Phenotyping and molecular characterization of Lysinibacillus sp . P-011 ( GU 288531 ) and their role in the development of Drosophila melanogaster

The bacterial strain Lysinibacillus sp. (P-011) was isolated from the midgut of the Drosophila melanogaster larvae. The bacteria were gram positive, spore forming, rod shaped ranging from 1.86 to 2.5 μm in length and 0.50 to 0.67 μm in diameter, positive for catalase, indole, oxidase, nitrate reduction, starch and gelatin hydrolysis, sensitive to tetracycline, chloramphenicol, doxycycline hydrochloride, gatifloxacin, ofloxacin, vancomycin, rifampicin, levofloxacin, ciprofloxacin, nalidixic acid, but resistant to ampicillin, streptomycin, gentamycin and kanamycin. The phylogenetic tree showed that the strain Lysinibacillus sp. P-011 (GU288531) branched with Lysinibacillus boronitolerans with 89% bootstrap support. Lysinibacillus sp. P-011 (×10 5 cfu/ml) played an important role on larval development of D. melanogaster under controlled environmental condition. Wild larvae when fed on normal food as well as normal food mixed with ineffective antibiotics, developed puparium within seven days whereas took more than 10 days when fed on normal food mixed with anti P-011 antibiotics and sterile food mixed with bacterial suspension and anti P-011 antibiotics. 94 to 98% cured larvae developed puparium within seven days when fed on only sterile food mixed with bacterial suspension (P-011) or sterile food mixed with bacterial suspension (P-011) and ineffective antibiotics.


INTRODUCTION
Insect guts act as reservoirs and fermentation vessel for a large variety of microorganisms.The enormous microbial diversity of insect gut may be originated from their different feeding habits, different gut structures and functions of different groups of insects promoting the establishment of different group of microbes (Dillon and Dillon, 2004).These gut microbes play important roles in various types of interactions ranging from pathogenesis to obligate mutualism (Dillon and Dillon, 2004).In various organisms, gut microbiota act as vital resource of novel bioactive compounds (Chernysh et al., 2002), enzymes (Zhang and Brune, 2004) and novel metabolites (Wilkinson, 2001).Proper scientific exploration of symbiotic gut microbes may be an alternative and effective strategy for controlling the spread of pathogens which utilize insects as hosts (Mickes and Ferguson, 1961;Lehane et al., 1997;Beard et al., 2002;Dillon et al., 2005).The presence and diversity of insect gut bacteria are influenced by the gut pH, redox conditions, digestive enzymes of insect gut and types of food ingested.The optimum pH for the growth of most bacteria ranges from 6 to 7, but some bacteria can grow at acidic pH.Anaerobic bacteria show their growth only at negative redox potentials whereas aerobic bacteria survive at positive redox potentials.Intestinal microorganisms help in digestion of food material and also produce essential vitamins for the host.Several experimental evidences revealed that the symbiotic gut bacteria of some beetles can provide vitamin B to their host (Blewett and Fraenkel, 1944).The role of symbiotic gut bacteria on the survival of fruit fly refers to obligate symbiotic relationships between insect larvae and their gut microbes with respect to larval nutrition, growth and development (Brummel et al., 2004).Drosophila melanogaster breeds in decaying organic matter or necrotic plant material in the presence of various microorganism and they have to interact with micro-organisms during all stages of their life cycle.Symbiotic microorganisms may be found in the gut (Douglas et al., 2011), gonad (Mateos et al., 2006) and some other parts of the fly body.It has been proposed that several fruit-feeding Drosophila species are nutritionally dependent on bacteria (Mateos et al., 2006).Laboratory experiments have revealed that sugar, essential amino acid, fat, cholesterol and some salts are important nutrients for the development of the D. melanogaster (Sang, 2006;Sang and King, 1961;Sang, 1956).Riboflavin, nicotinic acid and pyridoxin are the essential substances for the normal growth of Drosophila larvae which are known to be supplied by some micro-organism (Tatum, 1939).Symbiotic bacteria have different influences on different aspects of fly life-cycle such as contribution on host nutrition (Douglas, 1998), immunity (Hedges et al., 2008;Osborne et al., 2009;Teixeira et al., 2008) and reproduction (Serbus et al., 2008).Some bacteria can enhance the life-span of the Drosophila sp.(Brummel et al., 2004).Scanty information is available on the resident bacterial flora of the larval midgut of Drosophila sp.We used Drosophila sp. and their gut microbiota as an experimental model of insect microbial symbiosis.Present work was designed to study the phenotypic and molecular characterization of the gut bacteria in Drosophila sp. and to determine their effects on larval development.

