Mosquito collection and identification: Larvae of An. subpictus were collected from water bodies of some rural areas of Hooghly district by using standard dipper of 250mL capacity following standard protocols [30, 31]. Adult mosquitoes were collected within test tubes from cattle sheds and human habitations by hand collection method. All the collections were done once in summer, monsoon, post-monsoon & winter from february’2017-march’2018 during early morning or in the afternoon. After collection, the larvae and adult mosquitoes were brought to the Parasitology & Microbiology Research Laboratory, Department of Zoology, The University of Burdwan. Identification of larval and adult forms of mosquitoes were done under microscope following standard keys [32, 33]. Adult An. subpictus mosquitoes were maintained in a mosquito rearing cage (30cm x30cm x30cm) in optimum laboratory condition for egg laying and larval hatching. Some specimens were also sent to Zoological Survey of India, Kolkata for the reconfirmation of the species identification.
Isolation of bacteria: Both field collected and laboratory reared 3rd instar larvae (5-10 in number) of An. subpictus were surface sterilized by placing them in 70% alcohol for 3-5 mins. Then they were washed with double distilled water and mid-gut of the larvae were dissected carefully in aseptic condition under a binocular (2X magnification). Mid-gut content for laboratory reared and field caught larvae were mixed separately with 1mL sterile distilled water and from this mixture 100µL was mixed with sterilized moderately cooled nutrient agar media and poured on five sterile petri plates. The plates were incubated at 37±20C temperature in a B.O.D incubator for 24-48h. On the next day, total number of colonies that were formed over the nutrient agar plates were counted and bacterial load in the mid-gut of larvae were determined as colony forming unit (cfu/mL). Colonies that were formed on the nutrient agar plates were characterized by recording different colony characters (size, shape, colour, opacity, elevation & consistency). Pure cultures of the bacterial isolates were maintained on nutrient agar slants in a refrigerator at 4±1 ºC temperature for further analysis.
Characterizations of larval gut bacterial isolates
Phenotypic characterizations
Phenotypic properties of bacterial isolates such as Gram staining properties, presence of endospores were evaluated by staining with Gram’s stain and 5% Malachite green solutions respectively. Scanning electron microscopic analysis was performed to observe the surface morphologies of vegetative cells of the bacterial isolates [13].
Bio-chemical characterizations
Several bio-chemical properties of the bacterial isolates such as catalase; methyl red; indole; voges proskauer test; citrate test; oxidase test; urease test; growth on Triple Sugar Iron (TSI) agar; starch, protein & lipid hydrolysis test, nitrate reduction test and fermentation ability of twenty different carbohydrate sources such as dextrose (De), fructose (Fc), galactose (Ga), sucrose (Su), lactose (La), mannitol (Mn), cellobiose (Ce), dulcitol (Du), trehalose (Te), raffinose (Rf), sorbitol (Sb), xylose (Xy), melibiose (Mb), rhamnose (Rh), arabinose (Ar), mannose (Mo), lactose (La), adonitol (Ad), salicin (Sa) and inulin (In) were performed following standard methodologies [34-36].
Physiological characterizations
Physiological properties of the gut bacterial isolates like sodium chloride (NaCl) tolerance, growth at different pH scale and different temperature of the culture media were recorded following standard methodologies [34-36].
Molecular characterizations
Genomic DNA were isolated from the overnight grown liquid bacterial cultures following standard method [37] and 16S rDNA sequences were amplified by polymerase chain reaction by using 27F forward and 1492R primer. The PCR purified products were sequenced bi-directionally using universal primers. The obtained 16S rRNA gene sequences of the bacterial isolates were submitted to NCBI GenBank. Nucleotide percentages of 16S rRNA gene sequences of the bacterial isolates were calculated using Aqua software. Phylogenetic analysis was done by neighbour-joining method using MEGA X software [38].
