Knockout of ABC Transporter ABCG4 Gene Confers Resistance to Cry1 Proteins in Ostrinia furnacalis

Ostrinia furnacalis is an important borer on maize. Long-term and large-scale planting of transgenic corn has led O. furnacalis evolving resistance and reducing the control effect. Recently, high levels of resistance to Bt Cry1 toxins have been reported to be genetically linked to the mutation or down-regulation of ABC transporter subfamily G gene ABCG4 in O. furnacalis. In order to further determine the relationship between ABCG4 gene and the resistance to Cry1 toxins in O. furnacalis, the novel CRISPR/Cas9 genome engineering system was utilized to successfully construct ABCG4-KO knockout homozygous strain. Bioassay results indicated that an ABCG4-KO strain had a higher resistance to Cry1 proteins compared with a susceptible strain (ACB-BtS). The result indicates that the ABCG4 gene may act as a receptor of the Bt Cry1 toxin in O. furnacalis. Furthermore, the development time was significantly changed in the early stage ABCG4-KO larvae, and the population parameters were also significantly changed. In summary, our CRISPR/Cas9-mediated genome editing study presents evidence that ABCG4 gene is a functional receptor for Bt Cry1 toxins, laying the foundation for further clarification of the Bt resistance mechanism.

Trichogramma ostriniae Is More Effective Than Trichogramma dendrolimi As a Biocontrol Agent of the Asian Corn Borer, Ostrinia furnacalis

The Asian corn borer (ACB), Ostrinia furnicalis, is a serious corn pest in south-east Asia, causing huge economic losses every year. Trichogramma dendrolimi and Trichogramma ostriniae, two egg parasitoids, have previously been identified as key biological control agents. To determine the age impact of ACB eggs on their effective biocontrol potential, herein we compared the biological parameters (i.e., number of parasitized eggs, emergence, developmental time, and sex ratio) of both parasitoids on ACB eggs of various ages (i.e., 0–4, 4–8, 8–12, 12–16, 16–24, 24–36, and 36–48 h old), respectively. Our results showed that the age of ACB eggs had a significant impact on the parasitization activity of T. dendrolimi in both choice and no-choice conditions. Trichogramma dendrolimi preferred to parasitize 0–8-h-old ACB eggs, and its parasitization dramatically declined on ACB eggs older than 8 h under choice and no-choice conditions. On the other hand, T. ostriniae showed high preference to parasitize all tested ACB egg ages. The age of ACB eggs had no significant impact on the parasitization of T. ostriniae under choice and no-choice conditions. Furthermore, the female progeny of T. dendrolimi decreased as the age of ACB increased, while no differences were found in female progeny of T. ostriniae. Trichogramma ostriniae also developed faster on each ACB egg age group in comparison with T. dendrolimi. Overall, the age of ACB eggs had a significant impact on T. dendrolimi performance, leading us to conclude that T. ostriniae is more effective than T. dendrolimi as a biocontrol agent of the ACB.

Alteration of a Cry1A Shared Binding Site in a Cry1Ab-Selected Colony of Ostrinia furnacalis

The Asian corn borer, Ostrinia furnacalis (Guenée, 1854), is a highly damaging pest in Asia and the Pacific islands, and larvae feed mainly from corn crops. To determine the suitability of Bt-corn technology for the future control of this pest, understanding the potential to develop resistance to Cry1Ab and the basis of cross-resistance to other Cry1 proteins is of great interest. Here, we have explored the binding of Cry1A proteins to brush border membrane vesicles from two O. furnacalis colonies, one susceptible (ACB-BtS) and one laboratory-selected with Cry1Ab (ACB-AbR). The insects developed resistance to Cry1Ab and showed cross-resistance to Cry1Aa, Cry1Ac, and Cry1F. Binding assays with radiolabeled Cry1Ab and brush border membrane vesicles from susceptible insects showed that Cry1A proteins shared binding sites, though the results were not conclusive for Cry1F. The results were confirmed using radiolabeled Cry1Aa. The resistant insects showed a reduction of the specific binding of both Cry1Ab and Cry1Aa, suggesting that part of the binding sites were lost or altered. Competition binding assays showed full competition between Cry1Ab and Cry1Aa proteins in the susceptible colony but only partial competition in resistant insects, confirming the alteration of some, but not all, binding sites for these two proteins. The binding site model for Cry1A proteins in O. furnacalis is in agreement with the occurrence of multiple membrane receptors for these proteins.