MATERIALS AND METHODS
Wild type D. melanogaster flies were cultured in biochemical oxygen demand (B.O.D) incubator at 24  1°C using standard flyfood medium containing maize powder, sucrose, agar, yeast, sterile distilled water in the Department of Zoology of The University of Burdwan, Burdwan.

Bacteria isolation from the midgut of Drosophila
The third instar larvae of D. melanogaster were selected for the experiment.The larvae were sterilized with 70% ethanol for 3 min, washed thoroughly with sterile distilled water and their midguts were dissected out under the binocular microscope in laminar air flow.Each midgut was crushed separately on a sterile slide, gut extract was aspirated and diluted with 250 ml sterile distilled water and mixed with 100 ml nutrient agar (NA) medium (peptone-beef extract-NaCl-agar at 5:3:3:18 g/l) at pH 7.4, plated on five Petri plates and incubated in a biochemical oxygen demand incubator at 30  0.1°C for 24 h (Roy et al., 2010).The most prevalent colonies developed from the gut triturate of Drosophila sp. were then maintained on nutrient agar slants at 4  0.1ºC in refrigerator.

Scanning electron microscopy (SEM) of bacterial isolates
Bacterial smears were prepared on cover glasses, heat fixed over a flame for 1 to 2 s followed by 2.5% glutaraldehyde (aqueous) for 45 min.The slides were then dehydrated passing through 50, 70, 90% ethanol and finally with absolute alcohol for 10 min each.The specimens were gold coated and finally scanned and photographed under Scanning Electron Microscope (Model Hitachi S-530).

Molecular characterization and phylogenetic analysis of gut bacteria
Genomic DNA was isolated from the pure culture pellet using genomic DNA isolation kit.The ~1.5 kb rDNA fragment was amplified using high-fidelity PCR polymerase.The PCR product was sequenced bi-directionally through a genetic analyzer using the forward primer and reverse primer.The nucleotide sequence of the bacterial isolate P-011 has been submitted to the NCBI GenBank database and assigned accession number GU288531.Most similar strain sequences were retrieved from EzTaxon-e, a prokaryotic 16S rRNA Gene sequence database taking Lysinibacillus sp.P-011(GU288531) as a reference sequence.Alignment view and distance matrix table was constructed following Kim et al. (2012).Sequence was analyzed and restriction map was prepared with enzymes available in New England Biolab.The sequence data were aligned using the ClustalW submission form (http://www.ebi.ac.uk/clustalw) and analyzed by ClustalW software (Thompson et al., 1994).Evolutionary distances were calculated using the method of Jukes and Cantor (1969) and phylogenetic tree was constructed according to Tamura et al. (2007)'s research.

Evaluation of the role of gut bacteria on larval development
In order to observe the effect of the symbiotic bacteria on host body, we recorded the duration of larval development and formation of puparium in the presence and absence of the gut bacteria.For each test, 50 1 st instar larvae and three replications were used.All the tests were conducted in culture bottles holding standard Drosophila food medium, autoclaved at 121°C at 15 lb pressure.Third instar larvae were cultured for 24 h on food containing 100 µl mixture of antibiotics (chloramphenicol (10 μg/ml), tetracyclin (10 μg/ml), and doxycyclin (10 μg/ml) to which the bacterial isolate  30) Ampicillin ( 10) Chloramphenicol ( 30) Streptomycin ( 10) Gatifloxacin ( 5) Gentamycin ( 10) Ofloxacin ( 5) Kanamycin ( 30) Vancomycin ( 30) Rifampicin ( 5) Levofloxacin ( 5) Ciprofloxacin (5) Nalidixic acid (30) showed sensitivity.These axenically cultured D. melanogaster flies were transferred to each experimental culture bottle containing normal or sterile food medium.To assess the role of the bacteria P-011, on D. melanogaster larvae, 100 µl bacterial solution (10 5 cfu/plate) were mixed separately with food medium except the bottle containing only normal food and only sterile food.Duration of larval development to form puparium was recorded to show whether presence of bacteria have played any role in the development of D. melanogaster.Identical experiments were done with untreated D. melanogaster flies separately at 24 ± 1°C and were observed daily for the first 10 days and every other day thereafter, developmental duration of each stage being noted.