Antibiotic sensitivity of bacterial isolates
Susceptibility of the bacterial isolates to twenty different commercially available standard antibiotics namely bacitracin (10 µg/disc), nalidixic acid (30 µg/disc), kanamycin (30 µg/disc), ampicillin (10 µg/disc), amoxicillin (10 µg/disc), penicillin (10 µg /disc), levofloxacin (5 µg/disc), chloramphenicol (30 µg/disc), gentamicin (50 µg/disc), neomycin (30 µg/disc), tetracycline (30 µg/disc), ofloxacin (5 µg/disc), norfloxacin (10 µg/disc), rifampicin (5 µg/disc), ciprofloxacin (5 µg/disc), vancomycin (30 µg/disc), azithromycin (30 µg/disc), erythromycin (15 µg/disc), doxycycline (30 µg/disc) and streptomycin (10 µg/disc) were tested on Muller-Hinton agar plates by disc diffusion method [39]. Minimum inhibitory concentration of two broad spectrum antibiotics viz., doxycycline and levofloxacin against the bacterial isolates was determined using MIC strip (Himedia, India).
Role of larval mid-gut bacteria in survival and development of host
A batch of three hundred 1st instar larvae of An. subpictus were collected from the fresh water bodies and they were kept in a plastic tray (30cmX30cm) containing 1500mL of natural habitat water. Out of these total collected specimens, a group of thirty larvae were maintained in a separate tray containing 100mL natural habitat water. This group of larvae were provided with UV sterilized larval food and without any treatment which served as conventionally reared control group. Rest of the larvae were fed with UV sterilized larval food and a mixture of doxycycline and levofloxacin (1:1 ratio) at their minimum inhibitory concentration for 1day to clear the gut bacterial communities. Another group comprised of thirty 1st instar larvae were separated in a plastic tray containing sterile distilled water with UV sterilized larval food. This group served as axenic group. Rest of the antibiotic treated axenic larvae were separated in five trays (30 larvae in each) containing 95mL sterile distilled water with UV sterilized larval food. Axenic larvae in the 1st to 4th trays were provided with 5mL suspensions of previously isolated individual gut bacteria for their recolonization within larval gut and in the 5th tray axenic larvae were provided with 5mL suspension containing a mixture of all gut bacterial isolates. All the larvae were maintained in aseptic condition at 37±20C temperature, 75±5% relative humidity and 12:12h (light: dark) photo period. Five replicates were done for each of the treatment and control groups. Time of pupa formation for each group of larvae were noted down. After pupal emergence, pupae from each batch were kept in separate mosquito rearing cages (30cm X 30cm X 30cm) and were maintained at 37±20C temperature, 75±5% relative humidity and 12:12h (light: dark) photo period. Number of adult mosquitoes emerged from each group was noted down. Adult males and females emerged from each group were kept separately in test tubes with chloroform applied cotton plugging for 6-7 days. Dry weight of male and female mosquitoes from each group was measured. Wings of male & female mosquitoes were dissected out, mounted on glass slide with DPX solution. Then images of wings were captured by stereo zoom microscope (Olympus) and the length and breadth of each of the wing were measured with M Shot software.
Bio-chemical profile of larvae in presence and absence of gut bacteria
Total tissue carbohydrate, protein & lipid content of 4th instar larvae in each of the treated & control groups were evaluated. For that purpose, thirty synchronized 4th instar larvae were selected from each group. Total tissue carbohydrate and lipid were extracted [40]. Carbohydrate estimation was done by anthrone reagent [41]. Lipid quantification was done by vanillin-reagent method [42]. Total tissue protein was extracted [43] and estimated using foline-ciocalteau reagent [44].
Statistical analyses
One way ANOVA was done to compute any significant differences in pupation time, rate of survival up to adulthood, body weight & wing length of adult male & female An. subpictus mosquitoes and bio-chemical contents of 4th instar larvae among different treatment groups. Dunnett’s multiple comparison tests were performed to evaluate significant difference of each of the treatment group from control group. All the analyses were performed in GraphPad prism 9.0.0.