Reyaz Ahmad Lone and Indra Arul selvi P*

Plant and Microbial Biotechnology Laboratory, Department of Biotechnology Periyar University, India

(Received: 02 October 2017; accepted: 20 November 2017)

Abstract:

In an exertion to isolate natural non-harmful mosquitocidal bacteria, 158 samples of soil were collected from various habitats of Himalayan valley Kashmir. A total of 450 bacteria were screened for mosquitocidal activities against three epidemiological disease causing vectors:Aedesaegypti, Culexquinquefasciatus and Anopheles stephensilarvae/pupae. Out of 450 bacteria screened, none had shown pupicidal activity. However, two isolates KS2-15 and KS2-13 exhibited mosquito larvicidal activity against C. quinquefasciatus(LC50: 1.36× 103 spores/mL; 1.41× 103 spores/mL respectively) and A. stephensi(LC50: 2.14× 103 spores/mL; 2.11× 103 spores/mL correspondingly).These two isolateswere identified, morphologically, biochemically andcomparative investigation of 16S rRNA gene sequences, as Lysinibacillussphaericus(previously Bacillus sphaericus). Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis of spore crystal mixture of each strain revealed two major bands of around 51.4 kDa and 41.9 kDa. PCR of mosquitocidal toxin genes showed the presence of binA and binB genes in both the strains. Comparative amino acid sequence analysis revealed that the BinA (41.9 kDa) and BinB (51.4kDa) proteins of KS2-13 and L. sphaericus2362 differ by 3 (K89E, E104A, Y176D) and 6 (A69S, K70N, I110T, N248H, H314L and L317F) amino acids respectively. Similarly BinA and BinB proteins of KS2-15 and L. sphaericus2362 strains vary by 1(E104A) and 3 (H109P, N248H and P274S) amino acids respectively. The varied amino acid sequences could be reason for the difference in activity. These two strains can act as good candidates for insecticidal formulation. Moreover, we reported for the first time the isolation of mosquitocidalLysinibacillus strains from Kashmir valley.

Keywords:

Bin proteins;bin genes; Kashmir valley;Lysinibacillus; SDS-PAGE

INTRODUCTION

Vector-borne diseases cause main public health tribulations and their control is largely accomplished with usage of synthetic insecticides against carrier insects (Baird 2000).Although the application of chemical insecticides proved lucrative to effectively control mosquitoes for many decades, the usage of chemical pesticides in long term will result in malicious effect on human and environment. There are many health problems associated to pesticide usage which vary from abdominal pain, dizziness, headaches, nausea, vomiting, as well as skin and eye problems to cancer and developmental defects as well (Lorenz, 2009). Side effects on environment range from non-target organism killing (harmless insects, birds, amphibians and fishes) to increasing resistance to mosquito (Denholm et al. 2002). Effects on non-target organisms and concern about accumulation ofchemicals in the environment have hastened the requirement to develop substitutes. Control of mosquitoesby means of entomopathogenic bacteria is a potential environmental friendly alternative tochemical insecticides (Park and Federici. 2009).L. sphaericusis an aerobic, endospore-forming gram-positive bacterium (Suryadi et al. 2016).This bacterium shows high toxicity against mosquito larvae and has been used extensively in some countries as biopesticide (Poopathi andAbidha, 2010). The larvicidal propertiesof this bacteriumare mainly attributed to the presence of Binaryproteins(BinA 41.9 kDa and BinB 51.4 kDa) expressed during sporulation stage (Broadwell et al. 1990; Berry, 2012).Cry48/Cry49toxin expressed during sporulation in some strains play significant in the activity against Culex mosquitoes (Jones et al. 2007) and Mtxproteins (100-kDa toxin) formed during vegetative growth (Priest et al. 1997; Wirth et al. 2014).However Bin toxins are the key factors responsible for larvicidal activity.These Bin toxins afterintake bysusceptible larvae dissolve in the alkaline midgutand get activated by gut proteases. The 41.9 kDaBinAprotein is cleaved to 39 kDa, and 51.4 kDaBinB ischanged to 43 kDa (Baumann et al. 1991).BinBbinds to thespecific receptors present on larvalmidgutbrush border membranes (Silva-Filha et al. 1999), while activated BinAbrings thetoxicity by working together with BinB (Oei et al. 1992; Lekakarn et al. 2015).

