Análises moleculares de linhagens de Bacillus e Brevibacillus spp., isolados da Amazônia brasileira, ativas contra Aedes aegypti, Linnaeus, 1762

Abstract

The Aedes aegypti mosquito is considered the primary vector of many arboviruses that cause diseases such as dengue, chikungunya and Zika. The problems caused by this vector are immeasurable, and new control strategies are needed to mitigate the impacts on public health. Bacillus and Brevibacillus genus bacteria are the main entomopathogenic microorganisms used to control these mosquitoes. Considering the metabolic and genetic diversity of these bacteria, this study aimed to evaluate the chemical and biological potential of bacteria of the genus Bacillus and Brevibacillus, isolated from different Amazonian environments, with toxicity to A. aegypti. One hundred and forty three strains of bacilli were isolated in the culture media (NA, LB and ISP2) by means of heat shock, which was characterized by Gram stain, with 136 gram positive and seven gram negative bacilli found. Through the clusters of the phenotypic profile, 77 strains were selected as representatives and identified by the 16S rRNA gene sequence, being found six bacterial genera such as Bacillus, Brevibacillus, Achromobacter, Serratia, Klebsiella and Brevundimonas. The 77 characterized lines were tested in selective bioassays against A. aegypti, of which 21 (27.2%) had larvicidal activity. The strains that showed toxicity above 50% were used for bioassays of the supernatant and pellet fractions of bacterial cultures by means of selective and quantitative bioassays. Regarding the LC50 and LC90 values of the active strains (SPa07NA-Br. halotolerans and SX15LB-B. safensis) in the supernatant fraction (SUP), no statistically significant difference was observed (p>0.05) when compared to the standard Bti strain BR101NA, at intervals of 24, 48 and 72 hours. The results of the non-autoclaved pellet (PEL) at 48h showed that the SBC13LB-B. thuringiensis strain obtained a LC50 value (0.004 mg/l) lower than that of the standard strain Bti BR101NA (0.008 mg/l), with a statistically significant difference (p = 0.05), showing greater toxicity. Considering the results of autoclaved pellet + supernatant (APEL+SUP), the strains SP06LB-B. thuringiensis and SPa22LB-B. safensis did not show statistically significant differences (p>0.05) when compared to the standard Bti BR101NA strain at intervals of 24, 48 and 72h. The results of the autoclaved pellet (APEL) showed that the strain SPa22LB-B. safensis did not present statistically significant difference between the values of LC50 and LC90 in the intervals of 24, 48 and 72h. The active strains in the supernatant (SUP) and pellet (PEL) fractions were used for the study of secondary metabolites, in which two strains (SPa07NA-Br. halotolerans and SX15LB-B. safensis) showed toxicity in the extracts obtained from the supernatant ( SUP). The SPa07NA strain showed greater toxicity in LC50 and LC90, with a statistically significant difference (p<0.05) at intervals of 24, 48 and 72h. The study of metabolites of the SPa07NA-Br. halotolerans strain, allowed the tracking of active metabolites in the extracts. The results obtained by direct insertion in electrospray ionization mass spectrometry showed that during the 10 days of culture of the strains, no changes in the ions were observed, but the prevalence of major ions was observed. Furthermore, the biological activity against A. aegypti did not show significant differences. The isolation standardization technique by semi-preparative HPLC combined with biological assays and fractionation allowed obtaining three fractions and 12 active subfractions for the larvae. The chemical profile of metabolite extracts from promising strains needs further studies that can be carried out in future work in order to characterize and elucidate the active molecules. In addition to metabolite studies, the antagonistic behavior of two active strains that showed larvicidal activity only in the pellet (PEL) was evaluated. Studies of the potential of GD02.13NA-B. toyonensis strains and SBC13NA-B. thuringiensis showed similar results to the standard Bti BR 101 strain, in which bacterial colonization in the larvae's head and trunk was evidenced, and positive amplification for Cry4 and Chi genes with potential for production of chitinase enzymes. Thus, it is considered that the isolated strains have the potential to produce primary and secondary metabolites for different control strategies for these mosquitoes.