg2+ ions in this medium which is known to enhance sporulation [191, 192]. Hence NYSM has been considered as the best choice for the expression of larvicidal activity and therefore was used for all further studies. In a previous study, mosquitocidal bacteria were isolated from mangrove habitats of Andaman– Nicobar Islands, India by collecting 460 samples of soil, leaf and water. A total of 857 bacteria were screened, out of which three Bacillus strains showed mosquitocidal activity at promising levels. Morphologically and biochemical characterization was used to identify two of these as Bacillus subtilis, whereas one was identified as Bacillus thuringiensis [54]. It has been reported that Bacillus thuringiensis is naturally found in the soil and has proved to be a good larvicidal agent against mosquito larvae in the laboratory. Hence this organism and its product can be

156 further studied to search for novel compounds that can be used in the control of mosquito borne diseases [193]. The results of preliminary toxicity testing of the isolates obtained in the present study against 3rd instar larvae of Anopheles stephensi, Cx. quinquefasciatus, Aedes aegypti and Aedes albopictus showed the presence of 7 promising isolates in 4 soil samples indicating the ubiquitous nature of mosquito-pathogenic bacilli in the coastal environment of Goa, India. The grading of these bacilli on the basis of per cent mortality produced in the larvae enabled shortlisting of the most promising candidates for further studies and the remaining less promising bacterial isolates were not further pursued. Prior to their molecular identification, these candidates were code-named as BGUMS14, BGUMS16, BGUMS101 from salt pan sampling sites and C1A, C2A, C2C, B1C from beach sampling site in Goa. It is worth mentioning here that the criteria for selection of the promising isolates was purely arbitrary and only those isolates that produced > 50% mortality in the larvae at 24 hours of treatment were considered as highly pathogenic and worth studying further for characterization and evaluation [194]. No mosquito pathogenic isolates were however, obtained from the third site at Curca, Goa from mangrove as well as salt pan sampling sites. The results have suggested that on the basis of morphological and bio-chemical characteristics, the different strains obtained from the sampling sites were of Bacillus sp. Their identity was further confirmed at molecular level by phylogenetic analysis. For this, the variable sequence of 16S rRNA gene which has been widely used to identify unknown bacterium to the genus or species level was deployed [195]. The PCR amplification of the study bacterial isolates using 16S rRNA primers indicated a band of 1500 base pairs corresponding to the full-length size of Bacillus spp 16S rRNA gene which is approximately 1500bps in size hence confirming their identity [195]. Based on sequence homology using the BLASTn programme of NCBI, isolate BGUMS101 was identified as Sporosarcina sp. In another study, Kwon et al., 2007 reported the isolation of two Gram-positive, aerobic, spore-forming rods from upland soil in South Korea and based on analysis of 16S rRNA gene sequences as well as physiological and chemotaxonomic data, the isolates were identified as Sporosarcina koreensis sp. nov. and Sporosarcina soli sp. nov.[196]. However, this study is the first report of the larvicidal activity of

Sporosarcina sp. when the larvae of Aedes albopictus (LC50 = 0.18 ppm) and Culex quinquefasciatus (LC50 = 0.225 ppm) were found to be the most susceptible. Therefore,

157

Sporosarcina sp. is a new weapon for use against mosquito vectors which can be added to the arsenal of microbial agents currently in use. This has been the remarkable finding of this study. Further bioinformatic analysis indicated that the isolate BGUMS14 was also a Bacillus sp. while the isolate BGUMS16 was identified as a strain of Bacillus safensis. No prior reports exist of their mosquito larvicidal activity. However, the use of 16S rRNA gene for species identification could be limited as it is highly conserved among species of Bacillus hence other taxonomic marker genes, like gyrB, rpoD can be studied for confirmation of the species [47]. In this study, the isolate C1A which was identified at molecular level, as Aneurinibacillus sp. rd which showed high toxicity (LC50 = 0.08 ppm) to Culex quinquefasciatus 3 instar larvae. This is the first report of Aneurinibacillus sp. having mosquito pathogenic activity that could be developed as a novel mosquito pathogenic bacterium with high toxicity to Culex quinquefasciatus larvae. It may be mentioned that Shida et al., 1996 proposed the genus Aneurinibacillus as a novel genus arising from the reclassification of the Bacillus aneurinilyticus and related species of the genus Bacillus [85]. Alenezi et al., 2017 reported that the soil-borne gram-positive bacteria Aneurinibacillus migulanus strain Nagano shows considerable potential as a bio control agent against plant diseases. However, no prior reports exist of its mosquito larvicidal activity to date [197].

Earlier, Mohanty et al., 2017 isolated different strains of Lysinibacillus and grouped them based on their similarity [165]. In the present study isolates C2A and C2C were also identified as Lysinibacillus sp. Though the Lysinibacillus genus is a well explored one, yet its species such as fusiformis have not been commercially formulated as a microbial biocontrol agent hence gains importance in that context. Following field testing a suitable formulation using this agent could be developed against Culex quinquefasciatus as Lysinibacillus strain C2A and C2C showed high toxicity (LC50 = 0.30ppm and LC50 = 0.25ppm respectively).

The isolate B1C, on the other hand, was identified as a strain of Bacillus thuringiensis. This isolate showed a broad spectrum of mosquito pathogenic activity against the 3rd instar larvae of the 4 test vector species. It has been reported by Ammouneh et al., 2010 that screening the environment for new and highly potent strains of Bacillus thuringiensis has become inevitable as one of the strategies for insect resistance management [108]. In that sense, finding of this new strain of Bacillus thuringiensis is a welcome finding of this study. In addition, many reports on

158 the frequent occurrence of Bacillus thuringiensis isolates in the natural environment have suggested a high possibility of isolating a novel strain.

Nature has provided a wide variety of organisms including viruses, protozoans, fungi and bacteria, which are effective against mosquitoes [198]. Among the many bacteria that have been tested, the most promising and well known bacterial larvicidal strains are Bacillus thuringiensis israelensis (Bti) and Bacillus sphaericus (Bs) which are widely employed in vector control in public health settings. Bacillus strains are cheap, can be easily and locally manufactured, easy to handle, safe and can be applied at ease as compared to chemical insecticides [13]. Hence, there is tremendous scope of Biological control agents as part of the integrated vector management being advocated by the World Health Organization and routinely used in several countries including India. National Vector Borne Diseases Control Programme of India guidelines for vector control also recommend the use of wettable powders and aqueous formulations of Bacillus thuringiensis israelensis for vector control especially in the urban areas.

When the activity of the previously unreported mosquito larvicidal strains obtained in this study with commercial formulations of Bacillus thuringiensis H-14 and Bacillus sphaericius Neide 2362 with isolates of this study, it was observed that Sporosarcina sp. strain BGUMS101 showed pronounced broad spectrum larvicidal activity mainly against Culex and Aedes spp. However, its activity against Aedes albopictus was found to be comparatively lesser than the commercial Bacillus sphaericus strain in terms of LC50 values. Hence, this bacterial agent can serve as an alternative potent candidate in the wake of resistance development in the future to the already available bacterial bio control agents. However, against the other test vector species, the commercial strains performed significantly better as compared to the test isolates in spite of them being larvicidal.

The mode of action of these larvicidal strains when studied during the present investigation revealed that the larval mortality occurred because of lysis of the midgut due to the production of insecticidal toxins. This mode of action is well known and has been previously reported by Bauer et al., 1995 who described the midgut as the primary site of δ-endotoxin activity in Bacillus thuringiensis israelensis [199]. Similarly, Charles et al., 1996 and Rahman et al., 2012 described the association and binding of the activated BinA and BinB toxin of Bacillus

159 sphaericus to their receptor, which is a α-glucosidase on the midgut microvilli, resulting in lysis of midgut cells after internalization [200, 201].

Balakrishnan et al., 2015 observed larvicidal and pupicidal activity of the culture supernatants of B. subtilis, B. thuringiensis, B. sphaericus and B. cereus against the 3rd instar larve of An. stephensi and Ae. aegypti. The results showed that the 3rd instar larvae and pupae of A. stephensi and A. aegypti showed more susceptibility to the culture supernatant [107]. However, in the present study, the culture supernatant was most toxic to Culex quinquefasciatus 3rd instar larvae (40 - 92%) followed by Anopheles stephensi 3rd instar larve (4 - 44%) and Aedes aegypti 3rd instar larvae (4 - 12%) following 24 hours of treatment. Low pupicidal activity was observed with Sporosarcina sp. strain BGUMS101 in general. This isolate supernatant showed the highest pupicidal activity of 48% against Anopheles stephensi pupae. Earlier, Geetha and Manonmani., 2008 had reported that B. subtilis subsp. subtilis produced a Crude mosquitocidal toxin (CMT) to which the pupae of mosquitoes were more susceptible than the larvae [76]. However, in this study the CMT of Bacillus sp. strain BGUMS14, Bacillus safensis strain BGUMS16 and Sporosarcina sp. strain BGUMS101 as well as the commercial strains Bti and Bs was observed to be most toxic to Cx. quinquefasciatus and Ae. aegypti larvae at the highest tested dose (20ppm) and least to An. stephensi and Ae. albopictus larvae. The mode of action was observed to be direct action of the crude mosquitocidal toxin on the pupae resulting in its death. However, in certain cases of Anopheles stephensi and Culex quinquefasciatus, the fully formed adult mosquito was unable to emerge from the pupal casing resulting in its death. This mode of action has not been previously reported in the literature. In the present study, an attempt was made to detect the gene responsible for the toxicity for the larvicidal and pupicidal action of the previously unreported bacterial isolates by using PCR based methods. It was observed that Sporosarcina sp. strain BGUMS101 was negative for all the toxin genes tested (i.e., Cry 2(UN), Cry4 (UN), Cyt1 (UN), Cyt2 (UN), BINA, BINB, Mtx gene, Gyrase, Surfactin) indicating the presence of a novel toxic component responsible for its larvicidal and pupicidal activity. However, Aneurinibacillus sp. strain CIA were found positive for BinA, BinB and MtX toxin gene indicating that it has Bacillus sphaericus like mode of action.

160

Prior studies, as early as 1986, have reported sub lethal effects of Bti against Aedes aegypti. Studies showed that following the treatment with Bti, the duration of larval development period was prolonged as comparatively, a greater number of days were required for the offspring to emerge into adults from the egg stage [202].

In the present study the effects of sublethal exposure of Sporosarcina sp. strain BGUMS101 on egg hatch rate, pupation percentage and adult emergence rate of Anopheles stephensi, Aedes aegypti and Culex quinquefasciatus were observed and recorded.

A previous study revealed that treatment of Anopheles stephensi, Ae. aegypti and Culex quinquefasciatus with 500 μg/ml of whole plant crude extracts of Abutilon indicum and Citrullus colocynthis leaf extracts decreased the adult emergence by 49.45, 53.2 and 73.0%, and 53.0, 55.8 and 70.4% respectively [203, 204]. Similarly, 250 μg/ml of the acetone extract of the Egyptian plant Cupressus sempervirens leaves exhibited a reduction in the adult emergence of the mosquito Culex pipiens by 40.0% [205]. A 50% adult emergence inhibition was demonstrated using aqueous leaves extract of Calotropis procera at 277.90 and 183.65 μg/ml against Anopheles arabiensis and Cx. quinquefasciatus respectively [206]. With regards to effect of sublethal doses on female mosquito fertility, it has been reported that treatment of Cx. quinquefasciatus and Ae. aegypti with 50% EC50 of Calophyllum inophyllum and Rhinocanthus nasutus leaf extract reduced the fertility from 94.9 to 30.2 % and 82.2 to 37.7% respectively when compared to controls [207]. In addition, adults of An. stephensi exposed as larvae to sublethal concentrations of alkaloids isolated from Annona squamosa seeds were found to produce eggs with a 27.7% reduced hatchability in comparison to controls [208]. The reduction in fecundity was also observed in Ae. albopictus females indicating that the sub lethal dose of Ipomoea cairica leaf extract induced reproductive disadvantages in the exposed group [173]. With regards to insecticides, pyrethrin derived from Tanacetum cinerariaefolium which is a highly effective insecticide against Aedes species can cause a reduction in eggs production in emerging adults after exposure [209]. The naturally-derived insecticide ‘Spinosad’ has been shown to significantly decrease the fecundity in exposed females compared to unexposed females of Ae. aegypti [210]. In the present study the reproductive potential (fecundity) and egg hatchability of the F1 females of the 3 test vector species was decreased on treatment with sublethal dose of microbial agent

