Sub-lethal effects of a Bt-based bioinsecticide on the biological conditioning of Anticarsia gemmatalis.

Bioinsecticides based on Bacillus thuringiensis (Bt) Berliner, 1915 are widely used to control lepidopteran in several crops. However, surviving insects exposed to the sub-lethal concentration of Bt-based bioinsecticides can suffer a multitude of effects on the biological conditioning known as hormesis. Here, we aimed to provide a clearer understanding of the biological conditioning of Anticarsia gemmatalis (Hübner, 1818), exposed to different concentrations of a Bt-based bioinsecticide, by assessing life table parameters over three generations. We defined five sub-lethal concentrations (LC5, LC10, LC15, LC20, and LC25) from the response curve estimate of A. gemmatalis. Deionized water was used as a control. We assessed the parameters of eggs-viability and the duration of the stages, incubation, larval, pre-pupal, pupal, adult, pre-oviposition and total biological cycle. Data were used to construct the fertility life table using the two-sex program. The survival curves showed greater variation in the proportion of individuals at each development stage using the LC25. The sub-lethal concentrations did not influence the incubation-eggs period, pre-pupal and pupal. However, the larval and adult stages using LC25 and LC10 were the most affected. Changes in sex ratio were observed using LC20 and LC5. The toxic effect of Bt-based bioinsecticide interfered mainly in the parameters of fertility, sex ratio, net reproduction rate (R0), and gross reproduction rate (GRR).

Selection for high levels of resistance to double-stranded RNA (dsRNA) in Colorado potato beetle (Leptinotarsa decemlineata Say) using non-transgenic foliar delivery.

Insecticidal double-stranded RNAs (dsRNAs) silence expression of vital genes by activating the RNA interference (RNAi) mechanism in insect cells. Despite high commercial interest in insecticidal dsRNA, information on resistance to dsRNA is scarce, particularly for dsRNA products with non-transgenic delivery (ex. foliar/topical application) nearing regulatory review. We report the development of the CEAS 300 population of Colorado potato beetle (Leptinotarsa decemlineata Say) (Coleoptera: Chrysomelidae) with > 11,100-fold resistance to a dsRNA targeting the V-ATPase subunit A gene after nine episodes of selection using non-transgenic delivery by foliar coating. Resistance was associated with lack of target gene down-regulation in CEAS 300 larvae and cross-resistance to another dsRNA target (COPI ß; Coatomer subunit beta). In contrast, CEAS 300 larvae showed very low (~ 4-fold) reduced susceptibility to the Cry3Aa insecticidal protein from Bacillus thuringiensis. Resistance to dsRNA in CEAS 300 is transmitted as an autosomal recessive trait and is polygenic. These data represent the first documented case of resistance in an insect pest with high pesticide resistance potential using dsRNA delivered through non-transgenic techniques. Information on the genetics of resistance and availability of dsRNA-resistant L. decemlineata guide the design of resistance management tools and allow research to identify resistance alleles and estimate resistance risks.

First transcriptome of the Neotropical pest Euschistus heros (Hemiptera: Pentatomidae) with dissection of its siRNA machinery.

Over the past few years, the use of RNA interference (RNAi) for insect pest management has attracted considerable interest in academia and industry as a pest-specific and environment-friendly strategy for pest control. For the success of this technique, the presence of core RNAi genes and a functional silencing machinery is essential. Therefore, the aim of this study was to test whether the Neotropical brown stinkbug Euschistus heros has the main RNAi core genes and whether the supply of dsRNA could generate an efficient gene silencing response. To do this, total mRNA of all developmental stages was sequenced on an Illumina platform, followed by a de novo assembly, gene annotation and RNAi-related gene identification. Once RNAi-related genes were identified, nuclease activities in hemolymph were investigated through an ex vivo assay. To test the functionality of the siRNA machinery, E. heros adults were microinjected with ~28 ng per mg of insect of a dsRNA targeting the V-ATPase-A gene. Mortality, relative transcript levels of V-ATPase-A, and the expression of the genes involved in the siRNA machinery, Dicer-2 (DCR-2) and Argonaute 2 (AGO-2), were analyzed. Transcriptome sequencing generated more than 126 million sequenced reads, and these were annotated in approximately 80,000 contigs. The search of RNAi-related genes resulted in 47 genes involved in the three major RNAi pathways, with the absence of sid-like homologous. Although ex vivo incubation of dsRNA in E. heros hemolymph showed rapid degradation, there was 35% mortality at 4 days after treatment and a significant reduction in V-ATPase-A gene expression. These results indicated that although sid-like genes are lacking, the dsRNA uptake mechanism was very efficient. Also, 2-fold and 4-fold overexpression of DCR-2 and AGO-2, respectively, after dsRNA supply indicated the activation of the siRNA machinery. Consequently, E. heros has proven to be sensitive to RNAi upon injection of dsRNA into its hemocoel. We believe that this finding together with a publically available transcriptome and the validation of a responsive RNAi machinery provide a starting point for future field applications against one of the most important soybean pests in South America.

Interactions of Bacillus thuringiensis strains for Plutella xylostella (L.) (Lepidoptera: Plutellidae) susceptibility.

Bacillus thuringiensis (Bt) biopesticides are an environmentally safe alternative to the management of Plutella xylostella pesticide resistance evolution. We evaluated P. xylostella susceptibility to six Bt strains cultivated and applied individually, and 15 combinations of Bt strains mixed after cultivation. Three combinations resulted in synergism and one in antagonism. Promising results of larval mortality with synergistic effects were obtained with the combinations Bt var. thuringiensis strain HD-2 + Bt finitimus strain HD-3, Bt var. thuringiensis strain HD-2 + Bt dendrolimus strain HD-7 and Bt var. thuringiensis strain HD-2 + Bt var. aizawai strain HD-11.