Identificação do mecanismo celular de absorção de dsRNA em Diabrotica virgifera virgifera (LeConte) e seleção de cepas de Bacillus thuringiensis tóxicas contra Helicoverpa armigera (Hübner)

Abstract

RNA interference (RNAi) is a gene silencing mechanism triggered by double-stranded RNA (dsRNA) molecules, widely used in the entomological research, especially for the functional analysis of genes. Its potential for insect pest control has been demonstrated through several studies but for the effective application of the RNAi in the insect management is necessary the uptake of dsRNA from the intestinal lumen after it to be ingested by the insect and the RNAi signal needs to be systemically spread from one cell to another. Diabrotica virgifera virgifera LeConte (western corn rootworm) is one of the most important insect pest of maize in United States. In order to identify the mechanism involved in the cellular uptake of dsRNA in D. virgifera virgifera adults an “RNAi on RNAi” approach was used. To assess whether the transmembrane SID-1 protein (SID-1-like or SIL) participates of the dsRNA uptake in D. virgifera virgifera adults, the insects were injected with silA dsRNA, silC dsRNA or a mixture of silA dsRNA and silC dsRNA. Posteriorly, dsRNA of the marker gene vATPase-A was offered to the insects through feeding, and then the insect mortality and vATPase-A gene expression were evaluated. In addition, the participation of the clathrin-dependent endocytosis in dsRNA uptake was analyzed through the silencing effect of five important genes associated with the endocytosis (Clath, Vha16, AP50, Arf72A and Rab7) on RNAi of the marker gene laccase2. Our results demonstrated that the expression of vATPase-A gene was not significantly increased by silA and silC genes silencing compared to the control treatment, in which the insects were injected with GFP dsRNA and then fed with vATPase-A dsRNA. The silencing of silC gene did not affect the mortality of D. virgifera virgifera adults suggesting that SILC protein does not participate of the dsRNA uptake in this insect. The suppression of the silA gene affected significantly the D. virgifera virgifera adults mortality compared to control, but no effects on vATPase-A gene expression were observed suggesting that the SILA protein does not play a key role in dsRNA uptake. The silencing of Clath, Vha16 and AP50 genes inhibited the internalization of laccase2 dsRNA and induction of gene silencing suggesting that endocytosis plays a critical role in dsRNA uptake in D. virgifera virgifera adults. However, the suppression of the Arf72A and Rab7 genes did not affect the silencing of laccase2 gene. Unlike the RNAi which is considered a relatively recent technique, Bacillus thuringiensis based biopesticides and genetically modified plants expressing genes of this bacteria have been used as an efficient strategy for insect pest control over many years. However, the selection of resistant insects to B. thuringiensis toxins lead to identify new strains and genes to be used with this purpose. Toxicity bioassays were performed to select B. thuringiensis strains with insecticide activity against Helicoverpa armigera (Hübner). Eight strains, 426, 520B, 1636, 1644, 1648, 1657 and 1658, caused mortality higher than 75% in H. armigera larvae and showed LC50 beetwen 150.1 e 1543.3 ng/cm2. Through molecular analysis it was demonstrated that these strains harbor different cry and vip genes. In addition, they showed protein profile with major bands of 140 and 55 kDa. The insecticidal activity of the strains 426, 520B, 1636, 1641, 1644, 1648, 1657 and 1658 was also evaluated through bioassays with Anticarsia gemmatalis (Hübner), Diatraea saccharalis (Fabricius), Spodoptera cosmioide (Walker) and Pseudoplusia includes (Walker), demonstrating that some of these strains cause high levels of mortality in these insects. The results indicate that the selected B. thuringiensis strains have great potential to be used in the control of H. armigera and other important lepidopteran pests.