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Sporosarcina sp. strain BGUMS101 as compared to the control population with this difference being statistically significant. In Anopheles stephensi F1 females (n = 10), the overall fecundity and hatchability was highest in the first gonotrophic cycle followed by second and third cycle. No egg laying was observed in the treated mosquitoes in the 4th gonotrophic cycle. The difference in the fecundity (oviposition) of treated Anopheles stephensi F1 female’s v/s the control population was found to be non-significant (p = 0.485) as determined by Mann-Whitney Rank sum test. However, the mean egg hatchability was more in the control group than the test group and this difference was statistically significant (p = 0.052) as determined by t-test. In addition, the larval formation rate was although lesser in the test group compared with the control group, however, this difference was non-significant (p = 0.164). The pupation rate was significantly higher in the test group as compared to the control group (p < 0.001). Similarly, adult emergence was significantly lower in the test group than the control group (p = 0.0004) as indicated by the ^Chi square test. In addition, the present study indicated that there was no significant difference between survival time of treated control F1 females (p = 0.0606) using Log-rank test. However, for the test and control males the survival was statistically significantly longer in the test as compared to the control group (p = 0.0099) using Log-rank test. In the fecundity studies of Aedes aegypti F1 females (n = 10) a total of 10 gonotrophic cycles were observed. The overall fecundity and hatchability were highest in the first and third gonotrophic cycle, while the rest of the cycles showed variable fecundity and egg hatchability suggesting thereby that with the increasing age of the female, the number of eggs produced are lesser during each gonotrophic cycle and there appears to be variability in both in the numbers of eggs produced and larvae hatched in different generations. What could be governing this phenomenon besides the age, is however, not clear. No egg laying was observed in both the treated and control mosquitoes in the 10th gonotrophic cycle which indicates that the Aedes aegypti females continued to produce eggs until the 10th generation irrespective of whether treated or not showing thereby great degree of resilience. Also, the difference in the fecundity (oviposition) of the treated and the control population of Aedes aegypti F1 females was found to be statistically non-significant (p = 0.222) as determined by Mann-Whitney Rank sum test indicating that the biolarvicide did not affect the fecundity of the treated females. However, the mean egg hatchability of larvae from the eggs was more in the control group than in the test

162 group and this difference was statistically significant (p < 0.001) as determined by t-test showing thereby that although fecundity was not affected post-treatment but hatching of larvae was affected due to much lesser hatching of larvae from the treated eggs compared to the control eggs. In addition, the larval formation rate was slightly lesser in the test than in the control group. However, this difference was non-significant (p = 0.150) suggesting that there was no effect of the treatment. The pupation rate was significantly higher in the test group as compared to the control group (p < 0.001), however adult emergence was significantly lower in the test group than the control group (p < 0.001) using ^Chi square test. The present study indicated no difference between survival time of treated and the control Aedes aegypti F1 males and females (p = 0.074 and 0.070 for treated and control males and females respectively) using Log rank test suggesting thereby that treatment with sub-lethal dose of 1.014ppm did not affect longevity of Aedes aegypti.

In Culex quinquefasciatus F1 females (n = 10), a total of 8 gonotrophic cycles were followed. The overall fecundity was highest in the 5th gonotrophic cycle. No egg laying was observed in the treated and control mosquitoes in the 7th and 8th gonotrophic cycle. The fecundity (oviposition) of the treated Culex quinquefasciatus F1 females and the control females did not differ (p = 0.183) as determined by Mann-Whitney Rank sum test. However, the mean egg hatchability was more in the control group than the test group and this difference was statistically highly significant (p = 0.0003) as determined by Student’s t-test suggesting that treatment affected hatchability of larvae from eggs and the treatment induced sterility or adversely affected embryogenesis. In addition, larvae formation rate was nearly equal between test and control group, and it was also not statistically significant difference between groups (p = 0.805, using chi-square test). Pupation rate was higher in test than control group, it was also statistically significant (p < 0.001, using chi-square test). Adult emergence was lower in test group than control group, it was also statistically significant (p < 0.001) as suggested by the ^Chi square test the reason for which needs further exploration. The present study indicated non-significant difference between the survival time of treated and control F1 females (p = 0.754) using Log-rank test suggesting that sub-lethal dose of Sporosarcina sp. strain BGUMS101 did not make any difference to the longevity of the treated female population. However, for test and control males the survival duration was higher in test than in control group

163 males and the difference was significant between the two groups (p = 0.054) using Log-rank test. Kamiabi et al., 2015 have noticed reduction in the fecundity of the parental generation of Aedes aegypti and Aedes albopictus species following treatment with a sublethal dosage of the crude plant extract of Cyperus aromaticus. It was hypothesized that the extract of C. aromaticus might exert effects on fecundity and hatchability in mosquito species by its influence on the endocrine system due to the juvenile hormone III content [211]. Several other studies have also similarly addressed the reproductive responses of mosquito species towards plant derived extracts. Most of them have suggested reduction in the egg production in different mosquito species, following exposure to different concentrations of various phytoextracts [212, 213, 214]. Several available studies have also reported the fecundity and reproductive potential reduction in the mosquito species after exposure to the sublethal dose of chemical insecticides. Reports show that temephos and propoxur induced a marginal fecundity reduction in the An. stephensi. This may be due to the direct toxic effect of the insecticide which remained un-metabolized in the body of the parent and may have been transmitted to the eggs resulting in lower egg hatchability [215]. The exposure of Ae. aegypti females to higher concentrations of temephos, malathion and alpamenthtin has shown decreasing numbers of eggs production [216]. Also, a decreased fecundity upon exposure to entomopathogenic fungi, Leptolegnia chapmanii [208] and bacterial agents Bacillus thuringiensis H-14 has been observed in Ae. aegypti [217]. Reports suggest that the insect’s neuro-hormone system plays a key role in regulating a complex behavioural and physiological mechanisms related to their reproduction. Exposure to insecticide toxins can cause coordination disruption resulting in a diminished reproductive capability [218]. In the present study there was a reduction in the number of eggs produced by the treated population of F1 females as compared to the control. This reduction in the number of eggs produced by the female mosquito is of great advantage as a control tool in decreasing the total mosquito population

In the present study the developmental period i.e., the time required for completion of life cycle from eggs hatched till adult emergence was recorded as well.

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It was observed that developmental period is increased in the test population of Anopheles stephensi as compared to the control population of Anopheles stephensi mosquito (i.e., in the test population: L1-L4 = 18 days, pupal development and adult emergence = 11 days and in the control population: L1-L4 = 12 days, pupal development and adult emergence = 7 days). In Aedes aegypti and Culex quinquefasciatus populations no significant difference in developmental period between the treated and control populations was observed. The findings of the current study were in agreement with results of several previous research work on plant-based compounds.

Application of different concentrations of the crude leaf extracts from several plants such as Murraya koenigii, Citrullus colocynthis and Abutilon indicum against the early 3rd instar larvae of Ae. aegypti, An. stephensi and Cx. quinquefasciatus, significantly prolonged the development of the larvae and pupae stages [219]. Likewise, treatment of the 3rd instar larvae of Anopheles stephensi with 12.5, 25, 50 and 100 μg/ml of methanol extract of Azadirachta indica extended the developmental period from10 days in the control to 11, 12, 14 and 15 days respectively [220]. An extension in the larval developmental span of Ae. aegypti after exposure to 10 μg/ml of the acetone fraction of petroleum ether extract from the seeds of the Argemone mexicana plant also has been reported [221]. In addition, sublethal dosage of the methanolic extracts from Nerium indicum and Euphorbia royleana induced a significant delay in pupation in treated larvae of Cx. quinquefasciatus [222]. Similar findings were reported in this mosquito treated with 400 μg/ml of Momordica tuberosa leaf extract [223]. Such prolonged larval and pupal periods of insects could indicate interference by bioactive compounds on the normal hormonal activity coordination of the metabolic processes of the developing stages probably due to interference in the endocrine mechanism [221].

With reference to studies using microbial agents, in a study by Lee et al., 2005 Aedes albopictus larva, pupa and adult survival rate was significantly reduced (p < 0.05) after treatment with Bti [224] at the dosage of LC50 as compared to control . In this study, lower blood-engorgement rate was recorded by the treated group as well as lesser egg production by F1 females when compared to the control group. Longer life development period (number of day) was required by the offspring to emerge into adult from egg in this study after Bti treatment.

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In another study by Wang et al., 2005, the survival rate of Ae. aegypti larvae was significantly reduced (p < 0.05) up to 42% as compared to the control. For the adult emergence rate, more than 45% reduction was recorded after treatment with Bti and a lesser number of eggs were produced as compared to the control. The developmental period of immature stages increased by additional 5 days as compared to the control group [217].

It has been reported that larval development is slowed by reduced food availability hence, it is possible that the sublethal dose of Sporosarcina sp. strain BGUMS101 might have affected the nutrient assimilation of exposed larvae and lead to longer larval development period [225, 226]. The present study makes an attempt to explore the effect of sublethal dose of microbial agent Sporosarcina sp. strain BGUMS101 to study the fecundity of F1 females, life span and survival rate of treated populations, developmental period of the immatures, egg hatch rate, pupation percentage and adult emergence rate versus untreated control population of 3 test vector species i.e., Anopheles stephensi, Aedes aegypti and Culex quinquefasciatus.

Information about sublethal effects against vector mosquitoes is of utmost importance in control programmes. Control of vectors at larval stage is much easier when compared to adult stage since the movement area of larvae is restricted to the water habitat whereas adults are terrestrial and difficult to control. In addition, sublethal effects in prolonging the developmental period of malaria vector Anopheles stephensi immature stage will increase the chances of microbial bio- control agents to kill the larvae i.e., prolonging the immature stage development period could provide a longer time for the larval treatment by larvicides in addition to delaying the production of adults. Hence, in addition to the direct mortality caused, the advantages due to the reduction in the number of eggs produced and increased immature developmental period make Sporosarcina sp. strain BGUMS101 suitable as a biolarvicide for the control of mosquitoes.

In the present study, we found that soil samples from a marine environment in Goa contain novel strains of mosquito pathogenic bacilli. There is good potential for formulating a range of bacterial insecticides for more effective and environmentally safer control of common mosquito vectors and the diseases that they transmit. Therefore, there is a need to screen, isolate and characterize indigenous and more effective new bacterial isolates, which are not only locally

166 adapted but also provide a wide range of activity and pave way for bacilli based indigenous formulations which can then be utilized for vector control. Emergence of insecticide resistance and their harmful effects on non-target organisms and environment has necessitated an urgent search for development of new and improved as well as economical and effective mosquito control methods that are safe to non-target organisms and the environment [182]. In the recent years, one major challenge for achieving successful mosquito control has been the overcoming of insecticide resistance. Resistance to the microbial insecticides such as Bacillus thuringiensis subsp. israelensis (Bti) and Bacillus sphaericus (Bs) represents a serious potential threat to their success [109]. Available evidence indicates that the risk for resistance development to Bti is extremely low due to the complex makeup of its parasporal crystal, which contains Cyt1A, Cry4A, Cry4B, and Cry11A toxic proteins. Disrupting the toxin complex in Bti enables resistance to evolve, especially in the absence of the key factor, the cytolytic toxin, Cyt1A. Cross-resistance is widespread among mosquitocidal Bacillus thuringiensis Cry toxins and the mechanisms of Cry resistance in mosquitoes are, however, not known. Bacillus sphaericus (Bs) is at higher risk of resistance development in mosquito vectors due to its single-site action and there have been reports of resistance in Bs from a number of locations worldwide [197]. Until recently, chemical larvicides were the main armaments in the mosquito immature control strategy in most parts of the world and when compared with biolarvicides, the primary considerations are efficacy, safety and cost.

There are reports of extensive use of the Bacillus genus but these focused mainly on the control of agricultural pests [199]. Nevertheless, in the last few decades several formulations of biolarvicides for vector control of mosquitoes have become commercially available. The major advantage in the use of these organisms is their safety to non-target organisms including humans. However, their continued utilization in vector control programs including integrated vector management, would depend upon better screening methods, isolation of highly virulent indigenous strains showing broad spectrum of activity against mosquitoes, more effective formulations and mass production at an affordable cost [104]. Since this is the first report on the mosquitocidal activity of Sporosarcina sp. strain BGUMS101 and Aneurinibacillus sp. strain C1A these bacterial agents can serve as potent candidates in the wake of resistance development to the already available bacterial mosquitocides.