Valley Kashmir that is often referred as Terrestrial Paradise on Earth is situated at northern western tip of Himalayan biodiversityhotspot (Mittermeier et al., 2005). It is located roughly between 32˚.15′ and 37˚.05′ North latitude and 72˚.35′ to 83˚.20′ East longitude,with complicated geomorphologic characteristics (i.e. snow clad mountains, vast meadows full of flowers, thick forests, small mountains, valley lakes and numerous serpentine rivers). Thesedistinctive features associated with variety of animal forms ranging from higher groups such as vertebrates, including mammals, birds, reptiles, amphibians to lower groups like invertebrates including insects and even unicellular micro-organisms provide the opportunity to isolate novel mosquitocidal bacterial strains. In this study, we reported isolation and characterization oftwo highly mosquitocidalB.sphaericus strains from Himalayan valley Kashmir, active against larvae of C. quinquefasciatusand A. stephensiepidemiological disease causing vectors.

Materials and methods
Reference strain
  1. sphaericus2362was obtained from Bacillus Genetic Stock Center (Columbus, Ohio) which served as positive control.
Mosquito cultures

The cultures of Culexquinquefasciatus, A. stephensiandA. aegypti were procured from the Centre for Research in Medical Entomology (CRME),Madurai, TamilNadu andmaintainedat 28±2 ºC and 75% to 85% relative humidityunder a photoperiod of14L:10D in our laboratory. Larvae were reared in chlorine free water and fed with dog yeast and biscuits at 2:3 ratio.For all bioassays, third-instar larvae of the size and same age were used.

Sample collection

A total of 158 samples from different divisions (Anantnag, Kulgam, Pulwama, Shopian, Budgam, Srinagar, Ganderbal, Bandipora, Baramulla, Kupwara) of the Himalayan valley Kashmir, India, were used for isolation of mosquitocidal bacteria. To our best knowledge, microbial insecticides had not been formerly applied in the sampled areas. All the soil samples (each ~ 5 g) were collected from 2 to 4 cm below the surface after scrapping off the surface material with a sterile spatula.

Isolation of Bacteria

Isolation of bacterialstrains from the soil samples was performed according to the method described by Geetha et al (2007) with slight modification. In the laboratory, One gram of soil sample was suspended in 10 mL of sterile distilled water (10-1) in a boiling tube. One ml from this suspension was added to nine ml of waterwhich gives 10-2 dilution. Similarly, dilutions were made up to 10-5 and 0.1 ml from each dilution was spread on nutrient yeast salt mineral agar (NYSM) comprising  of 5 g peptone, 5 g yeast extract, 3 g beef extract, 5 g NaCl, 5 g glucose,103 mg CaCl2,10 mg MnCl2 and 203 mg MgCl2, (Hi-Media, India) perliter of distilled water.The bacterial suspensions were subjected to pasteurization before plating, expecting only gram-positive bacteria. The bacterial colonies which appeared on NYSM plates after incubation at 30ºC for 48 h and were further purified using NYSM agar. Purified colonies were then sub-cultured on NYSM agar slants and stored at 4ºC.These bacterial isolateswere tested for mosquito larvicidal activity.

Determination oflarvicidal activity

A loopful of bacterial culture fromeach NYSMmedium slant was inoculatedto 3 ml of NYSM broth. This was incubated ina shaking incubator (Hasthas Scientific Instruments, India) maintained at 30°C and 200 rpm till ≥90% sporulation ( ̴72 h). After incubation, 1 ml (about 109 spores) from the whole culture was utilized to screen for mosquito larvicidal activity. Bioassays with bacterial suspension (1 mL) were carried out in wax coated paper cups each containing25 third instar larvae and 25 pupae of C.quinquefasciatus/ A.stephensi/A. aegyptiin 125 ml chlorine-free tap water at a temperature of 28±2 ºC and 75% to 85% relative humidityunder a photoperiod of14L:10D. Incontrolcups 1 ml of un-inoculatedNYSM broth only was used and in another control 1ml of L. sphaericus2362was added.Mortalityin the individual cups was noted down by counting the number of live larvae or pupae present subsequent to 24 h of culture introduction.Based on the mosquitocidal activity two strains were selected, code named as KS2-13 and KS2-15 and further characterized.Further bioassays with larvicidal active isolates (KS2-13 and KS2-15) and L. sphaericus2362 were performed using sevendifferent concentrations(102, 103, 104, 105, 106, 107and 108) of bacterial spore crystal suspension obtained by serial dilution.All the bioassayswere performed in ten replicatesin wax coated paper cups each containing 25 third instar larvae of C.quinquefasciatus/ A. stephensiin 125 ml chlorine-free tap water at 28±2 ºC and 75% to 85% relative humidityunder a photoperiod of14L:10D. A control group, tested on NYSM broth without bacterial suspension, was included in each experiment. No source of food was added during bioassays. The number of dead larvae in each cup was counted after 24 h of culture introduction. Probit regression analysis was performed with SPSS 20.0 for windows software and LC50 and LC90 as well as their 95% fiducial limits were determined(Geetha etal. 2007; Tranchida et al. 2011).

