Lysinibacillus sphaericus is a spore-forming bacterium used in the biological control of mosquitoes and in bioremediation. Mosquito larvae exposed to heavy metals are tolerant to concentrations above the permissible limit for industrial residual waters. In this work, we characterize 51 L. sphaericus strains for metal tolerance and larvicidal activity against Culex quinquefasciatus. Lysinibacillus sphaericus OT4b.2, OT4b.20, OT4b.25, OT4b.26 and OT4b.58 were as toxic as the spores of the reference strain 2362 against C. quinquefasciatus larvae. 19 Mosquito-pathogenic L. sphaericus strains and 6 non-pathogenic strains were able to grow in arsenate, hexavalent chromium and/or lead. 16S rRNA gene sequences and phylogenetic analyses clustered 84 % of the metal-tolerant strains in L. sphaericus group 1, which encompasses the mosquitocidal strains. The larvicidal activity of vegetative and sporulated cells and its high tolerance to arsenate, hexavalent chromium and lead indicate that L. sphaericus OT4b.26 is a strong candidate for further studies examining its potential for biological control of mosquitoes in waters contaminated with metals.

The main toxicity mechanism of Lysinibacillus sphaericus, which is used in the control of mosquitoes, is its binary toxin produced during sporulation; additionally the Mtx1, Mtx2 and Mtx 3 toxins are expressed in vegetative cells. Mosquito larvicidal potency of the S-layer protein that is expressed in vegetative cells has been determined. The protein is similar to other S-layer proteins of mosquitocidal L. sphaericus strains. The LC50 values of the S-layer protein of the L. sphaericus OT4b25, OT4b26, and III(3)7 strains against third-instar larvae of Culex quinquefasciatus were 8.7, 24 and 0.68 μg/ml, respectively. To our knowledge this is the first study showing the mosquito larvicidal potency of the S-layer protein from Lysinibacillus sphaericus.

Lysinibacillus sphaericus CBAM5, was isolated from subsurface soil of oil well explorations in the Easter Planes of Colombia. This strain has potential in bioremediation of heavy-metal polluted environments and biological control of Culex quinquefasciatus. According to the phylogenetic analysis of 16S rRNA gene sequences, the strain CBAM5 was assigned to the Lysinibacillus sphaericus taxonomic group 1 that comprises mosquito pathogenic strains. After a combination assembly-integration, alignment and gap-filling steps, we propose a 4,610,292 bp chromosomal scaffold. The whole genome (consisting of 5,146,656 bp long, 60 contigs and 5,209 predicted-coding sequences) revealed strong functional and syntenial similarities to the L. sphaericus C3-41 genome. Mosquitocidal (Mtx), binary (Bin) toxins, cereolysin O, and heavy metal resistance clusters from nik, ars, czc, mnt, ter, cop, cad, and znu operons were identified.

Lysinibacillus sphaericus is used for the biological control of mosquitoes. The main toxicity mechanism of pathogenic strains is a binary toxin produced during sporulation. S-layer is a proteinaceous structure on the surface of bacteria; its functions have been involved in the interaction between bacterial cells and the environment, for example, as protective coats, surface recognition, and biological control. In L. sphaericus, S-layer protein (SlpC) is expressed in vegetative cells, and is also found in spore-crystal preparations; it has larvicidal activity in Culex spp. In this study, partial and completed sporulated culture toxicities were compared; also, S-layer protein and spore-crystal proteins were tested against Culex quinquefasciatus larvae for possible interactions. Larvicidal activity obtained with a combination of SlpC and spore-crystal proteins from strain III(3)7 showed no significant interaction, whereas, combinations of both preparations from strain 2362 showed synergistic effect. The highest synergistic activity observed was between spore protein complex from strain 2362 and SlpC from III(3)7. S-layer protein could be considered a good alternative in formulation improvement, for biological control of mosquitoes.