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CHAPTER 6: SUMMARY & CONCLUSIONS

This study highlights the mosquito pathogenic activity of bacterial strains isolated from the coastal region of Goa, India. Three sites in Goa (Ribandar, Miramar and Curca) were selected for screening for mosquito-pathogenic bacilli. Novel strains showing marked pathogenicity against 3rd instar larvae of Anopheles stephensi, Aedes aegypti, Aedes albopictus and Culex quinquefasciatus were obtained. Preliminary screening revealed that, the novel screening method for isolation of mosquito-pathogenic bacteria from soil samples proposed by Dhindsa et al., 2002 was experimentally more viable to the other screening methods. In addition, media optimization studies indicated that Nutrient yeast extract salt medium (NYSM) possessed the highest toxicity as compared to when grown on other types of media. Incubation of bacilli isolates for 96 hours in NYSM resulted in peak growth and sporulation. Following the preliminary screening, those isolates showed significant (> 50%) mortality were further bio assayed at different doses to determine their LC50 and LC90 values. Based on this criterion, seven isolates viz., BGUMS14, BGUMS16, BGUMS101 from Ribandar isolation site and C1A, C2A, C2C and BIC from Miramar beach site which produced > 50% mortality were shortlisted for most of the bioassays.

The morphological and biochemical characterization revealed that all the isolates were Gram positive rods and endospore producers belonging to Bacillus spp. Their identity was further confirmed at molecular level by phylogenetic analysis. Isolate BGUMS101 was identified as Sporosarcina sp. and this is the first report on its mosquitocidal activity in the larvae of Aedes albopictus (LC50 = 0.18 ppm) and Culex quinquefasciatus (LC50 = 0.225 ppm) being highly susceptible. Isolate BGUMS14 was identified as Bacillus sp. while the isolate BGUMS16 was identified as a strain of Bacillus safensis. No prior reports exist on their mosquito larvicidal activity. Hence this is the novel finding as far as these species are concerned.

In addition, the isolate C1A which was identified at molecular level, as Aneurinibacillus sp. rd showed high toxicity (LC50 = 0.08 ppm) to Culex quinquefasciatus 3 instar larvae. This is also the first report of Aneurinibacillus sp. having mosquito pathogenic activity. Isolates C2A and

C2C were identified as Lysinibacillus sp. showing high toxicity (LC50 = 0.30ppm and LC50 = 0.25ppm respectively) against Culex quinquefasciatus 3rd instar larvae. The isolate B1C was

168 identified as a strain of Bacillus thuringiensis showing a broad spectrum of mosquito pathogenic activity against the 3rd instar larvae of the 4 test vector species. On comparison of the activity of these previously unreported isolates with that of commercial formulations of Bacillus thuringiensis H-14 and Bacillus sphaericus Neide 2362, the larvicidal activity of Sporosarcina sp. strain BGUMS101 against Aedes albopictus was found to be comparatively higher in all the species but lesser than the commercial Bacillus sphaericus strain in terms of LC50 values. This high larval mortality occurred because of lysis of the mid gut due to the lethal activity of insecticidal toxins and this mechanism is well known. This could provide an avenue for the development of a potent indigenous strain based commercial formulations for vector control.

Further, the culture supernatant obtained by centrifugation of the bacterial culture of the active isolates was also tested against the 3rd instar larvae as well as pupae of the test vector species. The culture supernatant was found to be most toxic to Culex quinquefasciatus 3rd instar larvae however low pupicidal activity across the test vector species was also observed in general. The crude mosquitocidal toxin (CMT) of the bacterial isolates was obtained by acid precipitaion of the culture supernatant however, no significant mosquito larvicidal activity as compared to the commercial strains was observed. Sporosarcina sp. strain BGUMS101 showed best pupicidal activity against Anopheles stephensi and Culex quinquefasciatus pupae, while the remaining isolates showed extremely low pupicidal activity. The mode of action was observed to be both direct action of the CMT on the pupae resulting in its death as well as by causing emergence inhibition of the adults from the pupae. This mode of action has not been previously reported; therefore, this is new information.

An attempt was made to identify the toxin gene responsible for the larvicidal and pupicidal action of these previously unreported bacterial isolates by using PCR based methods. Sporosarcina sp. strain BGUMS101 was negative for all the toxin genes tested which are known in other mosquito pathogenic bacilli Bti and Bs [i.e., Cry 2(UN), Cry4 (UN), Cyt1 (UN), Cyt2 (UN), BINA, BINB, Mtx gene, Gyrase, Surfactin] indicating the presence of a novel toxic component responsible for its larvicidal and pupicidal activity. However, Aneurinibacillus sp.

169 strain CIA were found positive for BinA, BinB and MtX toxin gene indicating that it has Bacillus sphaericus like mode of action.

In addition, during the present study, adverse effects following treatment of the F1 generation of the test vector species with sub lethal dose of Sporosarcina sp. strain BGUMS101 on the fecundity, egg hatchability, pupal formation, adult emergence and life span as well as developmental period were also studied. A significant reduction in the fecundity and egg hatchability in the treated v/s control population was observed across the test vector species. It was observed that the larval hatch rate was lesser in the test group as compared with the control group, however, this difference was statistically non-significant. In addition, the pupation rate was significantly higher in the test group as compared to the control group (p < 0.001). However, the adult emergence was significantly lower in the test group than the control group as indicated by the ^Chi square test. This effect was common across the test vector species. All these observations suggested that sub-lethal doses have impact on fecundity, egg hatchability, pupal formation, adult emergence and life span of vector species.

There was no significant difference between survival time of treated and control F1 females (p = 0.0606) of Anopheles stephensi as indicated by Log-rank test. However, in the test and control males, the survival was surprisingly longer (statistically significant) in the test as compared to the control group. The present study also indicated no significant difference between survival time of treated and the control Aedes aegypti F1 males and females. With Culex quinquefasciatus treated and control population it was observed that sub-lethal dose of Sporosarcina sp. strain BGUMS101 did not make any significant difference to the longevity of the treated female population. However, in the test and control males, the survival duration was significantly more in the test than in control group males.

Another observation made was that the developmental period is increased in the test population of Anopheles stephensi as compared to the control population i.e., in the test population: L1 - L4 larval stage took up to 18 days, pupal development and adult emergence up to 11 days as against the control population: L1 - L4 stage took 12 days, while pupal development and adult emergence took up to 7 days. This means that even when sub-lethal dose is applied to the

170 breeding habitat or available, it will decrease the rate of development of larvae and pupae and the increase in generational time would adversely affect the population growth of this important malaria vector and in turn the transmission of malaria. In the other hand, no significant difference in developmental period was detected in the test and control populations of the other tested vector species. This research and knowledge about sub-lethal effects against vector mosquitoes are of utmost importance for disease control programmes. Further, studies are recommended to characterize the larvicidal toxins and the mode of action of these isolates. As there are no prior reports on these novel mosquito pathogenic bacteria from the coastal region of Goa, it could be advantageous to continue exploring this ecosystem for more potential bacterial isolates. Mass scale production of different formulation of these novel bacteria for further laboratory and limited field trials are necessary with appropriately designed experiments.

Although a variety of mosquito pathogenic bacteria have been isolated from various other geographic zones around the world, there is felt need to explore and deploy indigenous strains in vector control programmes due to restrictions imposed on the use of imported strains, environmental concerns, and prohibitive cost of the formulations and due to resistance development in the target mosquito species. As no mosquito larvicidal strains of Sporosarcina sp. strain BGUMS101 and Aneurinibacillus sp. strain C1A had been earlier described, these organisms may be considered potential novel agents for biological control of disease vectors.

In addition, the results from this study suggest that the search for new entomopathogenic bacteria should be continued and intensified.

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CHAPTER 7: BIBLIOGRAPHY

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APPENDIX

1. MEDIA

1.1 Nutrient Agar • Peptone 5g • NaCl 5g • Beef Extract 3g • Agar 15g • Distilled Water 1000ml • pH 6.8

1.2 Nutrient Broth (NB) • Peptone-10g • Beef extract- 10g • NaCl- 5 g • Distilled Water- 1000ml

1.3 NYSM Broth (Nutrient Yeast Extract Salts/ Sporulating Medium) Yousten (1985) • Nutrient Broth 13g • Yeast Extract 0.5g • Distilled Water 1000ml • pH 7 -4 -3 -5 • CaCl2 1.0304g (7x10 M), MgCl2 2.033g (1x10 M), MnCl2 9.895g (5x10 M) Dissolve each of the salts separately in 100ml each of distilled water and sterilize by autoclaving at 121°C and 15 psi for 12 minutes. To 970ml of sterile basal medium add 10ml each of the salt solutions and mix well [149].

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1.4 LB Acetate (pH 6.8, 0.25m Sodium Acetate) • Tryptone 1g • Yeast Extract 0.5g • NaCI 1g • Distilled water 100ml

• pH was adjusted to 6.8 with 100 m1 of 0.25M CH3COONa.3H20

1.5 Luria Bertani Broth • Tryptone 1g • Yeast Extract 0.5g • NaCI 1g • Distilled water 100ml

1.6 Nitrate Broth • Tryptone 1.5g • NaCl 0.5g • Potassium Nitrate 0.1g • pH 7.2 • Distilled water 100ml

1.7 Production of Indole • Tryptone 1.5g • NaCl 0.5g • pH 7.2 • Distilled water 100ml

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1.8 Glucose Phosphate Broth Peptone Broth • Peptone 5g

• K2HPO4 5g • Glucose 5g • pH 6.8 • Distilled water 1000ml

1.9 Gelatin Liquefaction • Nutrient Gelatin 128g • Distilled water 1000ml

1.10 Starch Hydrolysis • Nutrient Agar supplemented with 0.2- 1% starch.

1.11 Tyrosine Agar • Peptone 5g • NaCl 5g • Beef Extract 3g • Agar 15g • L-Tyrosine 5g • Distilled water 1000ml

1.12 Casein Agar • Skim Milk Powder 100g • Agar 20g • Distilled Water 1000ml Dissolve the agar in 500 ml of distilled water and sterilize at 121˚C for 20 minutes. Add the skim milk solution to the hot agar solution and mix well before pouring in sterile plates.

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1.13 Tween Agar • Peptone 1g • NaCl 0.5g

• CaC12 0.1g • Tween lml • Agar 1.5g • pH 7- 7.4

1.14 Thornley's Medium • Peptone 1g • NaCl 0.5g

• K2HPO4 0.0g • Arginine monohydrochloride 1g • Phenol Red 0.001g • Distilled water 100ml • Agar 0.3g

1.15 Hugh Leifson Basal Media • NaCl 5.0g

• K2HPO4 0.3g • Peptone 2.0g • Bromothymol blue 0.03g • Glucose 10g • Agar 3.0g • Distilled water 1000ml

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1.16 MD medium (Media D)

• Tryptone- 15g • Soya peptone- 5g • NaCl- 5g • Distilled Water- 1000ml

1.17 1/4th Nutrient Broth (NB) • Nutrient broth (HiMedia) at 1/4th concentration i.e., 6.25 grams in 1000ml distilled water.

1.18 NYSM broth supplemented with NaCl • NYSM broth (Appendix 1.3) containing 1%, 3%, 7% NaCl

2. REAGENTS 2.1 O’Meara’s reagent • Creatine 0.15g • NaOH 40g • Distilled Water 100ml Dissolve the NaOH in distilled water. To 3ml of this add 0.15g creatine.

2.2 Griess IIosvays reagent i) 8gm Sulphanalic acid in 1000ml 5N Acetic acid ii) 5gm α-Napthalamine in 1000ml of 5N Acetic acid

2.3 Kovacs Indole Reagent • Isoamyl alcohol 150ml • Para-dimethylaminobenzaldehyde 10g • Conc. HCl 50ml

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2.4 Methyl Red solution • Methyl red 0.5g • Ethanol (95%) 300ml • Distilled water 200ml

2.5 SDS-PAGE reagent 1. Lysis solution • 50mM Glucose • 25mM Tris Cl (pH 8) • 10mM EDTA 10ml of this solution was prepared

2. Extraction buffer • 1mM Phenylmethylsulphonyylflouride (PMSF) • 10mM EDTA • 2% SDS 50ml of this solution was prepared

3. Dissociation buffer (sample buffer) • 0.625 M TrisCl (pH 6.8) • 2% SDS • 5% β-mercaptoethanol • 10% glycerol • 0.005% Bromophenol blue

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Presentations

National 1. Participated in the National Conference ‘Frontiers in Bio pesticides And Bio fertilizers’ on 6th and 7th December, 2019 at P.E.S.’s College, Goa, India and won the 1st prize for poster presentation titled – “Biological control of mosquito larvae by using novel strains of halophilic bacteria – a new vector control tool.” 2. Participated in the Society for Vector Ecology (Indian region) inaugural International Conference held in Goa from 13th -16th February 2019 and made an oral presentation titled - “Sourcing and evaluation of the efficacy of mosquito pathogenic bacilli from coastal environment in Goa, India.” 3. Participated in the 28th National Congress of Parasitology from February 22nd - 24th 2018 at Belagavi, Karnataka, India and won the 1st place for a poster titled “Biological Control of vectors with novel mosquito pathogenic bacteria from the Coastal belt of Goa, India.” 4. Secured the 1st position in poster presentation on “Biological control of mosquito vectors” held on 1st October 2016 at the DST SERB School in Insect Biology, Punjabi University, India. 5. Oral presentation at the: National Seminar on “Life and Life processes: Sustainable Development” from February 19 – 21, 2015 at the Department of Zoology, Goa University on “Isolation, characterization and efficacy of mosquito pathogenic bacteria”.