Characterization of the potential bacterial strains

Two potential strains were studiedfor morphological, biochemical and physiological characteristicsaccording to Bergey’s Manual of Systematic Bacteriology(Sneath, 1986). Biochemical tests namely, fermentation ofarabinose, glucose,mannitol, xyloseand malonate, hydrolysis of starch, decarboxylation oflysine, ornithine, arginine dihydrolase, utilization of citrate, degradation of tyrosine, deamination of phenylalanine, nitrate reduction, decomposition ofurea,indole and acetylmethyl carbinolproduction were performed.The capability to grow on different concentrations (2%,5%, 7% and 10%)of NaCl was also studied.

Molecular characterization of the mosquitocidal bacterial strains

Genomic DNA was extracted from KS2-13 and KS2-15 using HiPurATM Bacterial and Yeast Genomic DNA Purification Spin Kit (Hi-Media, India). To determine the sequence of the 16S rRNA gene, a DNA fragment of ~ 1.5-kbwas amplified by PCR from the genomic DNA of the samples using universal eubacteria-specific primers:27F (5ꞌ-AGAGTTTGATCMTGGCTCAG-3ꞌ) and 1492R (5ꞌ-GGYTACCTTGTTACGACTT-3ꞌ), synthesized at Xcelris Labs Ltd, Gujarat, India.For polymerase chain reactions (PCR), 0.1 µg of total DNA from each isolate was mixed with 10µl of 2X PCR Master Mix  (GeNeiTMBengaluru, India) consisting  of dNTPs, Taq polymerase and  PCR buffer. Forward and reverse primers were used at a concentration of 1µM. The final volume was made upto 20µl with sterile double distilled water. PCR amplification was performed in a thermal cycler (cyber cycler-P series PCR peltier model p96+ USA)using the program: a 5 min denaturation step at 94 ºC, 30 amplification cycles of 1 min at 94ºC, 1 min at 56 ºC, and 1 min at 72 ºC, with a final extension step of 10 min at 72 ºC.The amplified PCR products were purified using GeneJETTMPCR purification kit (Fermentas life science Mumbai India) and sequenced by automated sequencer (ABI 3730xl Genetic) at Xcelris Labs Ltd, Gujarat, India. The forward and reverse sequences were edited using Bioeditprogram (Hall, 1999).The sequences obtained from strains KS2-13 and KS2-15 were compared to 16S ribosomal DNA(rDNA) gene sequences available in the databases of the National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov/) by BLASTN homology search, as described by Altschul et al. (1997).Phylogenetic analysis included the 16S rDNA gene sequences of the local isolates KS2-13 and KS2-15 and the reference strains of L. sphaericus, and L. fusiformis obtained from GenBank.Alycyclobacilluscycloheptanicus was used as an outgroup (Nakamura. 2000). Neighbor-Joining method (Saitou and Nei.1987) was used to infer the evolutionary history. Cluster support was assessed through 1,000 bootstrap replicates (Felsenstein. 1985). The branch lengths of the drawn tree were in the similar units as those of the evolutionary distances used to deduce the phylogenetic tree. A Kimura 2-parameter method was used to compute the evolutionary distances and is in the units of the number of base substitutions per site (Kimura. 1980). The analysis entailed 17 nucleotide sequences. All positions having missing data and gaps were removed. There were a total of 1366 nucleotides positions in the final dataset. Phylogenetic and Evolutionary analyses were conducted in MEGA5 (Tamura et al. 2011). Sequences were submitted in GenBank and accession numbers were obtained.

