The specificity of the double-stranded RNA (dsRNA) used in the RNA interference (RNAi) technique is crucial for the success of sequence-specific gene silencing.  Currently, RNAi-mediated insect control is a trending research topic.  However, the off-target effects of the dsRNA in RNAi are a major concern.  In this study, the dsHvβ´COPI (coat protein complex I, β´ subunit)-treated and untreated transcriptomes of the 28-spotted potato lady beetle (Henosepilachna vigintioctopunctata) were compared to understand its off-target gene silencing effects.  The RNA-seq results revealed that 63 and 44 differentially expressed genes (DEGs) were upregulated and downregulated, respectively, in the dsHvβ´COPI treated group as compared with the control.  Validation of the differential expressions of some selected DEGs via reverse transcription-quantitative PCR (RT-qPCR) analysis confirmed the reliability of the transcriptome analysis results.  Further downstream analysis revealed that there were no genes homologous with Hvβ´COPI in H. vigintioctopunctata.  Additionally, no genes with a >11 bp continuous match with dsHvβ´COPI were found in the H. vigintioctopunctata transcriptome.  Six genes (Hvcitron, Hvhelicase, Hvtransposase, Hvserine, Hvdynein, and HvE3 ubiquitin) were selected to examine the off-target activity of dsHvβ´COPI based on their potential involvement in various H. vigintioctopunctata metabolic pathways.  The severity of silencing these six off-target genes was evaluated by employing RNAi.  The RNAi results confirmed the downregulation of the expression of all six genes, although there was no significant lethality.  The findings of this study will be helpful in the risk analysis of future RNAi-mediated pest control experiments.

A recent breakthrough in agricultural biotechnology is the introduction of RNAi-mediated strategies in pest control.  However, the off-target effects of RNAi pest control are still not fully understood.  Here, we studied the off-target effects of two insecticidal siRNAs in both target and non-target insects.  The results revealed that off-target effects of insecticidal siRNAs occur widely in both target and non-target insects.  We classified the expression-changed genes according to their homology to the siRNA-targeted gene, related KEGG pathways with the siRNA-targeted gene and continuous matches with siRNAs.  Surprisingly, the unintended significant changes in gene expression levels did not strictly match with the number of contiguous nucleotides in the siRNAs.  As expected, the expression of small portions of the homologous and KEGG-related genes were significantly changed.  We calculated the Shannon entropy of the transcriptome profile of the insects after injecting them with insecticidal siRNAs.  Though hundreds of genes were affected in their expression levels post siRNA-treatment, the Shannon entropy of the transcriptome remained unchanged, suggesting that the transcriptome expression was balanced.  Our results provide evidence that siRNAs cross-reacted with individual genes in non-target species, but did not have significant effects on the integrity of the transcriptome profiles in either target or non-target species on a genomic scale.  The metric we proposed can be used to estimate the off-target effects of insecticidal siRNAs, which might be useful for evaluating the safety of RNAi in pest control.  

Using linker peptide LP4/2A for multiple gene transformation is considered to be an effective method to stack or pyramid several traits in plants. Bacillus thuringiensis (Bt) cry gene and epsps (5-enolpyruvylshikimate-3-phosphate synthase) gene are two important genes for culturing pest-resistant and glyphosate-tolerant crops. We used linker peptide LP4/2A to connect the Bt cry1Ah gene with the 2mG2-epsps gene and combined the wide-used manA gene as a selective marker to construct one coordinated expression vector called p2EPUHLAGN. The expression vector was transferred into maize by Agrobacterium tumefaciens-mediated transformation, and 60 plants were obtained, 40% of which were positive transformants. Molecular detection demonstrated that the two genes in the fusion vector were expressed simultaneously and spliced correctly in translation processing; meanwhile bioassay detection proved the transgenic maize had preferable pest resistance and glyphosate tolerance. Therefore, linker peptide LP4/2A provided a simple and reliable strategy for producing gene stacking in maize and the result showed that the fusion gene transformation system of LP4/2A was feasible in monocot plants.

