Distribution of cry and cyt Genes among Indigenous Bacillus thuringiensis Isolates with Mosquitocidal Activity

Bacillus thuringiensis strains isolated from Madurai, TamilNadu, India were evaluated for their mosquitocidal activity, as well as cry and cyt genes diversity. It revealed that 99% of the parasporal crystal morphology of these isolates was spherical in nature and a variable percentage (0% 100%) of toxicity was observed against Culex quinquefasciatus and Aedes aegypti. PCR analysis revealed that 53% of the isolates were positive for the various cry and cyt genes tested, whereas 47% did not produce any PCR product for the cry gene analyzed. Diverse pattern of cry and cyt genes distribution was observed even in the isolates from the same sample. B. thuringienis subsp. LDC-9 showed three-fold higher toxicity against Culex quinquefasciatus than that of B. thuringiensis var israelensis which might be used as a potential strain to control mosquitoes in near future after field evaluation.


Introduction
The search for native strains to control dipteran species have an impact on the control of mosquitoes worldwide, as vector borne diseases are major public health problems and their prevalence has dramatically increased worldwide [1].Chemical insecticides have been proved to be very effective in vector control programme, but their adverse environmental effects, insecticidal resistance and resurgence have prompted the search for alternative strategies for insect pest control [2].Among the various alternatives, B. thuringiensis and B. sphaericus are the most potent and successful group of organisms for effective control of pests among the microorganisms [3].The environmental safety of Bt. based products employed in pest control methods are well documented [4].B. thuringiensis is a gram positive organism that synthesizes crystalline inclusions (δ-endotoxins) during sporulation.These toxins are highly specific, completely degradable and harmless to humans, vertebrates and plants.Hence, researchers across the world are interested for screening new strains with increased levels of insecticidal toxicity with a broader spectrum of activity [5].The various screening programmes resulted in the number of B. thuringiensis strains not only active against Lepidoptera, Diptera, Coleoptera but also against Hymenoptera, Phthiraptera or Mallophaga, Acari, Nematheliminthes, Platyhelminthes and Sarcomastigophora [6][7][8].
The insecticidal activity of B. thuringiensis strains against Dipterans is attributed to the presence of Cry and Cyt proteins [9].Cry toxins are activated by host proteases, which interact with specific receptors located on the host cell surface, resulted in the formation of a pre-pore oligomeric structure that is insertion competent.In contrast, Cyt toxins directly interact with membrane lipids and insert into the membrane [10].The mosquitocidal activity of a B. thuringiensis strain is due to the additive effect of each toxin and a complex synergistic interaction among them.B. thuringiensis subsp.israelensis produces four Cry toxins such as Cry4Aa, Cry4Ba, Cry10Aa, and Cry11Aa, Cyt1Aa and Cyt2Ba respectively [11,12].The presence of the Cyt toxin synergizes and delays or prevents the development of resistance to Cry toxins by functioning as a Cry-membrane bound receptor [10].However, information on the diversity and distribution of cry and cyt genes among mosquitocidal B. thuringiensis isolates from southern part of India is negligible [13,14].Hence, the present study was envisaged to analyze the distribution of the cry and cyt genes of indigenous mosquitocidal B. thuringiensis isolates.ameter of 2.5 cm from different locations of Madurai district, Tamilnadu, India situated at an altitude of 100.58 meters above mean sea level, 9.3˚N latitude and 77˚E longitude with annual rainfall of 85 cm, relative humidity of 40% -70% with mean maximum and minimum temperature of 37.5˚C and 20.9˚C respectively.A total of 360 soil samples were collected which included 60 samples from the various ecological niches such as River bank, Subterranean, Urban, Agricultural land, animal contaminated soils and mountain regions as indicated in Table 1.Approximately, 10 g of soil samples were collected from four different sites of each location and pooled into one sample, homogenized by thorough mixing, sieved, air-dried up to 20% moisture content [15] and stored in sealed polythene bags within desiccators.A subsample of 1 g was used for further analysis.The storage time ranges from few days to three weeks with moisture level of 20%.
Leaf samples such as Murraya koengii and Ricinis communis were obtained from 2.0 to 2.5 m above the ground, 0.3 m inside the outer leaf canopy and from the east side of each tree or shrub.Cross contamination between samples was prevented by detaching leaves or needles while they were enclosed in standard plastic sandwich bags, which were immediately sealed for storage.
Samples containing leaf litters, leaf dust and animal droppings (contaminated samples) were collected and stored at 4˚C until use.A total of 60 samples of leaf litters were collected from Alagarkovil.Approximately 10 number of dead insect includes centipede, millipede, Spodoptera larva, pupa and Culex quinquefasciatus larva were assumed to be infected by B. thuringensis were collected from soil (sandy soil), plant surface (Ricinis communis leaf) and water bodies (stagnant water) using sterile forceps and was transferred in sterile polythene bags to the laboratory for analysis.