SDS PAGE analysis of spore crystal mixture

Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of spore crystal mixture was carried out by the method of Laemmli (1970) using 10% running and 4% stacking gels. The gels were stained with 0.4% Coomassie blue R250. The molecular mass of proteins was determined by using higher range protein molecular weight marker (myosin rabbit muscle 205kDa, phosphorylase b 97.4kDa, bovine serum albumin 66kDa, ovalbumin 43kDa and carbonic anhydrase 29kDa) obtained from GeNeiTM Bangalore, India.

the presence of L. sphaericus toxin genesScreening for 

Total cellular DNA isolated from indigenous L. sphaericusstrain (KS2-13 and KS2-15) was used for identification of the dipterans-specific binary genes. Primers (Table1) designed by Hire et al. (2010) for the detection of binary toxins was used. The PCR amplification was performed in a thermal cycler (cyber cycler-P series PCR peltier model p96+ USA) in a 20µL reaction volume containing 100 ng DNA, 0.5 mM of primers, 2X PCR Master Mix consisting ofdNTPs, Taq polymerase and PCR buffer (GeNeiTMBangalore, India).The amplified PCR products were purified using GeneJETTMPCR purification kit (Fermentas life science, Mumbai India).

Cloning and sequencing of binary genes

InsTAcloneTM PCR Cloning Kit (Fermentas Life Science, Mumbai India) was used for cloning of purified PCR products. The PCRamplified binA and binB sequences were ligated in pTZ57R/T vector as per instructions in user manual. The recombinant vectors pTZ57R/T-binA and pTZ57R/T-binBwere transformed intoEscherichia coli DH5α. Positive clones were identified by blue white screening. The nucleotide sequences of twopositive clones each from the pTZ57R/T-binA and pTZ57R/T-binBconstructs were confirmed by complete sequencingof binA and binB using an automated DNA sequencer(ABI 3730xl Genetic) at Xcelris Labs Ltd, Gujarat, India.The forward and reverse sequences were edited using Bioedit program (Hall. 1999) andblast performed using BLASTN. Sequences were submitted in GenBank and accession numbers were obtained.

Comparative analysis of deduced amino acid

The amino acid sequences were deduced from the complete DNA nucleotide coding sequences of binary genes (KS2-13 and KS2-15) using online ExPASy translation tool. These were compared with the sequences of BinA and BinB proteinsof reference strain L. sphaericus 2362 by Clustal Omega(www.ebi.ac.uk/Tools/msa/clustalo/) an online multiple sequence alignment program for amino acid variations.

Result

A total of450 bacteriafrom 158 sampleswere selected randomly and screened for mosquitocidal activity. Preliminaryscreening with 1 ml of culture demonstrated that two bacterial strains isolated from soil samples have mosquitolarvicidal propertieswith 100% mortality towardsC. quinquefasciatusand A. stephensiafter 24 hours of exposure.However, none of the isolate showed any activity against pupae of mosquito cultures. Subsequentbioassaysof these two strains against larvae ofC. quinquefasciatusand A. stephensiwith different culture concentrations obtained by serial dilutions showed promising activities at very low concentrations (Table2) which were comparable to control L. sphaericus2362strain. These strains, codenamed as KS2-13 and KS2-15were selectedand studied further.

 

Table 1. Primers used in this study

Gene Primer sequence ( 5ꞌ to 3ꞌ) Size Reference
binA gene F= AGC TAA AAC ATATGA GAA ATT TGG ATT TTA TTG 1.1 kb Hire et al., 2010
R= TTG TGG ATCCTTAGT TTT GAT CAT CTG TAATAATC
binB gene F= GAT GAA GAA CATATG TGC GAT TCA AAA GAC 1.3 kb
R= AGT TGG ATC CTT ACT GGT TAA TTT TAG GTA TTA A

 

 

Table 2.Larvicidal activity of Lysinibacillussphaericus strain KS2-13 and KS2-15 against third instar larvae of different mosquito species

 

 

Strain           Mosquito species                           LC50 (95% CL)            LC90 (95% CL)              X2

 

 

