Enterococcus durans with mosquito larvicidal toxicity against Culex quinquefasciatus, elucidated using a Proteomic and Metabolomic approach

Various bacteria from the Bacillus species have been used as pesticides against mosquito larvae for more than a decade. The prolonged use of these bacterial species by little alteration within their genome, using various permutations and combinations of mosquito-cidal toxins, has proven unsuccessful in controlling the mosquito population. In our current study we report Enterococcus sp. to be exhibiting similar kind of mosquito-cidal toxins alike those which are present in the mainly used Bacillus strains. Three Enterococcus species were isolated on a rich media selective for gram- positive bacteria from the mid-gut of dead mosquito larvae which were collected from the wild locations within and around the city of Mumbai, India. Their surface morphologies were studied by Scanning Electron Microscopy (SEM) and their identity was confirmed using the standard 16S rRNA sequencing method. Upon performing several repetitive toxicity assays of these three strains on the laboratory cultured third instar stage of Culex quinquefasciatus larvae, showed differential toxicities from a minimum of 20% (LC50: 59.6 CFU/ml), intermediate 35% (LC50: 48.4 CFU/ml) and a maximum of 60% (LC50: 35.7 CFU/ml). To justify the data in all the three similar strains of Enterococcus durans, we followed the differential proteomics using LCMS 6540 UHD Accurate Mass QTOF and differential metabolomics approach using both LCMS 6540 UHD Accurate Mass QTOF and 1H-NMR. The presence and significance of the obtained toxins were studied to elucidate the plausible reason for showing differential toxicities. This work helped in identifying Enterococcus durans as a new, potential and alternative strain to the Bacillus species in terms of mosquito larvicidal toxicity against Culex quinquefasciatus.