Phenotypic Characterization of Lysinibacillus sphaericus Strains Isolated From Mosquito Larvae (Diptera: Culicidae) and Genotypic Analysis by REP-PCR

Lysinibacillus sphaericus, is an obligate aerobic, mesophilic, round-spored shaped bacterium, naturally occurring in soil, which shows negative results for carbohydrate fermentation in phenotypic tests applied for Bacillus identification. Some strains may be highly toxic to some species of mosquito larvae such as Culex sp., Anopheles sp. and Psorophora sp., and can show low toxicity against Aedes sp. larvae. The aims of this study were to identify and characterize phenotypically and genotypically strains of L. sphaericus isolate from Culicidae larvae in Brazil. The larvae were collected from rivers of Sao Paulo state (Brazil) and then sent, on ice, to the laboratory where they were submitted to a sterilization process with baths of 70% ethanol, 10% sodium thiosulfate and 5% sodium hypochlorite, sequentially. Mixer was used to prepare a homogeneous suspension of the macerated larvae in sterile water followed by treatment at 80°C for 20 min. After heat treatment, 100µL were plated on BHI Agar (Brain Heart Infusion OXOID®), and incubated for 24 h at 30°C, according to Cavados et al. (2004). To identify L. sphaericus isolated strains cytomorphology, biochemical and physiological analysis were performed, and results were analyzed with the support of a dichotomous key. Qualitative bioassays were performed to evaluate the toxicity of isolated strains against Culex quinquefasciatus. Repetitive Extragenic Palindromic Elements - Polymerase Chain Reaction (REP-PCR) was used in order to differentiate pathogenic and non-pathogenic L. sphaericus isolates. Twenty-one strains isolated were identified as L. sphaericus by phenotypic features. In the qualitative bioassays, only the strain LFB-FIOCRUZ 1513 demonstrated toxicity against C. quinquefasciatus larvae. This study is in agreement with previous data reported by other authors, where the genetic variability in L. sphaericus population were analyzed and showed low similarity between the strains as well. Palavras-chave: Entomopathogenic bacteria; Culex quinquefasciatus; Bacillus sp.