2362       Culexquinquefasciatus                      1.34 (1.09-1.56)            2.48 (2.15- 3.08)         0.343

KS2-13Culexquinquefasciatus                        1.41 (1.14-1.65)            2.67 (2.29- 3.41)         0.190

KS2-15Culexquinquefasciatus                        1.36(1.13-1.58)             2.47 (2.15- 3.05)         0.313

2362       Anopheles stephensi                           2.10 (1.78-2.69)           3.77 (3.04-5.70)           0.543

KS2-13    Anopheles stephensi                         2.11 (1.74-2.91)           4.11 (3.18-7.06)           0.649

KS2-15  Anopheles stephensi                           2.14 (1.81-2.78)           3.86 (3.09-5.94)           0.729

 

 

Lethal concentrations (expressed in 103 spores mL-1) for 50% (LC50) or 95% (LC95) of larvae treated after 24 h


Table 3:- Morphological and biochemical characteristics of mosquitocidal bacterial strains

Property
KS2-13
KS2-15
Colony morphology
Cream colored, moist irregular
Cream colored , irregular
Spores
Cylindrical
cylindrical
Utilization of
Glucose
̶
̶
Starch
̶
̶
Lactose
̶
̶
arabinose
̶
̶
Xylose
̶
̶
Mannital
̶
̶
2% NaCl
+
+
4% NaCl
+
+
5% NaCl
W
W
7% NaCl
̶
̶
Erythromycin 1b
+
+
Erythromycin 2b
̶
̶
Tetracycline 2b
+
+
Tetracycline 5b
̶
̶
Chloramphenicol 8b
+
+
Citrate
+
+
Phenyl alanine
+
+
Arginine
̶
̶
Lysine
̶
̶
Ornithine
̶
̶
Tyrosine
̶
̶
H2S production
̶
̶
Decomposition of urea
W
W
Catalase
+
+
Oxidase
+
+
Indole
̶
̶
Methyl red
̶
̶
Voges-Proskauer
+
+
Identified as
Lysininbacillussphaericus
Lysinibacillussphaericus

+  indicates positive; – indicates negative  and W indicates weak

Characterisation of the potential mosquitocidal bacterial strains

Both the mosquitocidal bacterial isolates were found to be gram-positive, aerobic, motile, rod-shaped bacteria with terminal spherical sporangium. Table 3 summarizes results of the morphological andbiochemical tests performed.On the basisof morphological and physiological characters, strains KS2-13 and KS2-15 were identified as L. sphaericus.PCR amplification of the 16 S rRNAgenes of KS2-13 and KS2-15 yielded ampliconof ~1.5 kb size. BLASTN (NCBI) analyses of KS2-13 sequence indicated a 99% similarity with other L. sphaericusstrains (e.g., strainC5, accession no. KF523303.1; strain Ot4b.39,accession no. JQ744626.1 and strain VCRC B543, accession no. JN377786.1). Similarly, BLASTN result of KS2-15 also showed a 99% similarity with other L. sphaericusstrains (e.g. strain Y73,accession no. JX067902.1;strain C5, accession no. KF523303.1strain Ot4b.39, accession no. JQ744626.1).The 16S rRNA genes of KS2-13 and KS2-15 were submitted in GenBank under accession numbers KJ183073.1 and KJ183074.1 respectively. Next, we determined the degree of relatedness of our isolates to different L. sphaericus and L. fusiformis species through a phylogenetic analysis. The NJ tree shows a close relationship between the strains isolated in the present study and Group I of Nakamura (strains B-23269 and B-23287) (Fig. 1).

Fig. 1. Phylogenetic relationships are based on 16S rRNA gene sequence analysis of members of local isolates (KS2-13 and KS2-15) and the Lysinibacillusspecies. Evolutionary distances were calculated using the Kimura two parameters method, and the topology was inferred using the neighbor joining (NJ) method. Numbers above branches represent percentage bootstrap values based on 1,000 replicates. The 16S rRNA gene sequences of Bacillus cycloheptanicus was arbitrarily chosen as the out group.  Accession numbers are between parentheses

SDS -PAGE analysis

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of spore mixture revealed two major polypeptide chains of approximately 51.4kDa and 41.9kDa which correspond to known consistent sizes of BinA and BinB proteins (Fig. 2).