International 1. Participated in the Global Vector control response (GVCR-2019) International Conference held in Wageningen, The Netherlands from 11th -13th June 2019 and made a poster presentation titled - “Biological Control of mosquito vectors of global significance using novel mosquito pathogenic bacteria from the Indian coast.” 2. Participated and presented a poster titled “Detection of mosquito pathogenic bacteria from coastal belt of Goa, India” at the 7th International Congress of the Society for Vector Ecology (SOVE) from October 1st - 6th 2017 at Palma de Mallorca, Spain.

198

PUBLICATIONS

1. Almeida Joleen S1, Mohanty Ajeet K1, Dharini N1, Hoti SL2, Kerkar S3, Kumar Ashwani1*. Evaluation of entomopathogenic Bacillus spp. isolated from Miramar Beach, Goa, India against mosquito larvae. International Journal of Mosquito Research 2020; 7(2): 21-29. 2. Almeida JS1, Mohanty AK1, Kerkar S2, Hoti SL3., Kumar A.1* Current Status and future prospects of Bacilli based vector control. Asian Pac J Trop Med 2020; 13(12): 525- 534. 3. Almeida Joleen S1, Mohanty Ajeet K1, Kerkar S2, Kumar Ashwani1* (2020) A new species of mosquito larvicidal bacteria from marine ecosystem. (Communicated - Under review)

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International Journal of Mosquito Research 2020; 7(2): 21-29

ISSN: 2348-5906 CODEN: IJMRK2 IJMR 2020; 7(2): 21-29 A report on novel mosquito pathogenic Bacillus www.dipterajournal.com © 2020 IJMR Spp. isolated from a beach in Goa, India Received: 10-01-2020 Accepted: 14-02-2020 Joleen Almeida, Ajeet Kumar Mohanty, Dharini N, SL Hoti, Savita Joleen Almeida ICMR-National Institute of Kerkar and Ashwani Kumar Malaria Research, DHS Building, Campal, Panaji, Goa, Abstract India Nine Bacilli were isolated from 3 spatially diverse zones viz., tidal, intertidal and seashore at Miramar beach in Goa, India. The isolates were assessed for mosquito larvicidal activity against 3rd instar larvae of Ajeet Kumar Mohanty Anopheles stephensi, Culex quinquefasciatus, Aedes aegypti and Aedes albopictus. Colony and ICMR-National Institute of biochemical characteristics of the isolates showed similarity to Bacillus sphaericus Neide 2362 and Malaria Research, DHS Bacillus thuringiensis var. israelensis H-14 commercial strains. Preliminary toxicity screening indicated Building, Campal, Panaji, Goa, India that four isolates (B1C, C1A, C2A and C2C) possessed mild to high toxicity (60-100% mortality) and were further assayed to obtain LC50 and LC90 values following a 24 and 48 hour treatment period. Dharini N Phylogenetic analysis revealed that these isolates clustered with mild and highly toxic strains of Bacillus, ICMR-National Institute of Lysinibacillus and Aneurinibacillus sp. This is the first lead on the presence of mosquito pathogenic Malaria Research, DHS bacterial isolates native to Miramar beach in Goa which can be further explored for development of Building, Campal, Panaji, Goa, formulations for vector control. India Keywords: Seashore, larvicidal bacteria, high toxicity, formulations SL Hoti National Institute of Traditional 1. Introduction Medicine, Belagavi, Karnataka, India Mosquitoes transmit a variety of diseases that pose serious public health challenges worldwide [1]. The most important disease vectors belong to the following two subfamilies i.e. Savita Kerkar Anophelinae (Anopheles mosquitoes) which transmit malaria and Culicinae (Culex Department of Biotechnology, mosquitoes) that transmit filariasis, West Nile virus and Japanese encephalitis and Aedes Goa University, Goa, India mosquitoes responsible for dengue, chikungunya, yellow fever and Zika transmission. In order

Ashwani Kumar to prevent the proliferation of these mosquito borne diseases and to improve the quality of life, Ph.D., ICMR-National Institute vector control is essential. of Malaria Research, DHS Goa is a popular domestic and international tourist destination in India. Among the vector Building, Campal, Panaji, Goa, borne diseases, Goa has witnessed widespread outbreaks of malaria, dengue, chikungunya and India Japanese encephalitis, peaking during the monsoon season, especially along the coast since the [2] mid-1980s . Resurgence of vector borne diseases due to favourable environmental conditions, human lifestyle changes, rapid urbanization, development of resistance in vectors to routinely used public health insecticides, changes in public perception against mosquito nuisance favour adoption of safer and effective methods of vector control [3, 4]. The World Health Organization

advocates Integrated Vector Management (IVM), where emphasis is placed on the application of alternative cost effective, environment friendly and sustainable methods of vector control [5]. Among the alternate control tools, biological control agents such as larvivorous fish and several strains of spore forming bacteria (Bacillus sphaericus & Bacillus thuringiensis var. israelensis) have shown effectiveness for vector control in several field evaluations [6].

Besides, Bacillus subtilis, was also found to be a promising candidate for mosquito vector [7] control . Currently, these microbial agents are formulated as an aqueous suspension, wettable powder, water dispersible granules, briquettes as well as tablets to suit the application in the Corresponding Author: various breeding habitats of mosquitoes [8]. However, there is a risk of development of Ashwani Kumar resistance in the vectors to these bio control agents as a result of their long-term use. There are Ph.D., ICMR-National Institute of Malaria Research, DHS reports of development of resistance by the vectors against B. sphaericus and B. thuringiensis [9] Building, Campal, Panaji, Goa, var. israelensis , which calls for continued search for new bio control agents of mosquitoes. India Hence in the present study, we explored the coastal region of Goa i.e. ~ 21 ~

International Journal of Mosquito Research http://www.dipterajournal.com

Hence in the present study, we explored the coastal region of GTTTGATCCTGGCTCAG-3`) and (S-*-Univ1392-b-A-15: Goa i.e. Miramar beach for bio-larvicidal bacteria and it 5`-ACGGGCGGTGTGTNC-3`) [13]. PCR reaction mixture resulted in the isolation of some novel, previously unreported consisted of 15μl of PCR Master mix 2X concentrated strains. These strains were identified and characterised solution (0.05U/μl taq DNA polymerase, reaction buffer, through morphological, biochemical and molecular methods. 4mM MgCl2, 4x 1.25 ml nuclease free water), 1 μl of forward Their larvicidal efficacy was assessed against the four primer and 1 μl of reverse primer and 1.5 μl of the extracted mosquito vector species and the results are presented in this bacterial genomic DNA template of each sample was added in communication. sterile PCR tubes and the final volume was made up to 30 μl by using nuclease free water. Without DNA template control 2. Materials and Methods was also maintained. The samples were amplified in a thermal 2.1 Bacterial Isolation and Biochemical characterization cycler (Bioer XP Cycler). The PCR conditions were initial Soil samples were collected from 9 locations at Miramar denaturation at 94°C for 5 min, 30 cycles of denaturation at beach, Goa, India. The sampling location was at 15.4827° N 94° C for 1 min, annealing at 55°C and extension at 72°C for and 73.8074° E. Approximately, ten grams of soil samples 2 min respectively, followed by final extension at 72°C for 5 were collected from near the sea shore, intertidal and tidal min. To check for the amplification, 8μl of PCR amplified zones aseptically with a spatula and transferred into a sterile products were run on a 1% agarose gel. To verify the size of falcon tube. The top layer of the soil was collected aseptically the PCR product, 500bp ladder was loaded along with the and samples were transported to the laboratory for further sample. The amplified PCR products were purified using processing. For isolation of mosquito pathogenic bacteria Qiagen Q1A quick PCR purification kit (Cat. No. 28104) and from the soil samples, three methods were followed. Method sent for sequencing to Eurofins India Genomics Pvt. Ltd., 1 involved inoculation of 1 gram of soil sample in 10 ml Bangalore. The sequences obtained were edited using the Bio Nutrient broth (NB) followed by incubation on a shaker at edit sequence alignment editor version 7.0.4.1 software and 100 rpm for 48 hours. One ml of the supernatant was then compared with sequences of other closely related species screened for mosquito larvicidal activity. Method 2 consisted retrieved from the GenBank database of direct inoculation 0.5 grams of soil in 100 ml of Nutrient http://www.ncbi.nlm.nih.gov/BLAST and identified based on yeast sporulating (NYSM) medium followed by incubation on sequence homology. a shaker at 100rpm for 48 hours. Method 3 was that proposed by Dhindsa et al. in 2002 [10]. Accordingly, 0.5 g of soil 2.2.3 Phylogenetic analysis sample was added to LB broth (10ml) (buffered with sodium The phylogenetic analysis and tree construction was carried acetate) and incubated on a shaker at 200 rpm for 4 hours at out using neighbour-joining method. ClustalX version 2.0. 30˚C. One milliliter of this aliquot was heat shocked at 65˚C was used to generate multiple sequence alignment between for ten minutes in a pre-warmed 5 ml glass tube from which closely related species. The tree was obtained with 100 seeds 0.1 ml of sample was removed, added to 1 ml of LB broth and and 1000 bootstraps. The final tree was rooted and drawn incubated for 24 hours at 30˚C. To the same glass tube, one using MEGA4 [14]. ml of NYSM (sporulating medium) was added and incubated for two days at 30˚C. Sample (0.1ml) was withdrawn for 2.3 Bioassays preliminary toxicity testing against the 3rd instar larvae of the 2.3.1 Source of Mosquito Larvae mosquito vector species. The active soil samples were then The mosquito colonies of the 4 test vector species (An. processed by serial dilution and spread plated followed by stephensi, Cx quinquefasciatus, Ae. aegypti and Ae. picking up the morphologically different colonies. Pure albopictus) were maintained at ambient laboratory conditions colonies were maintained on Nutrient Agar (NA) plates. i.e. temperature of 27±2 ⁰C, relative humidity 70± 5% and a Colony, morphological and biochemical characteristics of the 12hrs day and night cycle. A pinch of larval food (Cerelac™ isolates were studied as per the tests listed in Bergey’s and finely ground fish meal in a 1:1 ratio) was provided to the Manual of Systematic Bacteriology [11]. Morphological larvae daily until the pupae stage. From the laboratory bred characteristics observed were colony morphology, Gram immature mosquitoes, healthy 3rd instar larvae were used for staining and endospore staining. Biochemical characteristics bioassays to screen the bacterial isolates for larvicidal activity. tested were nitrate, catalase, indole, oxidase, sugar fermentation, MR-VP, gelatin liquefaction, starch hydrolysis, 2.3.2 Preliminary toxicity screening tyrosine degradation, hydrolysis of casein, hydrolysis of Preliminary toxicity screening was carried out in sterile tween, hydrolysis of arginine, Hugh leifsons test and growth bioassay bowls containing ten laboratory reared larvae of each conditions. Sensitivity against antibiotics (Kanamycin, of the test species in 100 ml of water. 0.1ml of a bacterial Streptomycin and Erythromycin) was also assessed. culture grown in NYSM broth (sporulating medium) was used for screening for checking larvicidal activity. Uninoculated 2.2 Molecular Characterization NYSM broth was used in the control. Mortality was scored 2.2.1 DNA extraction after 24 hours and 48 hours of the treatment by counting the Pure colonies of the isolates were sub-cultured onto Nutrient number of the dead larvae in the respective bioassay bowls. Agar plates and following 24hrs incubation the bacterial The percentage mortality was calculated using the formula: genomic DNA extraction was carried out by phenol- chloroform method [12]. Number of dead larvae % Mortality = X 100 Total number of larvae 2.2.2 PCR amplification The 16S rRNA gene amplification was carried out using the If the % mortality was > 5% in the control larvae, the universal primers (S-D-Bact-0011-a-5-17: 5`- corrected mortalities were determined by Abbot’s formula [15]. 22 International Journal of Mosquito Research http://www.dipterajournal.com

2.3.3 Purification and storage of the isolates The isolates that showed potent activity were further maintained by streaking onto Nutrient Agar plates and slants which were kept in the refrigerator at 4 °C. They were purified by sub-culturing followed by Gram staining of individual colonies in order to confirm purity. A pure sample of each isolate was kept safe at -20 °C in 20% (v/v) glycerol.