Genome-Wide Analysis of WRKY Gene Family and the Dynamic Responses of Key WRKY Genes Involved in Ostrinia furnacalis Attack in Zea mays

WRKY transcription factors comprise one of the largest gene families and serve as key regulators of plant defenses against herbivore attack. However, studies related to the roles of WRKY genes in response to herbivory are limited in maize. In this study, a total of 128 putative maize WRKY genes (ZmWRKYs) were identified from the new maize genome (v4). These genes were divided into seven subgroups (groups I, IIa–e, and III) based on phylogenomic analysis, with distinct motif compositions in each subgroup. Syntenic analysis revealed that 72 (56.3%) of the genes were derived from either segmental or tandem duplication events (69 and 3, respectively), suggesting a pivotal role of segmental duplication in the expansion of the ZmWRKY family. Importantly, transcriptional regulation prediction showed that six key WRKY genes contribute to four major defense-related pathways: L-phenylalanine biosynthesis II and flavonoid, benzoxazinoid, and jasmonic acid (JA) biosynthesis. These key WRKY genes were strongly induced in commercial maize (Jingke968) infested with the Asian corn borer, Ostrinia furnacalis, for 0, 2, 4, 12 and 24 h in the field, and their expression levels were highly correlated with predicted target genes, suggesting that these genes have important functions in the response to O. furnacalis. Our results provide a comprehensive understanding of the WRKY gene family based on the new assembly of the maize genome and lay the foundation for further studies into functional characteristics of ZmWRKY genes in commercial maize defenses against O. furnacalis in the field.

Transcriptome Analysis of Ostrinia furnacalis Female Pheromone Gland: Esters Biosynthesis and Requirement for Mating Success

Female moths use sex pheromones to attract males, and corresponding regulatory mechanism underlying sex pheromone biosynthesis is species-dependent. However, the detailed mechanism involved in sex pheromone biosynthesis in Ostrinia furnacalis has not yet been fully addressed. In the present study, transcriptome sequencing of O. furnacalis pheromone glands screened a serials of candidate genes involved in sex pheromone biosynthesis. Our analysis showed that sex pheromone release in O. furnacalis females arrives its peak at the 2nd scotophase, consistent with its mating behavior. Pheromone biosynthesis-activating neuropeptide (PBAN) was confirmed to regulate sex pheromone biosynthesis, and Ca2+ is the secondary messenger of PBAN signaling in O. furnacalis. The functional analysis of candidate genes demonstrated that the decreased mRNA levels or activities of calcineurin (CaN) and acetyl-CoA carboxylase (ACC) led to significant decrease in sex pheromone production and female capability to attract males, as demonstrated by RNAi-mediated knockdown and pharmacological inhibitor assay. Most importantly, the activities of CaN and ACC depend on the activation of PBAN/PBANR/Ca2+. Furthermore, fatty-acyl reductase 14 was involved in PBAN-mediated sex pheromone biosynthesis. Altogether, our results demonstrated that PBAN regulates sex pheromone biosynthesis through PBANR/Ca2+/CaN/ACC pathway to promote sex pheromone biosynthesis in O. furnacalis and provided a reference for non-model organism to study neuropeptide signal transduction.

Cadherin Protein Is Involved in the Action of Bacillus thuringiensis Cry1Ac Toxin in Ostrinia furnacalis

Transgenic crops expressing Bacillus thuringiensis (Bt) insecticidal proteins have been extensively planted for insect pest control, but the evolution of Bt resistance in target pests threatens the sustainability of this approach. Mutations of cadherin in the midgut brush border membrane was associated with Cry1Ac resistance in several lepidoptera species, including the Asian corn borer, Ostrinia furnacalis, a major pest of maize in Asian–Western Pacific countries. However, the causality of O. furnacalis cadherin (OfCad) with Cry1Ac resistance remains to be clarified. In this study, in vitro and in vivo approaches were employed to examine the involvement of OfCad in mediating Cry1Ac toxicity. Sf9 cells transfected with OfCad showed significant immunofluorescent binding with Cry1Ac toxin and exhibited a concentration-dependent mortality effect when exposed to Cry1Ac. The OfCad knockout strain OfCad-KO, bearing homozygous 15.4 kb deletion of the OfCad gene generated by CRISPR/Cas9 mutagenesis, exhibited moderate-level resistance to Cry1Ac (14-fold) and low-level resistance to Cry1Aa (4.6-fold), but no significant changes in susceptibility to Cry1Ab and Cry1Fa, compared with the original NJ-S strain. The Cry1Ac resistance phenotype was inherited as autosomal, recessive mode, and significantly linked with the OfCad knockout in the OfCad-KO strain. These results demonstrate that the OfCad protein is a functional receptor for Cry1Ac, and disruption of OfCad confers a moderate Cry1Ac resistance in O. furnacalis. This study provides new insights into the mode of action of the Cry1Ac toxin and useful information for designing resistance monitoring and management strategies for O. furnacalis.