B. thuringiensis Isolation
The insect samples except Culex quinquefasciatus were surface sterilized following the methodology described by Alves [16] which eliminated external contamination.The larvae were passed first in 70% alcohol for 2 sec, followed in 5% sodium hypochlorite for 3 min and finally in sterile 10% sodium thiosulfate for 5 min.The specimens were then washed three times in sterile distilled water and transferred aseptically into a sterile mortar and macerated with a sterile pestle.The macerate was resuspended in 10 mL sterile distilled water and 5 mL was heated at 80˚C for 3 min followed by cooling on ice for 5 -10 min.The diluted samples (0.1 mL) were plated on nutrient agar and plates were incubated at 33˚C ± 2˚C for 24 h.In case of leaves, the leaf sections with an area of approximately 2 -3 cm 2 were trimmed and ground with a small mortar with 1 mL sterilized distilled water.Soil samples, animal contaminated soil samples, leaf litter samples, leaf dust, effluent samples were prepared as suspensions in 10 mL sterile distilled water.5 mL of the resulting suspensions were transferred in a fresh tube and incubated in the water bath at 80˚C for 3 min followed by cooling on ice for 5 -10 min.The aliquots (0.2 ml) were spread on plates of nutrient agar and incubated at 33˚C ± 2˚C for 24 h.

Genomic DNA Isolation
Total genomic DNA of B. thuringiensis was isolated by the method of Kalman et al., [17] with some modifications.Cultures were grown in Luria-Bertani broth (10 g tryptone, 5 g yeast extract, 10 g NaCl•L -1 ) to an optical density of 0.8 at 600 nm.The cells were washed once in 0.5 mL of TES (10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 100 mM NaCl) and resuspended in 2 mL of SET (25% sucrose, 25 mM EDTA, 25 mM Tris-HCl (pH 8.0) with 2 mg of lysozyme•mL -1 and were incubated at 37˚C for 1 h.Then SDS was added to a final concentration of 1% and incubated at 50˚C for 5 min and 4˚C for overnight.The supernatant was then extracted thrice with phenol-chloroform-isoamylalcohol (25:24:1) and precipitated with ethanol.Following centrifugation, the washed DNA pellets were resuspended in 100 µl of 1X TE (10 mM Tris-HCl (pH 8.0), 1 mM EDTA).

PCR Amplification
The family-specific primers (Table 1) for cry (cry2, cry 4-spe, cry10-spe and cry11-gen) and cyt (cyt1 gra1, cyt2 gra1) genes were used for screening cry and cyt genes through Polymerase Chain Reaction (PCR) following the method of Ibarra et al., [18].The PCR mix (25 µL) consisted of 30 ng of total genomic DNA, 1X buffer, 3 mM MgCl 2 , 0.2 mM each deoxynucleotide triphosphate, I U proofreading Taq DNA polymerase, 1.0 µM each primer set and 3% DMSO as PCR additive.Amplification was done in an Eppendorf PCR system (Master cycler Personal 5332, Eppendorf, Hamburg, Germany) with a 3 min denaturation step at 95˚C, followed by 30 amplifications cycles of 95˚C for 1 min, 52˚C for 1 min, 72˚C for 1 min and an extra extension step of 10 min at 72˚C.The amplified products were electrophoresed on agarose gel [19] and filed using Gel-Doc photodocumentor device (Geneline, Spectronics, India).B. thuringiensis subsp.kurstaki HD-1 and B. thuringiensis subsp.israelensis (gift from Bacillus Genetic Stock Center, Ohio) were used as positive controls and B. subtilis as a negative control.The distribution frequency of cry gene in B. thuringiensis strains from a certain origin is defined as the percentage of B. thuringiensis isolates containing this gene among all the isolates from that origin.

Preparation of Spore-Crystal Suspensions and Morphological Characterization
B. thuringiensis isolates were transferred to 50 ml SCG media [20] in 250 ml Erlenmeyer flasks and incubated for 3 days at 33˚C ± 2˚C with shaking at 250 r•min -1 [21].Spore-crystal mixtures were harvested after complete autolysis of the cells and were centrifuged at 12,000 g (4˚C) for 20 min.The pellet was washed three times in 0.5 M NaCl, thrice in distilled water to eliminate extra cellular components, including proteases and β-exotoxins, known to accumulate in the cell culture supernatant and finally resuspended in sterile distilled water as 50-fold concentrate [22].The morphology of parasporal body was analyzed as described [23] by Phase Contrast microscopy (Olympus DP-12, CX46, Tokyo, Japan) and Scanning electron microscopy for the spore-crystal complex as explained [24].