Fig. 2:SDS-PAGE analysis of spore crystal mixture ofL. sphaericusstrains: Lane M:High molecular weight marker; Lane 1-2:L.sphaericusstrains, KS2-13 and KS2-15 respectively.

Fig. 3: PCR amplification of binA and binB genes from L. sphaericusstrainsKS2-13 and KS2-15. Lane M, molecular weight marker; Lane C1 and C2: negative controls; Lane 1-2:binA genes (KS2-13 and KS2-15 strain); and Lane 3-4, binB genes (KS2-13 and KS2-15 strain).

Screening for the presence of L. sphaericus toxin genes

Primers for binA and binB gave rise to fragments of 1.1kb and 1.3kb amplicons, corresponding to the binAtoxin (41.9 kDa) gene and binB toxin(51.4 kDa) gene (Fig. 3) respectively. The sequences of binA and binB genes have showed 99% and 100% identity with thesame genes found in L.sphaericus.The sequences were submitted to the GenBankunder accession no. KJ547612, KJ547613, KJ547614 andKJ547615.

Comparison of deduced amino acid sequences

The deduced amino acid sequences of binA and binBgenes ofKS2-13 and KS2-15 strainswhen compared with deduced amino acid sequences of binA and binBgenesof L. sphaericus 2362 revealed that the BinA (41.9 kDa) and BinB (51.4kDa) proteins of KS2-13 and L. sphaericus 2362 differ by 3 (K89E, E104A, Y176D) and 6 (A69S, K70N, I110T, N248H, H314L and L317F) amino acids respectively. SimilarlyBinA and BinB proteins of KS2-15 and L. sphaericus2362 strains vary by 1 (E104A) and 3 (H109P, N248H and P274S) amino acids respectively.  The varied amino acid sequences may be the reason for difference in activity.