2.3.4 Preparation of lyophilized powder for bioassay The bacterial isolates were grown in a 250ml conical flask containing 100ml of NYSM broth at 28°C on a rotary shaker for 72-96 hours [16]. Spore crystals were harvested by centrifugation at 8000rpm for 20 minutes and the pellet was washed twice with sterilized distilled water. The final pellet Fig 1: Screening of soil sample by Method 1 using Nutrient broth obtained was lyophilized and stored at 4 ˚C until use. (NB)

2.3.5 Main Bioassays The bioassays of the bacterial isolates were carried out against laboratory reared 3rd instar larvae of An. stephensi, Cx. quinquefasciatus, Ae. aegypti and Ae. albopictus mosquitoes according to WHO protocol [17]. Stock dilutions in parts per million (ppm) were prepared by dissolving the weighed lyophilized powder in sterile distilled water. A series of doses were prepared based on the preliminary toxicity screening results. Four replicates of 25 3rd instar larvae from each mosquito species were taken in 500ml plastic bowls containing 250ml of distilled water and different doses were administered. Concurrent controls were maintained under similar conditions. The mortality was scored by counting the number of dead larvae after 24 hours and 48 hours of treatment. Efficacy of the isolates in terms of LC and LC 50 90 was calculated by analyzing dose mortality responses of individual strains by Probit analysis using SPSS version 16 Fig 2: Screening of soil sample by Method 2 using Nutrient Yeast software [18]. Sporulating medium

3. Results 3.1 Isolation and Characterization of potential larvicidal bacterial strains A total of 9 soil samples from three different zones i.e. tidal (Sample A1, B1, C1), intertidal (Sample A2, B2, C2) and sea shore (Sample A3, B3, C3) were collected and processed further. Among the three methods used for screening the soil samples, the method proposed by Dhindsa et al. [10] showed the best results in terms of mosquito pathogenic activity with sample B1 collected from the tidal zone showing 100% mortality against the 3rd instar larvae of all the test vector species. Sample C1 obtained from tidal and C2 from the intertidal zone showed 100% mortality against 3rd instar Culex quinquefasciatus larvae. The rest of the soil samples showed mortality of 40% and below. All the samples processed by

Method 1 and 2 showed a mortality of 20% and below against the tested vector species (Figure 1, 2, 3). Fig 3: Screening of soil sample by Method 3 using Dhindsa et al. 2002 method

23 International Journal of Mosquito Research http://www.dipterajournal.com

The 3 soil samples (B1, C1, C2) which showed significant C1A, C2A, C2C and B1C showed moderate to high toxicity larvicidal activity were considered to be ideal for isolation of (60-100%) against the 3rd instar larvae of An. stephensi, Cx. mosquito pathogenic Bacilli. From these soil samples nine quinquefasciatus, Ae. aegypti and Ae. albopictus. morphologically distinct bacterial isolates were obtained and Subsequently, these isolates were bio assayed at different coded as B1A, B1B, B1C, C1A, C2A, C2B, C2C, C2D, C2E. doses to determine their LC50 and LC90 values as seen in Preliminary screening showed that 4 out of the 9 isolates i.e. Table 1.

rd Table 1: LC50 and LC90 values in ppm of the most active bacterial isolates following 24 hours and 48 hours of treatment against the 3 instar larvae of test vector species

Isolate No. Test Vector species LC50 24 hours 48 hours LC90 24 hours 48 hours 39.42 30.68 303.70 261.28 Anopheles stephensi (17.38-89.36) (12.36-80.88) (133.96-688.49) (184.36-461.46) 0.08 0.032 0.87 0.45 Culex quinquefasciatus (0.038-0.169) (0.012-0.078) (0.423-1.854) (0.174-1.183) Isolate C1A 13.18 3.65 61.15 23.22 Aedes aegypti (7.81-22.26) (2.01-6.63) (36.21-103.24) (12.78-42.17) 23.58 20.593 636.723 190.259 Aedes albopictus (12.54-50.08) (9.57-44.273) (151.24-1156.54) (88.497-409.034) 95.74 38.28 408.76 299.17 Anopheles stephensi (39.43-232.42) (9.85-148.68) (280.28-820.07) (149.44-445.80) 0.30 0.16 1.32 0.62 Culex quinquefasciatus (0.174-0.502) (0.102-0.300) (0.77-2.24) (0.34-1.00) Isolate C2A 50.98 14.21 399.95 96.31 Aedes aegypti (24.82-104.68) (7.73-26.10) (194.7-821.27) (52.42-176.93) 16.500 12.729 89.769 60.629 Aedes albopictus (9.56-28.46) (7.68-21.07) (52.03-154.88) (36.62-100.37) 54.22 29.35 323.78 144.33 Anopheles stephensi (17.39-89.37) (12.36-80.89) (133.96-688.49) (54.81-358.68) 0.25 0.15 1.37 0.70 Culex quinquefasciatus (0.13-0.45) (0.08-0.28) (0.75-2.51) (0.37-1.33) Isolate C2C 42.30 17.17 145.74 60.52 Aedes aegypti (27.89-64.29) (11.21-26.32) (95.88-221.53) (39.49-92.73) 41.848 24.907 124.802 92.703 Aedes albopictus (28.61-61.20) (16.40-37.82) (85.334-182.523) (61.044-140.780) 2.011 0.37 50.2 3.99 Anopheles stephensi (0.83-4.85) (0.18-0.75) (20.79-121.17) (1.96-8.11) 0.162 0.13 0.528 0.461 Culex quinquefasciatus (0.11-0.23) (0.091-0.197) (0.36-0.76) (0.31-0.68) Isolate B1C 0.14 0.13 0.384 0.315 Aedes aegypti (0.10-0.19) (0.09-0.17) (0.28-0.52) (0.23-0.43) 0.189 0.161 0.660 0.530 Aedes albopictus (0.131-0.272) (0.112-0.233) (0.458 - 0.951) (0.368-0.763) 0.34 0.21 1.83 0.63 Anopheles stephensi (0.19-0.61) (0.09-0.46) (1.02-3.25) (0.38- 1.02) 0.0002 0.02 Culex quinquefasciatus 0.00009 (0.00002-0.00031) 0.003 (0.001-0.012) *Bti commercial (0.0001-0.0021) (0.002-0.31) strain 0.00252 0.09 0.020 Aedes aegypti 0.00104 (0.00021-0.00508) (0.00057-0.01121) (0.02-0.48) (0.0042-0.101) 0.00252 0.00015 0.271 0.048 Aedes albopictus (0.00057-0.01121) (0.0002-0.00129) (0.06-1.20) (0.005-0.421) 1.86 0.88 20.23 18.25 Anopheles stephensi (0.72-4.82) (0.31-2.45) (7.83-52.26) (7.45-44.68) 0.00002 0.007 0.0049 Culex quinquefasciatus 0.00006 (0.00001-0.00029) *Bs commercial (0.00001-0.0001) (0.001-0.034) (0.00074-0.03267) strain 0.12 0.05 2.411 0.995 Aedes aegypti (0.04- 0.35) (0.01- 0.16) (0.872- 6.663) (0.319- 3.100) 0.23 0.02 3.594 0.380 Aedes albopictus (0.09- 0.59) (0.008- 0.091) (1.439- 8.977) (0.110-1.313) *All values are in parts per million (pp) *Bti- Bacillus thuringiensis israelensis strain H-14* Bs- Bacillus sphaericus Neide 2362

Microscopic examination following Gram staining and were performed along with commercial strains of B. endospore staining showed all isolates to be gram positive, thuringiensis H-14 and B. sphaericus Neide 2362. The rod shaped and endospore forming. The scanning electron morphological and biochemical characteristics of these microscopy (SEM) images also revealed similar isolates are presented in Table 2. Based on Gordon (1973) and morphological features i.e. rod shaped and endospore formers Berkeley (1984) simplified key for Bacillus species and as depicted in (Figure 4a, 4b, 4c and 4d). Biochemical tests Bergeys Manual of Systemic Bacteriology Vol 4, Bacteria: 24 International Journal of Mosquito Research http://www.dipterajournal.com

Firmicutes, these isolates were found to be belonging to Bacillus sp. [11, 19, 20].

Fig 4 (a, b, c, d): Scanning Electron micrographs of mosquito-pathogenic bacilli isolated from a coastal region sampling site showing long rods and endospores (a) Isolate C1A (b) Isolate C2A (c) Isolate C2C (d) Isolate B1C

Table 2: Biochemical characteristics of Isolates

Isolate TEST C1A C2A C2C B1C Bti H-14 Bs 2362 Gram staining + + + + + + Endospore + + + + + + Indole ------Hugh Leifson ------Oxidase + + + + + + Catalase + + + + + + Motility + + + + + + Growth at 65° ------MR - - - + + - VP - - - + + - Nitrate + - - + + - Sugar (Glucose) fermentation - - - + + - Arginine hydrolysis - - - + + - Tyrosine + - - - - - Casein Hydrolysis - + + - + + Growth in 2% NaCl - + + + + + 5% NaCl - + + + + + 7% NaCl ------Growth at 50°C - - - + + + Sensitivity to Streptomycin(10mcg) + - - + + + Sensitivity to Kanamycin (30mcg) - + + + + + Sensitivity to Erythromycin (15mcg) + + + + + + Tentative identification Bacillus sp. Bacillus sp. Bacillus sp. Bacillus sp. Bacillus thuringiensis Bacillus sphaericus *Bti- Bacillus thuringiensis israelensis strain H-14 Bs- Bacillus sphaericus Neide 2362 +: Positive-: Negative

Further molecular characterization of these isolates by PCR 99.93% sequence similarity with Aneurinibacillus sp., while amplification of the 16S rRNA gene yielded amplicons of the Isolate C2A and C2C showed high similarity (99.2% and size 1500 bps. The PCR amplicons were custom sequenced 100% respectively) with Lysinibacillus sp. Isolate B1C and the nucleotide sequences obtained were blasted against showed 100% sequence similarity with B. thuringiensis strain. the NCBI database using BLASTN. Isolate C1A showed All sequences have been submitted to the NCBI GenBank

25 International Journal of Mosquito Research http://www.dipterajournal.com database with accession id’s MN595035, MN606113, organisms. The isolate C1A formed a coherent branch with MN606137 and MN606138 respectively. The phylogenetic Aneurinibacillus group. While the isolates C2A and C2C tree was constructed based on the comparison of the 16S formed a branch with Lysinibacillus group. Isolate B1C rRNA sequences generated in this study with sequences of aligned with B. thuringiensis group (Figure 5). species belonging to Bacillus genus, and other closely related

Fig 5: Phylogenetic tree showing the phylogenetic relationships among isolate Anuerinibacillus sp strain C1A., Lysinibacillus sp. strain C2A, Lysinibacillus sp. strain C2C, Bacillus thuringiensis strain B1C and their close relatives inferred from 16S rRNA gene sequences from NCBI GenBank.

3.2 Larvicidal activity of the bacterial isolates with Lysinibacillus sp., strain C2C. Culex species showed All the toxic isolates showed a similar pattern with the highest mortality (LC50 = 0.25ppm) followed by Aedes mortality increasing with an increase in the dose (ppm). species (LC50 = 41.84ppm and 42.30ppm against Ae. Aneurinibacillus sp., showed the highest toxicity against the albopictus and Ae. aegypti respectively. An. stephensi showed rd 3 instar larvae of the test vector species with Cx. the least susceptibility (LC50 = 54.22ppm) on 24 hours of quinquefasciatus being most susceptible (LC50 = 0.08ppm) treatment. Lastly, Bacillus thuringiensis sp., strain B1C followed by Ae. aegypti (LC50 = 13.12ppm), An. stephensi showed highest toxicity against Ae. aegypti larvae (LC50 = (LC50 = 39.42ppm) and Ae. albopictus (LC50 = 23.58ppm). 0.14ppm) followed by Cx. quinquefasciatus and Ae. When treated with Lysinibacillus sp., strain C2A, Culex albopictus larvae with LC50 = 0.162ppm and 0.189ppm species showed the highest mortality (LC50 = 0.30ppm) respectively and LC50 of 2.011ppm against An. stephensi followed by Aedes species (LC50 = 16.50ppm and 50.98ppm larvae. The mode of action of the bacterial isolates was against Ae. albopictus and Ae. aegypti, respectively). An. observed to be due to lysis of the midgut of the larvae which stephensi showed least susceptibility (LC50 = 95.74ppm). A is well known, resulting in death (Figure 6, 7, 8, 9). similar pattern of susceptibility was observed upon treatment Treated with novel isolate Fig. Mosquito species Untreated Aneurinibacillus sp. strain C1A 26 International Journal of Mosquito Research http://www.dipterajournal.com

6. Anopheles stephensi

7. Culex quinquefasciatus

8. Aedes aegypti

9. Aedes albopictus

Fig 6-9: Indicating midgut lysis of the 3rd instar larvae following treatment with Aneurinibacillus sp.