Bioassay
The 50-fold concentrated sporulated cultures of B. thuringiensis isolates were examined for qualitative toxicity against early fourth instar larvae of Culex quinquefasciatus and Aedes aegypti by one-dose assays, according to the method described previously [25].Twenty larvae as triplicates were maintained in distilled water for the control experiment.Bioassays were repeated thrice with the 30 isolates that have shown mortality above 50%.Lethal concentrations (LC 50 ) were determined by probit analysis [26].B. thuringiensis var.israelensis was employed as the positive control.The data were expressed as arithmetic mean ± standard error.Statistical analysis involved one-way analysis of variance (ANOVA) followed by Tukey's multiple pair wise comparison test.
The levels of significance were expressed as P-value less than 0.05.

Results and Discussion
The results of the experiments performed in this study are presented and discussed in the following sections.

Environmental Distribution of B. thuringiensis
The strains with typical B. thuringiensis colony morphology (fried egg appearance) were noted in 495 of the 540 samples collected from various ecological niches as shown in Figure 1.B. thuringiensis occurrence was highest in the agricultural soil (93%).Similar observation of the ubiquitous distribution of B. thuringiensis was previously reported [21].B. thuringiensis abundance in soil might be due to the high levels of insect activity and a large amount of nutrients in the soil, allowing optimum survival and enrichment as previously reported [28].While comparing B.t diversity from various samples, many isolates were selected from soil samples (89%) than from insect and leaf samples (67%) with respect to the biological origin as noted in Table 2.This observation deviated from studies of Wang et al. [29] wherein they have reported that the average frequency of B. thuringiensis isolates from soil samples were 29.8% only.As B. thuringiensis is an insect pathogen, the soil samples were further analyzed based on the presence and absence of plants and insects, in order to correlate how environment influenced the densities of B. thuringiensis.The analysis of samples sites based on the presence and absence of plant communities revealed that B. thuringiensis index of 0.5 was observed in agricultural fields, which included plants such as Oryza sativa (Rice), Brassica oleracea (Cabbage), Zea mays (Corn), Pennisetum glaucum (pearl millet), Pisum sativum var.sativum (Garden pea), Vigna mungo (Black gram) and Brassica napus (Rapeseed) (Table 2).On the other hand, analysis of soil samples based on the presence and absence of intense insect activity revealed that B. thuringiensis index of 0.89 was isolated from soil samples without insects (Table 3).This observation did not correlate with the relationship between insect environments and densities of B. thuringiensis as indicated in the earlier studies [30,31].
The isolated B. thuringiensis strains were screened for crystal inclusions using Phase Contrast Microscopy.A total of 417 strains were selected among 1956 spore formers based on the crystal inclusions after Phase Contrast Microscopic observation.99% of these isolates showed spherical parasporal inclusions (Figure 2).This observation was different from the earlier reports of    Bernhard et al., [32] wherein strains with bipyramidal crystals were predominant in Eastern Asia, except Southeast habitats.The differences in the distribution of morphology of parasporal body might be due to the genetic variation caused by the differences in the environmental conditions or to habitat effects [33].

Identification of cry and cyt Gene Composition of B. thuringiensis Isolates and Their Distribution in Different Sources
Identification of B.t cry and cyt genes by PCR has proven to be a very useful method for strain characterization, offering several advantages in terms of rapidity and reproducibility [34].Selected 417 strains were characterized for the presence or absence of the specific cry2, cry4, cry10, cry11, cyt1 and cyt2 genes by PCR.The most frequent cry genes were cry4 (50%) [53% cry4a + 47% cry4b] and cry2 (25%) followed by cry11 (10%) and cry10 (15%) genes.Similar occurrence of cry4 and cry2 gene diversity was reported by Ibanez et al. [35].Our study revealed that cry11 (10%) whereas Bravo et al., [36] indicated that 38% of the tropical strains were with cry11 and cyt genes.Cytolytic genes, cyt1 (9%) and cyt2 (7%) genes were the least identified.Strains with multiple cry genes were found at a lesser frequency (less than 3%) than strains with single cry genes.Isolates with a cry gene harbouring cyt1 and or cyt2 gene were less than 2% (Figure 3).Interestingly, few strains such as B. thuringiensis LDC-9, LDC-14 and LDC-21 in our B. thuringiensis collection harbored more than one cry gene as reported earlier [37].On the other hand, 47% of the strains with crystal inclusions failed to give PCR product when assayed with the primers used in this study.It did not necessarily imply that these strains were devoid of genes coding for insecticidal properties, as all of them did produce crystals [36].These strains might contain other cry, cyt or non insecticidal parasporal inclusions as suggested by Uemori et al. [37].Further, the cry gene frequency of these isolates were correlated to the source of the sample and classified into three groups.The first group contained the most common cry genes (cry2, cry4 and cry11) with high frequencies noted in the soils, compared to other sources.The second group included cry10 gene, found only in the samples derived from the insects, while absent in other samples.The third group contained the cytolytic cyt1 and cyt2 genes, present only in soil and insect samples (Figures 4 and 5).These results are in close agreement with Wang et al., [29], where the strains from different sources differed in their cry gene content.