Conogethes punctiferalis (Guenée) (Lepidoptera: Crambidae) was originally considered as one species with fruit-feeding type (FFT) and pinaceae-feeding type (PFT), but it has subsequently been divided into two different species of Conogethes punctiferalis and Conogethes pinicolalis. The relationship between the two species was investigated by phylogenetic reconstruction using maximum-likelihood (ML) parameter estimations. The phylogenetic tree and network were constructed based upon sequence data from concatenation of three genes of mitochondrial cytochrome c oxidase subunits I, II and cytochrome b which were derived from 118 samples of C. punctiferalis and 24 samples of C. pinicolalis. The phylogenetic tree and network showed that conspecific sequences were clustering together despite intraspecific variability. Here we report the results of a combined analysis of mitochondrial DNA sequences from three genes and morphological data representing powerful evidence that C. pinicolalis and C. punctiferalis are significantly different.

Domain I of the activated Crystal protein from Bacillus thuringiensis has a seven α-helix bundle structure, which is responsible for membrane channel formation in its insecticidal mechanism. Cry1Ie is toxic to Asian corn borer, Ostrinia furnacalis (Guenée), and plays important roles in insect biological control. The domain I from Cry1Ie has been expressed and purified in its normal conformation, as embedded in the full length homologous toxin structure. The membrane insertion ability of this single domain was compared with the full length homologous toxin using a monolayer insertion experiment. The results indicated that the Cry1Ie-domain I had the ability to insert into the lipid monolayer, and this ability is greater than that of the IE648 toxin. However, the state of insertion is not stable and remains for only a short period of time. The Cry1Ie-domain I plays no role in receptor binding as it had a nonspecific binding with the brush border membrane vesicles of the Asian corn borer.

A novel insecticidal gene cry1Ah was cloned from Bacillus thuringiensis isolate BT8 previously for plant genetic engineering improvement. Truncated active Cry1Ah toxin has a toxicity level similar to that of the full-length Cry1Ah toxin. In this study, plant expression vector pMhGM harboring truncated cry1Ah gene was transformed into maize (Zea mays L.) immature embryos by Agrobacterium tumefaciens-mediated transformation at which maize alcohol dehydrogenase matrix attachment regions (madMARs) were incorporated on both sides of the gene expression cassette to improve gene expression. A total of 23 PCR positive events were obtained with a transformation efficiency of 5% around. Bioassay results showed that events 1-4 and 1-5 exhibited enhanced resistance to the Asian corn borer (Ostrinia furnacalis). These two events were further confirmed by molecular analysis. Southern blot suggested that a single copy of the cry1Ah gene was successfully integrated into the maize genome. Western blot and ELISA showed that the foreign gene cry1Ah was expressed stably at high level in maize and could be inherited stably over generations. The results of a bioassay of T1-T4 transgenic maize plants indicated that the transgenic plants were highly toxic to the Asian corn borer and their resistance could be inherited stably from generation to generation. Thus, events 1-4 and 1-5 are good candidates for the breeding of insect-resistant maize.

Wolbachia are maternally inherited endosymbiotic bacteria infecting a wide range of arthropods and filarial nematodes. They can induce various reproduction alterations in their hosts, including thelytokous parthenogenesis, cytoplasmic incompatibility (CI), feminization of genetic males and male killing. Here we investigated diversity and prevalence patterns of Wolbachia infection in 43 geographical populations of the Asian corn borer, Ostrinia furnacalis, in China and one population in North Korea. Based on Wolbachia surface protein gene (wsp) sequences, nine strains of Wolbachia (wFur1-wFur9), belonging to supergroups A and B, were identified in populations of O. furnacalis with an average infection rate of 10.5%. Superinfection commonly appeared in individuals of O. furnacalis and coinfection patterns were very complex. There was no specific pattern for the prevalence and distribution of the nine Wolbachia strains suggesting an intricate evolutionary history of Wolbachia infection in this species. The genetic similarity of the wFur1-wFur9 strains with those detected in two parasitoids of O. furnacalis, Macrocentrus cingulum and Lydella grisescens, strongly suggests host-parasitoid horizontal transmission.