RESULTS AND DISCUSSION
The colonies of the bacteria (P-011) were spherical, cream colour, opaque and elevated (Table 1).The bacteria were rod shaped.Length of the organisms ranged from 1.86 to 2.5 μm and 0.50 to 0.67 μm in diameter (Plate 1).The bacteria were positive for Gram staining, spore forming and could tolerate up to 60°C and up to 6% NaCl (Table 1).The organism was positive for catalase, indole, oxidase, nitrate reduction, starch and gelatin hydrolysis but negative for citrate utilization, methyl red, vogues-Proskauer test, casein and chitin hydrolysis.Response of the organisms to the recommended doses of different antibiotics showed that all of them were sensitive to tetracycline (30 µg/ml), chloramphenicol (30 µg/ml), doxycycline hydrochloride (30 µg/ml), gatifloxacin (5 µg/ml), ofloxacin (5 µg/ml), vancomycin (30 µg/ml), rifampicin (5 µg/ml), levofloxacin (5 µg/ml), ciprofloxacin (5 µg/ml), nalidixic acid (30 µg/ml), but resistant to ampicillin (10 µg/ml), streptomycin (10 µg/ml), gentamycin (10 µg/ml), kanamycin (30 µg/ml) (Table 1).The nucleotide composition is shown in Figure 1.AT and GC content were 46.55 and 53.45%, respectively.Restriction map has been displayed by Figure 2. Phylogenetic affiliation of the bacterium (P-011) was done by 16S rRNA gene sequence analysis.Alignment view and distance matrix table (Table 2) depicted that Lysinibacillus sp.(P-011) showed 96.30% similarity with Lysinibacillus macroides (AJ628749) and 95.92% with Lysinibacillus boronitolerans (AB199591).To assign the taxonomical affiliation of this bacterium, the phylogenetic tree was constructed through multiple sequence alignments followed by a neighbor-joining analysis (Saitou and Nei, 1987) (Figure 3).The phylogenetic  3).When the wild type larvae were fed on normal food, it developed puparium within seven days in the B.O.D incubator at controlled environmental condition.Similar result was found when the wild type larvae were fed on normal food with ineffective antibiotics.Wild type and cured larvae took more than 10 days to develop puparium when fed on normal food mixed with anti P-011 antibiotics and sterile food mixed with bacterial suspension and anti P-011 antibiotics, respectively (Table 3).Previous published works show that Lysinobacillus sp. can promote plant growth (Vendan et al., 2010) and nitrogen fixation (Vendan et al., 2010;    along with other 16S rRNA genes.Sgroy et al., 2009), which supports the growth of the insects (Rajagopal, 2009).It has also been reported that several midgut bacteria like Acetobacter pomorum, Gluconobacter morbifer, Lactobacillus plantarum, Lactobacillus brevis and Commensalibacter intestine, have beneficial role on larval development.Absence of these bacteria has been shown to lengthen time duration to reach puparium formation in D. melanogaster larvae (Ryu et al., 2011;Douglas et al., 2011).The results clearly indicate that the time to puparium formation is delayed due to the elimination of Lysinibacillus sp.(P-011) from larval midgut.Lysinibacillus sp.(P-011) has been isolated from all the larval stages in all the seasons throughout the year.So, it is proved that it is not a mere transient flora inhabiting the midgut rather an important resident symbiotic flora of D. melanogaster playing an important physiological role in larval development.

Table 1 .
Phenotypic and biochemical characterization of the Lysinibacillus sp.P-011.

Table 2 .
Alignment view and distance matrix table taking Lysinibacillus sp.(GU288531) as reference sequence.

Table 3 .
Effect of Lysinibacillus sp.(P-011) on the duration of larval development of the D. melanogaster *.
*For each test, 50 1 st instar larvae and three replications were used.Data are means of three replications ± SE.