To determine and evaluate the virulence of the bacterial isolates against Cx. quinquefasciatus, the bioassay tests were carried out at 30 ± 1°C; against 100 late third instar larvae kept in 1000 ml sterile distilled water in glass beakers. Mortality was recorded at 24h intervals.
5 ml suspension (containing ≈109 bacteria/ml) of each of the bacterial isolates was mixed with each 1000 ml of water in the beakers containing 100 larvae within. Each of the tests was replicated three times along with a control and the mortality (%) was determined using Abbott's formula.
After preliminary screening, the LC50 and LC90 values of the most potent biocide bacteria i.e; Bacillus thuringiensis DSB27 against Culex quinquefasciatus was determined using PROBIT analysis (Finney 1971) and compared with the standard Bti strain H14. This bioassay tests were carried out using 25 late 3rd instar larvae (average 0.5 cm length) in 100 ml distilled water contained in a 250 ml glass beaker taking five replications for each experimental set. Different concentrations (102, 103,104, 105, 106, 107, 108, 109 bacteria/ml) of DSB27 were tested along with a control set in each replicate (containing the larvae alone). Bioassay tests were performed at 37±1˚C and mortality (%) of the larvae was recorded for each dose up to 72 h.
The inoculum of DSB27 and the standard Bti strain each of them containing ≈109 bacteria/ml bacteria/ml, was applied (100 ml/m3 water of the breeding sites) to four drains (two drains for each of the strains) having Cx. quinquefasciatus (average density 59.2 larvae/dip). To maintain the control batch, two drains with an average larval density of 50.4 per dip were left untreated. A 250 ml capacity dipper was used to determine the larval population taking 25 dips per each breeding habitat. After treatment, the mosquito larval density were estimated in both treated and untreated sites using the dipper method at 24 h intervals up to 7 d during July 2015 to determine larval mortality. Average reduction of larval population (%) of Cx. quinquefasciatus was calculated (%) in the breeding habitats (%) with the bacterial isolate DSB27.
Isolation and characterization of the Bacteria from Dead larval mid gut:
After the field application, the moribund larvae were collected and the mid guts were dissected out to scan the gut wall by scanning electron microscopy and to isolate the bacteria attached with it. The gut walls were teased carefully under microscope and processed for Scanning electron microscopy. The guts of both the untreated and treated moribund larvae were crushed separately on sterile watch glasses and mixed with 1 ml sterile distilled water. The gut extracts (100 µl) were mixed with 100 ml nutrient agar (NA) medium separately, plated on five Petri plates and incubated in a BOD incubator at 30±0.1ºC for 24 h. The colonies were checked and one of them found to be different from the colonies of untreated larval gut triturate. This colony was isolated and maintained on NA slants at 4 ±0.1 ºC in a refrigerator. The morphological, biochemical and molecular characterization of the isolated colony were done following previously explained standard methodologies.
Similarity checking of the isolated gut bacteria with DSB27:
To check the similarity of the bacterial isolate Bacillus thuringiensis DSB27 and isolated gut bacteria CGB1, the 16S rRNA gene sequences of CGB1 and DSB27 were pair wise aligned using ClustalW software.
The bacterial strain DSB27 produced circular, off-white, convex, entire and gummy colonies having 4±0.02 mm diameter. Bacteria were rod shaped, motile, gram positive, facultative anaerobic spore formers (Fig 1). The bacteria produce polymorphic crystals. Dimensions of vegetative cells were (1.9-2.4) x (0.93-1.05) µm. The ellipsoidal spores were formed in a central or paracentral position without exhibiting swollen sporangium. Average diameter of the spores was (1.35-1.47) x (0.75- 0.80) µm. The crystals produced by DSB27 were of different shapes such as bipyramidal, polymorphic and spherical (Fig 1). The organism was positive for catalase, oxidase, nitrate reductase and methyl red tests, but negative for indole production Vogues-Proskauer, urease, and citrate tests. DSB27 could hydrolyse starch and gelatin producing amylase and gelatinase respectively, but unable to hydrolyse lipase, tween20, tween80, lecithin and casein. DSB27 could ferment glucose, fructose, arabinose, xylose, lactose and trehalose but could not ferment sucrose, mannose and manitol (Table 1). Total DNA, protein and carbohydrate content of DSB27 were 44 µg/ml, 47.2µg/ml and 24.25 µg/ml NB respectively. SDS-PAGE electrophoresis revealed that DSB27 contained 21 bands of protein having molecular weight of 13.387 to 92.324 KDa (Fig 2). Phylogenetic analysis revealed that DSB27 branched with the cluster containing other strains of Bacillus thuringiensis (Fig 3). On the basis of morphological, physiological and biochemical characters the bacterial strain DSB27 was characterized as Bacillus thuringiensis (KX130958). Bioassay tests showed that the bacteria DSB27 (KX130958) was effective against late 3rd instar larvae of Culex quinquefasciatus. The LC50 and LC90 values of DSB27 against late 3rd instar larvae of Culex quinquefasciatus was 3.34 X 105 and 2.93 X 107 respectively (Table 2). The field assay results revealed that after 7 days of treatment, the reduction in larval density of Culex quinquefasciatus has been upto 96% whereas the standard strain was able to reduce the larval density of Culex quinquefasciatus upto 75% (Table 2). The scanning electron microscopy of the dead larval gut showed that rod shaped bacterial vegetative cells, spores and crystals were attached with gut wall causing disruption of the gut epithelium (Fig 4). The isolated gut bacteria CGB1 was found to be similar to Bacillus thuringiensis DSB27 morphologically and biochemically. The clustalW alignment of the 16S rRNA gene sequences of Bt DSB27 (KX130958) and CGB1 showed that the 16S rRNA gene sequence of CGB1 completely identical with that of Bt DSB27 (KX130958) (Fig 5). This finding proved that the bacteria CGB1 isolated from the treated larvae was the same bacteria DSB27 (KX130958) that was used for the control of Culex quinquefasciatus larvae.
