Histopathological and ultrastructural effects of delta-endotoxins of Bacillus thuringiensis serovar israelensis in the midgut of Simulium pertinax larvae (Diptera, Simuliidae)

The bacterium Bacillus thuringiensis (Bt) produces parasporal crystals containing delta-endotoxins responsible for selective insecticidal activity on larvae. Upon ingestion, these crystals are solubilized in the midgut lumen and converted into active toxins that bind to receptors present on the microvilli causing serious damage to the epithelial columnar cells. We investigated the effect of these endotoxins on larvae of the Simulium pertinax, a common black fly in Brazil, using several concentrations during 4 h of the serovar israelensis strain IPS-82 (LFB-FIOCRUZ 584), serotype H-14 type strain of the Institute Pasteur, Paris. Light and electron microscope observations revealed, by time and endotoxin concentration, increasing damages of the larvae midgut epithelium. The most characteristic effects were midgut columnar cell vacuolization, microvilli damages, epithelium cell contents passing into the midgut lumen and finally the cell death. This article is the first report of the histopathological effects of the Bti endotoxins in the midgut of S. pertinax larvae and the data obtained may contribute to a better understanding of the mode of action of this bacterial strain used as bioinsecticide against black fly larvae

Identification of entomopathogenic Bacillus isolated from Simulium (Diptera, Simuliidae) larvae and adults

Entomopathogenic bacteria isolated from Simulium larvae and adults from breeding sites in the states of São Paulo and Rio de Janeiro, Brazil, were identified as 18 strains of Bacillus thuringiensis and one of B. sphaericus. Most of these strains were serotyped according to their flagellar antigens. However, nine of the B. thuringiensis samples, could not be serotyped and were designated as "autoagglutinating"; they were also shown to be toxic in preliminary tests against Aedes aegypti larvae. Additionally, B. sphaericus was also shown to be toxic towards Culex quinquefasciatus larvae

Isolation and genetic characterization of Lysinibacillus sphaericus strains found in mosquito larvae (Diptera: Culicidae)

Clara de Fátima Gomes Cavados,1 Eder Soares Pires,1 Jeane Quintanilha Chaves,1 Danielle Nunes Alvarez,1 Helio Benites Gil,2 Iris Braz Ribeiro de Oliveira,2 Andrea de Barros Pinto Viviani Cunha,2 Carlos José Pereira da Cunha de Araújo-Coutinho2 1Laboratory of Bacterial Physiology, Fundação Oswaldo Cruz, Instituto Oswaldo Cruz, Rio de Janeiro, 2Superintendência de Controle de Endemias – SUCEN, São Paulo, Brazil Introduction: Lysinibacillus sphaericus is a highly effective and specific bioinsecticide used for the control of Culicidae larvae. Objective: This study aimed to identify and characterize L. sphaericus strains isolated from Culex quinquefasciatus larvae in Brazil. Methods: C. quinquefasciatus larvae were collected from streams in the urban area of São Paulo state. L. sphaericus strains were identified through cytomorphology, biochemical, and physiological analyses. Qualitative bioassays were performed to evaluate the toxicity of the strains against C. quinquefasciatus. The crystal compound protein pattern of L. sphaericus strains was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Five reference strains were used as standards in all tests performed. Repetitive extragenic palindromic elements-polymerase chain reaction (REP-PCR) was utilized in an attempt to differentiate pathogenic and nonpathogenic isolates. Results: Twenty-one strains were isolated. Only one presented toxic activity against C. quinquefasciatus. REP-PCR results identified 23 patterns among the 26 strains used in the study, and the fragment analysis showed low similarity (16%) between L. sphaericus isolates and the five reference strains. Conclusion: Comparison of strains isolated in this study using REP-PCR showed a low similarity to other strains, demonstrating the high intraspecific variability for L. sphaericus. Keywords: Lysinibacillus sphaericus, Culicidae, SDS-PAGE, qualitative bioassays, REP-PCR, mosquitoes, entomopathogenic bacteria&nbsp

Bacillus thuringiensis isolates entomopathogenic for Culex quinquefasciatus (Diptera: Culicidae) and Anticarsia gemmatalis (Lepidoptera: Noctuidae)

Samples of the Bacillus thuringiensis (Bt) were collected from soil and insects. Eight isolates were selected from rural soil, 15 from urban soil and 11 from insects. These were evaluated for entomopathogenicity against larvae of Anticarsia gemmatalis and Culex quinquefasciatus. The pathogenicity tests showed that a higher percentage of isolates were active against A. gemmatalis (60%) compared to C. quinquefasciatus (31%). Probit analysis (LC50) indicated that against A. gemmatalis four of the isolates presented values similar to the reference strain against A. gemmatalis, while against C. quinquefasciatus one isolate showed an LC50 similar to the reference strain (IPS-82). SDS-PAGE characterisation of two isolates showed a 27 kDa protein fraction related to the Bt subspecies israelensis cytolytic toxin (cyt) gene. One 130 kDa protein, possibly related to the Bt crystal inclusions (cry1) gene, was identified in the other two isolates, which were more toxic for lepidoptera; another isolate presented a protein of 100 kDa. Some new local Bt isolates had similar LC50 probit values to the reference strains