Discussion

In the course of diverse microbial pesticides, Bacillus thuringiensisandL. sphaericus are the most widely used alternative control agents for mosquitoes (Geetha et al. 2007; Hayes et al. 2011; Prabhu et al. 2013). B. sphaericus strains have been isolated worldwide from diverse habitats, including the soil (Radhika et al 2011; Suryadi et al. 2016), aquatic habitats (Foda et al 2013), excreta of arid birds (Poopathi  et al 2014) and dead larvae (Tranchida et al. 2011). In this study,for the first time,we have reported the isolation and characterization of themosquitocidal strains from soilsamples of Kashmir valley that is situated at northern western tip of Himalayan biodiversity hotspot (Mittermeier et al. 2005). A total of 450 bacterial isolates from 158 samples were screened for mosquitocidal activity. However, only two isolates code named as KS2-13 and KS2-15, being toxic were further studied. Level of toxicity was higher in case of C. quinquefasciatusthan Anopheles stephensi.The isolate KS2-13 and KS2-15did not produce mortality in A. aegypti larvae. Earlier it had been reported thatfluorescent labeledL. sphaericus toxin binds proficiently with cultured C. quinquefasciatuscells and larval midgut cells in contrast to A. aegypti cultured cells and larval midgut cells where it binds slightly or is undetectable (Davidson et al.1987; Davidson 1988). However, Tranchida et al. (2011) have reported isolation of Lysinibacillus strains from Culexpipiens larvae active against larvae of Culex, Aedes, Culex, Ochlerotatus, and Anopheles species.Identification of B. sphaericus like organisms is hard and arduous as they cannot be differentiated from each other by conventional phenotypic tests. The sequence of 16S rRNA genes has been widely used to identify an unknown bacterium to the genus or species level (Geetha et al. 2008). In present study,two potentialmosquito larvicidal toxic strains were identifiedas B. sphaericus by their morphological,biochemical features and 16S rRNA gene sequence. Blast analysis of 16S rRNAgene sequences of KS2-13 and KS2-15 showed 99% identity with other already reported B. sphaericus. A typical feature used in the depiction of the L. sphaericus species is the lack of ability to utilizepentoses and hexoses as the solitary carbon source. This is due to the absence of thepgi genes encoding enzymes for breaking and transporting these sugars (Hu et al. 2008).Our two isolates KS2-15 and KS2-13 exhibited promising mosquito larvicidal activity against C. quinquefasciatus(LC50: 1.36 × 103 spores/mL; 1.41 × 103 spores/mL respectively) and A. stephensi(LC50: 2.14 × 103 spores/mL; 2.11 × 103 spores/mL correspondingly) which was comparable to control L. sphaericus2362 (C. quinquefasciatusLC50: 1.34 × 103 spores/mL; A. stephensi2.10 × 103spores/mL) under our laboratory conditions.Tranchida et al. (2011) reported two B. sphaericus strains C107 and C207 that had showed highest mosquitocidal activity against Culexpipiens and Ochlerotatusalbifasciatus with LC50: 4×104 spores/mL and LC50 of 3.4×106 spores/mL respectively. Recently, Suryadi et al. (2016) reported four mosquitocidalL. sphaericus strains MNT, SKT, TJL2, and SLG. L. sphaericusstrain MNT showed LC50 of 3.70 × 105 cell/mL, 1.76 × 107 cell/mLand 4.45 × 107 cell/mLagainstCulex, Anopheles and Aedes respectively. Similarly strains SKT, TJL2 and SLG showed LC50 of 1.13 × 105 cell/mL, 2.85 × 105 cell/mL, 1.78 × 107 cell/mL; 1.03 × 105 cell/mL, 8.94 × 104 cell/mL, 1.72 × 107 cell/mL and 9.41 × 105 cell/mL, 2.39 × 105 cell/mL, 2.08 × 107 cell/mL against Culex, Anopheles and Aedes respectively.The toxicity of L. sphaericus to mosquitos larvae mainly results from binary toxins (41.9 kDaand 51.4 kDa) encoded by the binA and binB genes expressed during sporulation stage(Broadwell et al. 1990; Tangsongcharoen et al. 2015). Another toxin called Mtx (100 kDa,) is present in L. sphaericus strains of both low and high toxicity. According to Thanabalu et al (1991), the existence of the mtx genes does not per se confer toxicity to this bacterial strain against mosquito larvae. Therefore, the low toxicity in some strains could result from either low expression or shortlived stability of the binary toxins during sporulation. Recently Prabhu et al (2013) reported the molecular characterization of forty two L. sphaericusstrains isolated from Tamil Nadu, India. Their results revealed genetic heterogeneity between both toxic and non-toxic isolates and pointed out there is a good correlation between the existence of toxin genes and toxicity of the strains. In current study SDS-PAGE analysis of spore crystal revealed two major protein bands of 41.9 kDaand 51.4 kDa in size which correspond to the BinA and BinB proteins. The PCR forbinA and binBgenes and their sequence analysis confirmed the presence of two bin genes in each strain.Deduced amino acid sequences of binA and binB genes from KS2-13 and KS2-15 strains when compared with amino acid sequences of binA and binB genes fromL. sphaericus 2362revealed that the BinA and BinB proteins of KS2-13 and L. sphaericus 2362 are different by 3 (K89E, E104A, Y176D) and 6 (A69S, K70N, I110T, N248H, H314L and L317F) amino acids respectively. Similarly BinA and BinB proteins of KS2-15 and L. sphaericus2362 strains contrast by 1 (E104A) and 3 (H109P, N248H and P274S) amino acids respectively.  The varied amino acid sequences may be reason for the difference in activity.

The high larval toxicity existing in some L. sphaericus strains, such as 2362 (Weiser. 1984) and IAB59 (de Barjac et al. 1988), has resulted into their commercial use as biopesticides against populations of mosquitos. Recent reports of resistance development devoted the attention of researchers worldwide towards the discovery of new isolates from natural resources as an alternative to the existing biopesticides globally. As variability was found between the new isolated L.sphaericus strains as well as withL. sphaericus strains 2362, we concluded that thestrains (KS2-13 and KS2-15) isolated and identified in this studyembody good candidates for exploiting in the control of mosquito within the situation to which both the host and the parasite are evenly well adapted.

Acknowledgement:We would like to thank Dr.Daniel R. Zeigler (Ph.D),director ofBacillusGenetic Stock Center (Columbus, Ohio) for providing L. sphaericus 2362.We also extend our thanks to Director of ‘Centre for Research in Medical Entomology (CRME)’ Madurai, Tamil Nadu for providing mosquito cultures: Culexquinquefasciatus, A. stephensiandA. aegypti.

 

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