The colony morphology of the native Bt isolates was highly in agreement with that of other Bt isolates described by many authors (Rampersad and Ammons 2005; Chatterjee et al. 2007; Thaphan et al. 2008; Gobatto et al. 2010; Maheswaran et al. 2010; Ranganathan et al. 2011). The biochemical properties exhibited by the native Bt isolate found to be somewhat similar with the Bacillus sp. Isolated from different regions (Chatterjee et al. 2007; Azmi et al. 2014; Mondal et al. 2014 a,b). High salt tolerance activity of the Bt isolates could be due to the possibility of their adaptation to saline dynamics and environment. This characteristic might be helpful for this isolate in effective vector management in the saline environment where other salt-stress sensitive bacteria would not be as much effective as it. The isolate found to be sensitive against most of the antibiotics of selective doses (Table 1); this might be due to the poor exposure to anthropological interventions and thus no resistance development to broad spectrum antibiotics. Occurrence of B. thuringiensis strains having mosquitocidal activity have been reported from almost every corner of the world except, the Americas and Australia (Soares-da-Silva et al. 2015; Bravo et al. 2011; Lo’pez-Meza et al. 1996; Seleena et al. 1995) and so far around 36 subspecies/serotypes of mosquito-toxic B. thuringiensis strains were reported from different parts of the world (Balaraman. 2005). An archetypal feature of all the mosquitocidal B. thuringiensis strains is that they all possess a large transferable plasmid which is accountable for the toxicity and it carries cry and cyt genes that code for the cry and cyt toxins (Gonzalez and Carlton, 1984).
The cosmic variation observed in expressing the toxicity of different strains may likely be due to the presence or absence differential activities of the cry and/or cyt gene(s) present in them (Balaraman 2005). Insect mid gut is the main target for B. thuringiensis crystalline toxin (Knowles 1994). Four genes, Cry IVA, Cry IVB, Cry IVC and Cry IV, present in B. thuringeinsis var. morrisoni and israelensis are responsible for encoding mosquitocidal toxins (Bechtel and Bulla 1976). The crystalline protein first produced as a protoxin and later gets solubilised in the mid-gut at alkaline pH producing activated proteins named delta-endotoxins. The protein’s N-terminal part is dedicated to express toxicity, while the C-terminal portion is mainly involved in the production of the parasporal inclusion bodies that is typically hydrolysed into small peptides (Choma et al. 1990). Along with the Cry or cyt toxins Bt possesses additional virulent factors such as, phospholipase C (Palvannan and Boopathy 2005; Martin et al. 2010), enterotoxins, chitinases, proteases (Brar et al. 2009; Infante et al. 2010) and hemolysins (Nisnevitch et al. 2010) that are also responsible in exhibiting mosquito toxicity. Mosquitocidal Bt isolates belonging to different serovars such as thompsoni, malaysiensis, canadensis, jegathesan, israelensis and morrisoni that are toxic to Cx. quinquefasciatus, Ae. Aegypti and An. hyrcanus were isolated and evaluated by different scientists all over the world (Ming et al. 1996; Weiser et al. 1984;).
Among the 18 B. thuringiensis isolates recovered from intertidal brackish water sediment samples of mangroves, two isolates of Bt var israelensis/tochigiensis (H14/19) produced high toxicity to Cx. p. molests (Maeda et al. 2001). Lee et al. (2001) obtained an isolate of Bt var kurstaki (H3a:3b:3c) from Korea exhibiting toxicity against Cx. pipiens. In India, potent mosquitocidal strains of Bt var israelensis (H14) pathogenic to Culex mosquitoes were isolated (Manonmani et al. 1987). One strain of Bt var thompsoni (H-12), showing high toxicity to mosquito larvae was isolated from India which protein profile of parasporal body was comparable with that of Bt var israelensis (Manonmani and Balaraman 2001). Patil et al. (2012) determined the insecticidal potency of Bacillus thuringiensis SV2 against immature Cx. quinquefasciatus, Ae. aegypti and An. stephensi. The Bt SV2 isolate showed 100% mortality against early fourth instars of Culex quinquefasciatus, Aedes aegypti and Anopheles stephensi in parallel to Bti H14 standard strain. Kovendan et al. (2011) reported the excellent prospective of B. thuringiensis israelensis as cidal agent against the all the larval instars of Culex quinquefasciatus having values of LC50 as 9.332%, 9.832%, 10.212%, and 10.622%, and LC90 as 15.225%, 15.508%, 15.887%, and 15.986% against the first, second, third and fourth instar larvae respectively. The study of different researchers regarding biocontrol of mosquito species revealed that the efficacy of Bacillus thuringiensis and other mosquitocidal bacteria was dependent on the application concentration, dose, larval age, time of exposure, resistance capability of the mosquito and environmental factors (Chenniappan and Ayyadurai 2011; Mwangangi et al. 2011; Singh and Prakash 2009;). DSB27 (KX130958) was more effective than the standard bacterial strain H14 showing lower LC50 value. This may be due to the higher activity of the spore-crystal mixture present in the bacterial culture. Bacillus thuringiensis DSB27 (KX130958) might be more adaptable to its native environment and might be more acclimatized to the surrounding environmental cues, and thus exhibited higher efficacy than standard Bt isolate.
From the present investigation it can be concluded that Bacillus thuringiensis DSB27 (KX130958) has been proved to be a potential biocontrol agent against filarial vector Culex quinquefasciatus by exhibiting higher efficacy than standard Bt isolate and sustainability in coastal environment.