4. Discussion from a coastal region of Goa (i.e. Miramar beach) yielded Vector mosquitoes pose a significant threat to human health valuable mosquito pathogenic bacilli isolates. as they have the ability to efficiently transmit disease causing Nabar et al. [27] had earlier reported that the extreme natural pathogens causing a variety of diseases which afflict millions environments have been consistently generating microbial of people worldwide. They are responsible for infecting over species which contribute to the control of diseases and their 700 million people every year in more than 80 countries and transmission. It has been previously reported by Manonmani approximately 20% of the world’s population is at risk of et al. that Bacillus spores are known to settle rapidly in watery acquiring infections of a vector borne-disease [21]. Malaria is areas [28]. In the present study, that of the 3 different sampling the largest single component of disease burden, epidemic zones, mosquito pathogenic isolates were obtained only from form of malaria, in particular, remains a major public health tidal and intertidal zones. However, no mosquito pathogenic concern in the low income tropical countries [22]. In the past bacteria could be detected in the soil sample collected from few years, resistance to insecticides and chemical agents has the sea-shore zone as pointed by Manonmani et al. [29]. been increasing rapidly, hence there is a persistent demand for It is noteworthy that the protocol devised by Dhindsa et al. [10] development of new insecticidal agents from natural for the screening of soil samples for the presence of mosquito environments. pathogenic bacilli prior proved successful in the present study. In this regard, soils have proven to be an excellent source of This method involves the use of LB broth buffered with microbes which can be explored for their bio control potential Sodium acetate and a heat shock step at 65°C. Using this [23]. Bacillus sp., Streptomyces sp., Penicillium sp., and technique for screening, in the present study, three out of nine Trichoderma sp. are being developed as effective microbial soil samples were found to contain microbial isolates control agents which can act as successful alternatives to pathogenic to An. stephensi, Cx. quinquefasciatus, Ae. aegypti chemical compounds [24]. and Ae. albopictus larvae. Further their identity was In a recent study by Suryadi et al. [25] four toxic isolates of B. confirmed at molecular level following standard method [30]. sphaericus were isolated from the beach area of Lombok However, in the recent years, one major challenge for Island, Indonesia. They showed mild toxicity against larvae of achieving successful mosquito control is the overcoming of three mosquito species such as Cx. quinquefasciatus, An. insecticide resistance to the commonly used microbial aconitus and Ae. aegypti with Culex sp. being the most insecticides such as B. thuringiensis israelensis and B. susceptible to all the isolates. In another study, Poopathi et al. sphaericus which is a serious threat to their success as bio reported the isolation of Bacillus cereus strain VCRC-B520, a control agents. Available evidence indicates that Bti has a novel mosquitocidal bacterium from marine soil collected lower risk for resistance development in the target vector from coastal areas at Pondicherry in Eastern India. The LC50 species due to the complex makeup of its parasporal crystal, and LC90 values for Cx. quinquefasciatus were 0.30 and 2.21 which contains Cyt1A, Cry4A, Cry4B, and Cry11A toxic mg/L, respectively [26]. Similarly, in this study soil samples proteins. Disrupting the toxin complex enables resistance to

27 International Journal of Mosquito Research http://www.dipterajournal.com evolve, especially in the absence of the cytolytic toxin, use of these organisms is their safety to non-target organisms Cyt1A. B. sphaericus (Bs), on the other hand shows a higher including humans [38]. However, their continued utilization in risk for resistance development due to its single-site of action vector control programs including integrated vector and therefore, operational failures have been reported from management, would depend upon better screening methods, several locations worldwide [31]. isolation of highly virulent indigenous strains showing broad In the present study, the isolate C1A was identified as spectrum of activity against mosquitoes, more effective Aneurinibacillus sp. having highest activity against Culex formulations and mass production at an affordable cost [9]. rd quinquefasciatus 3 instar larvae (LC50 = 0.08ppm) among the This study for the first time reports the mosquitocidal activity isolates tested. This is the first report of Aneurinibacillus sp. of Aneurinibacillus sp. in the world and this bacterial agent having mosquito pathogenic activity that could be developed can add to the existing armament of bacilli based larvicides. as a novel mosquito pathogenic bacterium with high toxicity to Cx. quinquefasciatus larvae. It may be mentioned that 5. Conclusion Shida et al. proposed the genus Aneurinibacillus as a novel Although a variety of mosquito pathogenic bacteria have been genus arising from the reclassification of the Bacillus isolated from various geographic regions of the world, there is aneurinilyticus and the related species in the genus Bacillus a pressing need to explore and deploy indigenous strains in [32]. Alenezi et al. [33] reported that the soil-borne gram- vector control programs due to the restrictions imposed on the positive bacteria Aneurinibacillus migulanus strain Nagano use of imported strains, the prohibitive cost of formulations shows considerable potential as a bio control agent against and resistance development in target mosquito species. As no plant diseases. However, no prior reports exist of its mosquito mosquito larvicidal strains of Aneurinibacillus sp. have yet larvicidal activity. been described, it is important to add this microbial organism Earlier, Mohanty et al. had reported isolation of different to the list of new agents for bio control of mosquito vectors. strains of Lysinibacillus (formerly named as B. sphaericus) Besides, the results from this study suggest that a search for from Goa and grouped them based on their similarity [13]. In new entomopathogenic bacteria should continue and intensify. the present study two isolates, C2A and C2C were identified Further work is needed in order to study the mode of action as Lysinibacillus sp. Though the Lysinibacillus genus has and nature of the toxin of the new bacterial isolates as well as been well explored, yet its species have not been validation of the results obtained in the laboratory by small commercially formulated as microbial bio control agents and scale field trials followed by phase II and Phase III field hence gain importance in that context. Following their field evaluation of the formulation based on the promising testing, a suitable formulation using these Lysinibacillus sp., Aneurinibacillus sp. and Lysinibacillus sp. isolates discovered could be developed for the control of Cx. quinquefasciatus as in this study for their possible deployment in the public health these isolates showed high toxicity (LC50 = 0.30ppm and LC50 setting. = 0.25ppm respectively). The isolate B1C identified as a strain of B. thuringiensis 6. References showed a broad spectrum of activity against the 3rd instar 1. Pravin Y, Mohanraj RS. Bio efficacy of Spathodea larvae of the 4 test vector species An. stephensi, Cx. campanulata P. Beauv. (Bignoniaceae) hexane leaf quinquefasciatus, Ae. aegypti and Ae. albopictus. It has been extract against dengue and Zika virus vector Aedes reported by Ammouneh et al. [34] that screening the aegypti (Diptera: Culicidae). International Journal of environment for new and highly potent strains of B. 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Asian Pacific Journal of Tropical Medicine 2020; 13(12): 525-534 525

Review Article Asian Pacific Journal of Tropical Medicine

journal homepage: www.apjtm.org

Impact Factor: 1.94 doi: 10.4103/1995-7645.296720 Current status and future prospects of bacilli-based vector control Joleen Savianne Almeida1, Ajeet Kumar Mohanty1, Savita Kerkar2, Sugeerappa Laxmanappa Hoti3, Ashwani Kumar4

1ICMR-National Institute of Malaria Research, DHS Building, Campal, Panaji, Goa-403001, India 2Department of Biotechnology, Goa University, Goa-403206, India 3National Institute of Traditional Medicine, Nehru Nagar, Belagavi-590010, Karnataka, India 4ICMR-Vector Control Research Centre, Indira Nagar, Puducherry-605 006, UT, India

ABSTRACT Aedes mosquitoes which primarily transmit dengue, chikungunya, yellow fever and Zika. These diseases account for more than Mosquito-borne diseases such as malaria, filariasis, dengue, 17% of all infectious diseases, causing 7 00 000 deaths annually chikungunya, Japanese encephalitis, yellow fever and Zika with 80% of the world’s population at risk of one or more vector- contribute significantly to health problems of developing as well borne diseases . In recent years, changes in public health policy [2] as developed nations. Vector control is central to control of vector and social factors as well as reports of resistance in both vector borne diseases. In the last four-five decades, biological control mosquitoes and the pathogens transmitted by them have caused a methods have been inducted in the integrated vector management resurgence in the incidence of mosquito borne diseases . [3] strategy, advocated nationally as well as globally by the World Vector control is a key strategy to control these diseases. In Health Organization. Currently, biological control of vectors is India, vector control is primarily based on the use of long-lasting globally acknowledged as the best available strategy in the wake insecticide treated nets (LLINs) in addition to indoor residual of growing concerns about vector resistance as well as adverse spraying of insecticides in rural areas and anti-larval operations in effects of insecticides on the environment and non-target fauna urban areas . Larval control may be particularly valuable in regions [4] co-inhabiting the same ecological niches as vectors. In India and where the eradication or elimination of vector borne diseases elsewhere, efforts are ongoing to screen newer isolates to bring is being targeted, as a means of reducing the mosquito larval forth new biolarvicidal products of public health importance. In populations before they emerge to the adult stage . [5] this review, by carrying out extensive literature survey, we discuss However, with regards to mosquito control strategies, chemical advances thus far and the prospects of bacilli-based control of control agents still play a major role. Insecticides applied with vectors and vector borne diseases. the aim of eliminating mosquitoes have given rise to other serious problems . Not only have mosquitoes developed resistance, but [6] KEYWORDS: Mosquito-borne diseases; Vector control; Biological these insecticides also pose threat to human, health and control; Biolarvicide the ecosystem as a whole. Chemical insecticide exposure among

To whom correspondence may be addressed. E-mail: [email protected] This is an open access journal, and articles are distributed under the terms of the 1. Introduction Creative Commons Attribution-Non Commercial-ShareAlike 4.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as appropriate credit is given and the new creations are licensed under the identical Mosquitoes are associated with transmission of pathogens to terms. For reprints contact: [email protected] humans and other vertebrates resulting in significant morbidity and ©2020 Asian Pacific Journal of Tropical Medicine Produced by Wolters Kluwer- mortality due to the difficulty of controlling mosquitoes . The most Medknow. All rights reserved. [1] How to cite this article: Almeida JS, Mohanty AK, Kerkar S, Hoti SL, Kumar A. important disease vectors belong to the subfamily Anophelinae Current status and future prospects of bacilli-based vector control. Asian Pac J Trop (Anopheles mosquitoes) which transmits malaria; Culicinae i.e. Culex Med 2020; 13(12): 525-534. Article history: Received 11 September 2019 Revision 16 December 2019 species transmit filariasis; West Nile virus, Japanese encephalitis and Accepted 11 July 2020 Available online 19 October 2020 [Downloaded free from http://www.apjtm.org on Tuesday, October 20, 2020, IP: 10.232.74.27]