Correlation with Insect Toxicity
All the selected B. thuringiensis isolates with dipteran specific cry genes such as cry2, cry4, cry10 and cry11 were tested for its mean toxic mortalities using three replicates against Culex quinquefasciatus and Aedes aegypti as noted (Table 4).The spore-crystal complex of B. thuringiensis isolates, which promoted 50% or more larval mortality, were considered as active strains as reported by Hossain et al., [30].Toxicity tests revealed that only 7% of the B. thuringiensis isolates were pathogenic (>50%) to dipteran larvae.Nontoxic B. thuringiensis is more common than toxic B. thuringiensis as reported by earlier studies of Ohba [38].The variation in toxicity was not related to cry gene content in all cases, as some strains sharing the same cry gene but significantly differed in their insecticidal potency.In this study, for ex-  ample, strains (B.thuringiensis LDC-14 and B. thuringiensis LDC-21) displayed the cry4 gene but the mortalities produced by the spore-crytsal complex were 96% and 24% respectively.This might be explained by a variation in the level of gene expression, which can strongly influence the insect toxicity as reported by Martinez and Caballero [39].Strain B. thuringiensis LDC-9 alone with unique combinations of cry (cry4a, cry4b, cry10, cry11) and cyt genes (cyt1 and cyt2) demonstrated three-fold higher mosquitocidal activity (6 ng•mL -1 ) than B. thuringiensis israelensis (19 ng•mL -1 ).

Protein Profiling
B. thuringiensis LDC-9 was noted to be the toxigenic strain based on the mosquitocidal activity and SDS-PAGE of spore-crystal suspensions of selected strain as shown 6).B. thuringiensis LDC-9 exhibited protein profile which is distinct from B. thuringiensis sp.
israelensis as reported earlier [18].The present study resulted in identification of a novel B. thuringiensis LDC-9 with higher mosquitocidal activity than the earlier reported B. thuringiensis strains.Hence, this strain is likely to be a viable mosquito control agent after field evaluation and toxicity analysis against other aquatic insects including dragonflies, damselflies, mayflies, stoneflies, caddisflies, water beetles or bugs and other invertebrates such as Daphnia, Cyclops, rotifers and crustaceans.

Conclusion
B. thuringiensis presents great genetic and molecular diversity even in isolates from the same soil sample.Moreover, the diversity and activity of the isolates might have a relationship with the geographical origin of the samples.The results obtained here indicate that the B. thuringiensis LDC-9 may be a potential control agent    that could be used in control programmes against mosquitoes.

Figure 1 .
Figure 1.Map showing sample collected sites; closed circle blue dots indicates sample collected sites.

Figure 3 .
Figure 3. Frequency of single mosquitocidal cry or cyt genes in indigenous Bacillus thuringiensis isolates.

Figure 4 .
Figure 4. Frequency of multiple mosquitocidal cry or cyt gene in indigenous B. thuringiensis isolates.

Figure 5 .
Figure 5. (a) Mosquitocidal gene distribution based on the nature of the soil samples; (b) Mosquitocidal gene distribution based on the origin of the samples.

Table 1 . Characteristics of primers employed for screening cry1, cry2, cry4, cry10, cry11 and cyt genes.
a Position at 5' end of direct and reverse primers for each PCR primer pair.b d and r, direct and reverse primers, respectively.

Table 2 . Distribution of B. thuringiensis in different habitats of Madurai. Habitat No. of samples examined No. of samples with atleast one B. thuringiensis Percentage of samples with atleast one B. thuringiensis B. thuringiensis Index*
River bank refers to the margin of the land along lakes and rivers where sediments are reworked or deposited.It includes regions from Tiruparankundram, Sholavandan; b Subterranean-underground samples (below 10 cm) from Ladan Cave Temple, Tiruparankundram and Jain cave temples; c Urban-Samples collected from sites wherein there is no human intervention or human interference; Mountain region-samples collected from mountains of Tiruparankundram; a d Agricultural fields-samples were collected included.rice, corn, cabbage, millet, pulses, cotton, oilseed, sugarcane, cotton, maize, green gram, sorghum and tomato; e Animal contaminated soils-soils with animal feces such as cow, cat, dog, goat; f Waste products-tannery effluent, diary effluent, molasses and sewage sludge; g Mountain-samples collected from Tiruparankundram and Alagarkovil; h Insects-centipede, millipede, Spodoptera larva & pupa and Culex quinquefasciatus larva; i leaves-Murraya koengii, Ricinis communis; *The B. thuringiensis index indicates the number of B. thuringiensis isolates recovered divided by the total number of sporulated bacilli.