Joleen Savianne Almeida et al./ Asian Pacific Journal of Tropical Medicine 2020; 13(12): 525-534 526 humans has been linked to immune dysfunction, neurological subspecies e.g. B. thuringiensis subsp. israelensis . [11] disorders, various forms of cancer, birth defects, liver damage Cyt proteins have been studied less in comparison to Cry and infertility . These adverse effects have led to the discovery proteins. Based mainly on studies of Cyt1Aa, their importance [7] of alternatives to these insecticides. Microbial control agents are is in the biology of mosquitocidal strains as they synergize with effective and proven to be a method effective against mosquito other mosquitocidal Cry proteins (Cry4Aa, Cry4Ba, and Cry11A) immatures of both Anophelines and Culicines. Commercial resulting in delay in the phenotypic expression of resistance which biolarvicide formulations of gram positive and spore forming would require multiple mutations at different loci . [12] bacteria, Bacillus (B.) thuringiensis israelensis and B. sphaericus The high degree of host specificity and the complexity of B. are now being widely used across the globe in the vector control thuringiensis mode of action results from the interaction of the programmes. These strains have been well characterized both at the mosquitocidal toxins within the complex environment of the insect’s microbiological and molecular level. Based on these two bacilli, midgut lumen. Although researchers discovered relatively early that there are several effective and well tested formulations commercially the midgut was the primary site of δ-endotoxin activity as seen in available including the wettable powder, slow release granules, (Figure 1A and 1B), the molecular mechanisms of Bt intoxication briquettes, tablets and emulsifiable concentrates. These formulations have continued to be the subject of intensive research . [13] are often deployed as an integral components of the integrated vector Although Bti is proven to be effective against many mosquito management strategy advocated by the World Health Organization species, operational application showed that it is more suitable and adopted by vector borne disease endemic countries. against Aedes species. Aedes (Ae.) aegypti and Ae. albopictus were In this review article, extensive literature search was done to collect most susceptible to B. thuringiensis H-14 in comparison to other and collate published information on bacilli-based biolarvicides and vector mosquitoes . More advantages for Aedes control may be [14] their control in different parts of the world, especially the articles due to their feeding behaviour, as most of the Bti toxins sediment published on the recent advancements in the field of bacilli-based to the base of the container during treatment where Aedes larvae vector control. frequently feed . On the other hand, Anopheles (An.) balabacensis [15] and Mansonia (Mansonioides) indiana were found comparatively less susceptible to the Bti (H-14) . [16] 2. Bacilli as bio-control agents Cx. quinquefasciatus was also found to be highly susceptible to B. thuringiensis H-14 . This mosquito species, however, is more [17] In nature, a wide variety of organisms including viruses, protozoans, susceptible to B. sphaericus, the latter being more effective in fungi and bacteria, effectively control mosquitoes . Among many polluted water with high organic contents where Cx. quiquefasciatus [8] bacteria that have been tested, strains of B. thuringiensis (Bt) and prefers to breed as B. sphaericus is known to recycle in polluted B. sphaericus (Bs) are the most promising for vector control so far. water and persists longer than B. thuringiensis H-14 . [18] B. thuringiensis var. israelensis (Bti) has an advantage of a broader host range. While B. sphaericus has a narrow spectrum, it has an 2.2. B. sphaericus advantage of increased duration of larvicidal activity against specific mosquito species like Culex (Cx.) quinquefasciatus and possess B. sphaericus is a common aerobic, rod-shaped, endospore forming recycling ability within mosquito cadavers . There are options gram positive soil bacterium with a few entomopathogenic strains. [9] available for ‘stand-alone’ and combined formulations of these two The first discovery of a strain toxic to mosquito larvae was reported Bacilli species and their strains for vector control programmes. by Kellen et al. in 1965 . The biolarvicide based on B. sphaericus is [19] unique in that it consists of two binary proteins BinA (42 kDa) and 2.1. B. thuringiensis BinB (51 kDa), both of which are required for toxicity to mosquito larval midgut. These binary proteins are cleaved by mosquito B. thuringiensis is a ubiquitous, gram positive, sporulating aerobic gut proteases, forming the active toxin by yielding peptides of bacterium which can be easily grown and cultured on routinely 39 kDa and 43 kDa respectively. These associate and bind to the used media like nutrient agar. It can be isolated from a variety of α-glucosidase receptor located on the midgut microvilli, resulting in sources . On sporulation, it produces two types of insecticidal lysis of midgut cells upon internalization . It is suspected that [10] [20,21] crystal proteins or δ-endotoxins, Cry (for crystal) and Cyt (for reported loss of toxicity i.e. resistance in target mosquito species cytolytic) proteins and further variations of each of these types. Cry to B. sphaericus may be due to the reduction or loss of interactions proteins target lepidopteran insects, while few are toxic to dipteran between BinA and BinB or BinB and its receptor . In addition, [22] or coleopteran insects. Cyt proteins show moderate toxicity to another 100 kDa mosquitocidal protein appears to be synthesized mosquitoes and black fly larvae occurring mostly in mosquitocidal in lesser toxic and some highly toxic strains. This polypeptide is [Downloaded free from http://www.apjtm.org on Tuesday, October 20, 2020, IP: 10.232.74.27]

Joleen Savianne Almeida et al./ Asian Pacific Journal of Tropical Medicine 2020; 13(12): 525-534 527 A

B

Figure 1. (A) Diagrammatic representation of mode of action of Bacillus thuringiensis israelensis toxin; (B) Cartoon showing death of mosquito larvae due to toxin action of biolarvicide.

expressed during the vegetative phase and is not homologous with to mosquito pupae . Its mosquitocidal activity is associated with [24] the 51 and 42 kDa proteins . an exotoxin identified as surfactin, a cyclic lipopeptide highly active [23] at both acidic and basic pH, temperature range of 25 -42 , and 曟 曟 2.3. B. subtilis UV stability, suitable features for the development of a biolarvicide. Preliminary toxicity studies with crude surfactin showed that it A B. subtilis strain producing mosquitocidal (larvicidal and is non-toxic to mammals . The arsenal of biocontrol agents is [25] pupicidal) toxin was isolated from mangrove forests of Andaman further augmented with this potential mosquitocidal bacterium. The and Nicobar Islands of India and found to kill larval and pupal stages overview of different bio-control agents, their strains, activity profile of three species of mosquitoes viz., An. stephensi, Cx quinquefasciatus against target species, toxin genes and strain modifications with and Ae. aegypti. It is the first gram-positive bacterium highly toxic recombinant technology is shown in Table 1. [Downloaded free from http://www.apjtm.org on Tuesday, October 20, 2020, IP: 10.232.74.27]

Joleen Savianne Almeida et al./ Asian Pacific Journal of Tropical Medicine 2020; 13(12): 525-534 528 Table 1. Overview of different bio-control agents, their strain, activity profile against target species, toxin genes and strain modifications with recombinant technology. Sr. Bacteria reported Strain Activity against mosquito species Toxin gene identified Recombinant technology Reference No. 1 Bacillus thuringiensis israelensis (Bti) H-14 Ae. aegypti, Cx. quinquefasciatus, Cry (crystal) and Cyt 130 kDa toxin from Bti introduced [26-28] and VCRC B17 An. stephensi, Ae. nigromaculis, (cytolytic) proteins into plasmid pRK248 expressed in An. quadrimaculatus, Cx. tarsalis Caulobacter crescentus CB15. kurstaki (Btk) Ae. aegypti Cry protein NK [28] morrisoni NK Cry protein NK [29] jegathesan (Btj) An. stephensi, Cry protein NK [30] Cx. pipiens, (Cry19Aa1, Ae. aegypti Cry11Ba1) medellin Ae. aegypti, An. albimanus, Cry protein NK [31] Cx. quinquefasciatus (Cry11Bb1, Cyt1Ab1) higo Cx. pipiens Cry protein NK [32] (Cry 21Aa1) fukuokaensis Ae. aegypti Cry protein NK [32] (Cry 20Aa1) kyushuensis Ae. aegypti, An. stephensi, Cx. Cry protein NK [32] pipiens (Cyt2Aa1) tochigiensis Ae. aegypti, Cx. quinquefasciatus Cry protein NK [32] 2 Brevibacillus strains 921 and 615 Ae. aegypti, An. stephensi and Cx. Toxic factors likely Enhanced larvicidal activity by bio- [33-36] laterosporus pipiens. to be proteins encapsulation in Protozoa. of about 14 kDa 3 Bacillus sphaericus Neide H5a5b and Culex sp., Anopheles sp., Aedes sp. Binary (Bin) toxins (1) Use of cyt1A promoters + mRNA [37-41] VCRC of 42 (BinA) and 51 stabilizing sequence to synthesize high B42 (BinB) kDa levels of Bs binary toxin in Bti strains. + MtX (2) Cry4Ba and cyt1Aa genes (mosquitocidal) expressed in Bs2362 produced stable toxins transformants 10 times more toxic to Aedes larvae than the host strain. 4 Clostridium malaysia An. sp., Ae. detritus, Ae. caspius, Toxic extract Toxin encoding gene cloned and [42] bifermentans Ae. aegypti, Cx. pipiens, blackfly contains three induced to be expressed by transformed larvae major proteins Bt, exhibited toxicity against of 66, 18 and 16 kDa mosquitoes. 5 Bacillus alvei NK Culiseta longiareolata NK NK [43] 6 Bacillus brevis NK Culiseta longiareolata NK NK [43] 7 Bacillus circulans NK Cx. quinquefasciatus, An. gambiae Toxicity due to spores NK [44] 8 Pseudomonas Migula strain VCRC An. stephensi, Cx. quinquefasciatus Mosquitocidal NK [45-47] fluorescens B426 and Ae. aegypti exotoxin (44 kDa) 9 Bacillus subtilis subtilis An. stephensi, Cx. quinquefasciatus Exotoxin- surfactin NK [23] and Ae. aegypti 10 Bacillus strain VCRC B483 An. stephensi, Cx. quinquefasciatus Production of NK [48] amyloliquefaciens and Ae. aegypti lipopeptide (s) 11 Peanibacills macerans NK Ae. aegypti 80 kDa parasporal NK [49] protein 12 Chromobacterium spp Chromobacterium Ae. aegypti Colonize the insect NK [50] strain midgut and displays Csp-P entomopathogenic and anti-pathogen properties 13 Bacillus cereus VCRC B540 An. stephensi, Ae. aegypti NK NK [51,52] 14 Pseudomonas strain KUN2 Ae. aegypti Extracellular toxins NK [53] aeruginosa *NK-not known, Bs-Bacillus sphaericus, Bt-Bacillus thuringiensis, Bti-Bacillus thuringiensis israelensis, An-Anopheles, Ae-Aedes, Cx-Culex, sp-species.

3. Bioassays, isolation, characterization and pathogenic Bacillus strains was cumbersome and time consuming, identification hence Dhindsa et al. in 2002 devised a new soil screening method that could reveal the presence of mosquito pathogenic bacilli in the Microbial isolates are constantly screened and isolated from soil samples. This method involves the use of LB broth (buffered terrestrial and aquatic environments for mosquito control with Sodium acetate) and a heat shock step at 65 . Using this 曟[55] programmes . The earlier method of isolating mosquito method, eight different Bacillus strains, B. pumilus (KSD-1), B. [54] [Downloaded free from http://www.apjtm.org on Tuesday, October 20, 2020, IP: 10.232.74.27]

Joleen Savianne Almeida et al./ Asian Pacific Journal of Tropical Medicine 2020; 13(12): 525-534 529 sphaericus (KSD-2), B. brevis (KSD-3), B. sphaericus (KSD-4), B. insect. If mixtures are used, there is inherent risk of resistance build subtilis (KSD-5), B. stereothermophilus (KSD-6), Bacillus sp. (KSD-7) up to multiple classes of insecticides rendering them eventually and B. sphaericus (KSD-8) were successfully isolated, identified and useless. But there is a drawback in this approach for their practical evaluated for their larvicidal activity in Goa, India . application mainly due to higher cost and practical difficulty as [55] In a screening assay carried out by Radhika et al. in 2011, ten both compounds need to be present in equally high and persistent bacilli were isolated from Tamil Nadu, India and tested for larvicidal concentrations . In such a scenario, the use of two or more [62] activity against Ae. aegypti mosquito. Two microbial isolates (B. interventions has been advocated so that mosquitoes that survive megaterium and Acinetobacter sp.) effectively caused 97% larval contact with one (e.g. LLINs) are killed due to exposure to the mortality at 48-hour incubation at bacterial concentrations of second (e.g. indoor residual spraying). In such a scenario, the use of (4.1±0.39) and (3.6±0.71) mg/L . Another study by Allwin et al. biolarvicides where feasible, can also delay the onset of resistance [56] in 2007 showed native strains of B. thuringiensis were isolated from and ease selection pressure of insecticide on target vectors. soil samples collected from different locations and characterizations in India . Samples collected from mangroves of Vellar estuary [57] 4.3. ULV and thermal application in India yielded mosquitocidal bacteria B. subtilis with increased activity against An. stephensi and Ae. aegypti . Many other reports The dengue vector Ae. aegypti is a container breeder, hence use [58] on the frequent occurrence of mosquito pathogenic bacterial isolates of Bti for its control is limited due to difficulty in its effective in the natural environment showed high possibility of isolating novel application. In this respect, ULV cold fogging can be used effectively strains . for larviciding purposes when the agent is applied correctly and [59] under required conditions . Seleena et al. 1996 found that ULV [63] fogging of B. thuringiensis H-14 was highly effective in Aedes larval 4. Resistance phenomenon and overcoming resistance control and when used together with malathion it induced complete adult mortality . [64] Since insecticide resistance can undermine efforts to control vector In addition, the effectiveness of the thermal application of an borne diseases, effective mosquito control can be successfully aqueous suspension of Bti with and without pyrethroids using achieved only by overcoming insecticide resistance. Resistance a thermal fogger has been reported without loss of its larvicidal is a complex genetic, evolutionary and ecological phenomenon. activity . [65-67] Resistance to microbial insecticides formulations is a serious threat to their success in public health settings . [60] 4.4. Application of ice granules containing endotoxins of microbial agents 4.1. Strategies for management of resistance to biolarvicides A novel method for the aerial delivery of microbial mosquito Some measures to counter resistance include: (1) rotation or control agents into vast aquatic sites in the form of ice granules alternation of bacterial biolarvicidal toxins with other toxins, was developed by Becker et al. in 2003. The solutions containing insecticides or biological control strategies; (2) less frequent powder formulations of Bti or Bs were transformed into ice pellets biocide treatments; (3) use of slow-release, ultra-low volume (ULV) (named IcyPearls) using a special ice-making machine and applied and thermal formulations which are active for longer durations; aerially. Successful field tests using IcyPearls applied at the rate of (4) use of source reduction methods and (5) constant resistance 5 and 10 kg/ha containing various dosages of 100, 200, and 400 g of surveillance and monitoring. These principles when combined are VectoBac® WDG (3 000 ITU/mg) were conducted against larvae of essentially a blueprint for integrated pest management which will Ae. vexans with mortality rates of 91%-98% . [68] successfully delay or prevent the development of resistance in vector populations . [61] 5. Commercial bio-larvicide formulations and their 4.2. Insecticide mixtures field efficacy

Studies have shown that by combining different classes of Two biolarvicide formulations-Bacticide® and VectoBac® insecticides or their application by mosaic design can effectively containing viable endospore and delta endotoxin of Bti H-14 were overcome resistance in the target insects. However, unless evaluated in 2001 in Surat city, India against An. subpictus and Cx. insecticides of different classes are combined and judiciously quinquefasciatus. Both formulations were equally effective on larvae used, there is possibility of cross resistance if insecticides induce after second application . [69] similar mechanism of resistance and have mode of action in target Field testing and evaluation of the efficacy of bio-larvicide, [Downloaded free from http://www.apjtm.org on Tuesday, October 20, 2020, IP: 10.232.74.27]

Joleen Savianne Almeida et al./ Asian Pacific Journal of Tropical Medicine 2020; 13(12): 525-534 530 ® Bactivec SC (Bti H-14) was carried out in Bengaluru, India. It was habitats of An. stephensi was carried out in order to control malaria found to be operationally feasible and easy to handle . in Goa, India. This was found to successfully replace DDT and [70] Kumar et al. 1995 and 1996 tested a formulation of Bactoculicide pyrethrum fogging . [74] ® (Bti strain 164) in construction sites, abandoned overhead tanks and In addition, the efficacy of various formulations of Bti (Bactimos , ® ® ® ® ® curing waters and a formulation of Spherix (B. sphaericus H5a5b) in Teknar , VectoBac , Bactisand , VectoPrime , VectoMax ) and Bs Goa, India respectively and found them highly effective . (HIL-9® & HIL-10®, VectoLex®) in the form of tablets, granules, [71,72] The weekly application of biolarvicide B. sphaericus (Strain 101, wettable powder, pellet, aqueous suspension, etc. were tested against Serotype H5a5b) in Panaji, Goa, India helped in malaria control mosquito vectors and found to be highly effective. and was identified as a useful biocontrol agent of An. stephensi . Table 2 provides a list of the available commercial bio-larvicide [73] 2 Similarly, application of biolarvicide Bti strain 164 at 1 g/m and formulations, their type, potency and field evaluation of these introduction of larvivorous fish Aplocheilus blocki in major breeding formulations.

Table 2. A list of the available commercial bio-larvicide formulations, their type, potency and field evaluation of these formulations. Sr. Formulation Active ingredient Type Potency Field evaluation Reference No. 2 1 HIL-9 & Bacillus sphaericus strain Dust NK V/s An. culicifacies (doses-0.05, 0.1 and 0.5 g/0.1 m ) [75] HIL-10 1593 100% mortality in third and fourth instar larvae. ® 2 Bactimos Bacillus thuringiensis PT 3 000 ITU/mg against High mortality (96%-100%) in late larval instars of Ae. [76] israelensis strain AM 65- Ae. aegypti larvae albopictus and Cx. quinquefasciatus from lab and field 52 24 hours after application. 3 Bactisand® Bacillus thuringiensis FG 112 ITU/mg against NK [77] israelensis H-14 Ae. aegypti larvae 4 Bactoculicide Bti (strain 164) Suspension 993 ITU/mg against Culex, Aedes and Anopheles larvae breeding controlled 2 [78] Ae. aegypti larvae (96%-100%, for up to 5 weeks, dose-0.5 g/m ) in industrial scrap in UP, India. 5 Spherix Bacillus sphaericus, WDG NK In lab evaluation in Assam, India 90% mortality [79] serotype H5a5b, strain observed in Cx. quinquefasciatus third instar larvae at 0.6 B101 ppm. ® ® ® 6 VectoBac Bacillus thuringiensis WDG 12 AS, 3 000 ITU/mg (1) In a study in Malaysia, VectoBac G and VectoBac [80,81] israelensis H-14 strain SCG 1 200 ITU/mg 12AS effective for 24 hrs v/s Ae. albopictus in discarded AM 65-52 200 ITU/mg tires with > 80% mortality. ® against Ae. aegypti (2) VectoBac WDG evaluated in the lab and field in larvae Bangalore, India v/s An. culicifacies and An. stephensi revealed increased efficacy against An. stephensi.

7 Teknar® Bacillus thuringiensis SC 1 200 ITU/mg against Larvicidal efficacy v/s Cx. quinquefasciatus was [82] israelensis, strain SA3A Ae. aegypti larvae determined in lab and field in Pondicherry, India. In cesspits >80% reduction of pupae up to day 6 post- treatment and in unused wells >80% reduction of pupae for 17 days post treatment was observed. ® 8 VectoLex Bacillus sphaericus 2362, FG 50 BSITU/mg against Cx. Efficacy against third instar larvae of Culex sp. and Ae. [83] Serotype H5a5b strain quinquefasciatus larvae aegypti was studied in Queensland, Australia. Both ABTS 1743 WDG 650 BSITU/mg against formulations were most effective against Culex spp, Cx. quinquefasciatus with the WDG 10-100 times more effective than the larvae FG on an ITU/mosquito basis.

9 VectoMax® Bacillus sphaericus 2362, FG 50 BSITU/mg against A trial v/s Ae. albopictus larvae in Spain took place over [84] Serotype H5a5b, Strain Cx. quinquefasciatus 2 seasons in the same water at dosages of 10, 50, and ABTS 1743 + Bacillus larvae 577 kg/ha. At all 3 concentrations the efficiency was thuringiensis israelensis close to 100% for up to 345 days post-treatment. Serotype H-14 Strain AM WSP 50 BSITU/mg against Residual effectiveness of VectoMax® WSP when [85] 65-52 Cx. quinquefasciatus applied to septic tanks against 3rd and 4th stage larvae larvae of Cx. pipiens was evaluated in a study in Turkey, at operational application rates of 1 pouch (10 g) and 2 pouches (20 g) per septic tank. Both application rates resulted in >96% control of larvae for 24 days. 10 VectoPrime® Bacillus thuringiensis FG 400 ITU/mg NK [86] israelensis strain AM 65- 52 + (S)-methoprene *FG=Fine granule, G=Granule, SC=suspension concentrate, WDG=water-dispersible granule, WSP=water soluble pouch, SC=Suspension concentrate, PT=pellet, AS=aqueous suspension, NK= not known, An-Anopheles, Ae-Aedes, Cx-Culex, sp-species. [Downloaded free from http://www.apjtm.org on Tuesday, October 20, 2020, IP: 10.232.74.27]

Joleen Savianne Almeida et al./ Asian Pacific Journal of Tropical Medicine 2020; 13(12): 525-534 531 6. Future prospects mosquito populations and has been recently combined with auto- dissemination i.e., adult females contaminated with dissemination Future prospects for the use of biolarvicide formulations stations of juvenile hormone to treat breeding habitats, especially against mosquito vectors will depend on low cost production and for the control of Aedes species, but this technique has not been development of cost-effective formulations. Cheaper formulations used in large scale at present. Recently, a new control concept has designed from the seeds of legumes, dried cow blood and mineral been devised, named “boosted SIT” that might enable the area- salts as well as the use of potato-based culture medium, bird wide eradication of mosquitoes . In addition, the exploitation [98] feather waste and de-oiled rice bran waste as culture medium when of cytoplasmic incompatibility can be an advantageous mosquito assessed for growth and production of insecticidal toxins of Bti were control method . Cytoplasmic incompatibility is induced by the [99] shown to be more economical and effective against Ae. aegypti, Cx. bacterial endosymbiont Wolbachia which is widespread and its use quinquefasciatus and An. gambiae . This is very important from is a promising tool for mosquito control either alone or associated [87-89] the point of media optimization for the economical production of with SIT . Lately, mosquitoes modified with gene drive systems [100] Bacillus based insecticides in mosquito control programs . are being proposed as new tools that will complement the existing [90] In addition, enhanced activity of protein toxins by use of ones . The synergistic utilization and application of these [101] recombinant bacteria containing a mixture of endotoxins having control measures to protect against mosquito borne diseases could different modes of action shows great promise. A few examples have a major impact on the socio-economic health of populations include newly discovered mosquitocidal proteins and peptides such particularly in developing countries. These methods are currently as Mtx proteins and trypsin modulating oostatic factor which can in the pipeline and could complement the integrated vector be genetically engineered for development and use in vector control management programmes when available. programs . [91] Research is also underway with respect to transgenic algae and cyanobacteria by expressing larvicidal endotoxins of Bti and B. 7. Conclusions sphaericus to allow the toxins to persist in the feeding zone for a longer duration as well as providing increased protection from Vector borne diseases transmitted by mosquitoes are a major public sunlight (UV light). The most promising results were obtained health concern. Effective vector control requires the deployment of a when Cry4Aa and Cry11Aa alone or with Cyt1Aa were expressed range of integrated interventions. This review focuses on the current in the filamentous, nitrogen-fixing cyanobacterium Anabaena PCC status and future prospects of bacilli-based vector control to explore 7120 . A transgenic strain of Anabaena PCC 7120 was reported to additional options and potentially augment existing strategies. It is [92] protect the expressed δ-endotoxins of Bti from damage inflicted by of immense importance to focus on the development, evaluation and UV-B. This organism has an added advantage as it has the ability to deployment of alternative vector control products and strategies. multiply in the breeding sites as well as serving as a food source to However, for effective control and elimination of the mosquito vector mosquito larvae . and vector borne diseases, these strategies will have to be locally [93] Recently there has been focus on the development of novel adapted to account for vector biology and the intensity of disease biolarvicides and their applications. The use of entomopathogenic transmission keeping in mind both human and financial resources. In bacteria and fungi mainly ascomycetes fungi such as Metarhizium addition, we are waiting for the discovery of a novel bacterium from anisopliae and Beauveria bassiana, for control of both larval and nature which could be developed into an ideal biolarvicide having adult stages of mosquito vectors such as Aedes . The use of spatial a broad spectrum of activity at very low concentrations without [94] repellents has been advocated to release volatile chemicals into the developing resistance in the target mosquito species. air, to induce modifications in insect behaviour and to reduce human- vector contact thereby reducing pathogen transmission . [95] Although mosquito traps have been used effectively as surveillance Conflict of interest statement tools in order to capture vector mosquitoes for population and disease transmission studies, they have recently been considered The authors declare that there is no conflict of interest. a control strategy by the introduction of the lethal ovitrap. These traps such as attractive baited lethal ovitrap are being developed to attract and kill the egg-bearing females. They have shown promise Authors’ contributions in both lab and field settings for significant reduction in Aedes populations . The use of attractive toxic sugar baits which work A.K. designed the study, J.S.A and A.K.M. carried out the data [96] by attracting mosquitoes and having them feed on toxic sugar meals collection, data analysis and interpretation. J.S.A and A.K.M. drafted could also be a potential vector control tool. the article. A.K, S.K and S.L.H. edited the article. All authors read The future vector control includes the use of sterile insect technique and approved the final article. (SIT) which has been successfully demonstrated against Ae. albopictus mosquitoes . SIT appears very promising to control [97] [Downloaded free from http://www.apjtm.org on Tuesday, October 20, 2020, IP: 10.232.74.27]

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