An OBP gene highly expressed in non-chemosensory tissues affects the phototaxis and reproduction of Spodoptera frugiperda.

Insect odorant binding proteins (OBPs) were initially regarded as carriers of the odorants involved in chemosensation. However, it had been observed that a growing number of OBP genes exhibited broad expression patterns beyond chemosensory tissues. Here, an OBP gene (OBP31) was found to be highly expressed in the larval ventral nerve cord, adult brain and male reproductive organ of Spodoptera frugiperda. An OBP31 knockout strain (OBP31-/- ) was generated by CRISPR/Cas9 mutagenesis. For OBP31-/- , the larvae needed longer time to pupate, but there was no difference in the pupal weight between OBP31-/- and wild type (WT). OBP31-/- larvae showed stronger phototaxis than the WT larvae, indicating the importance of OBP31 in light perception. For mating rhythm of adults, OBP31-/- moths displayed an earlier second mating peak. In the cross-pairing of OBP31-/- and WT moths, the mating duration was longer, and hatchability was lower in OBP31-/- group and OBP31+/- ♂ group than that in the WT group. These results suggested that OBP31 played a vital role in larval light perception and male reproductive process and could provide valuable insights into understanding the biological functions of OBPs that were not specific in chemosensory tissues.

A nonsensory odorant-binding protein plays an important role in the larval development and adult mating of Spodoptera frugiperda.

Odorant-binding proteins (OBPs) play key roles in the perception of semiochemicals in insects. Several OBPs in insect olfactory systems have been functionally characterized, and they provide excellent targets for pest control. The functions of some OBPs that are highly expressed in the nonsensory organs of insects remain unclear. Here, the physiological function of an OBP (OBP27) that was highly expressed in the nonsensory organs of Spodoptera frugiperda was studied. OBP27 was nested within the Plus-C cluster according to phylogenetic analysis. The transcription of OBP27 steadily increased throughout the development of S. frugiperda, and transcripts of this gene were abundant in the fat body and male reproductive organs. An OBP27 knockout strain with an early frameshift mutation was obtained using the clustered regularly interspaced palindromic repeats (CRISPR) / CRISPR-associated protein 9 (Cas9) system. The development time of OBP27-/- larvae was significantly longer than that of other larvae. Both male and female OBP27-/- pupae weighed significantly less than wild-type (WT) pupae. In crosses of OBP27-/- males or females, the mating rate was lower and the mating duration was longer for OBP27-/- male-WT female pairs than for WT-WT pairs. By contrast, the mating rate, hatching rate, and number of eggs of OBP27-/- female-WT male pairs and WT-WT pairs were similar. These findings indicate that OBP27 plays an important role in the larval development and mating process in male adults. Generally, our findings provide new insights into the physiological roles of nonsensory OBPs.

Plasticity of the Gene Transcriptional Level and Microbiota in the Gut Contributes to the Adaptability of the Fall Armyworm to Rice Plants.

Insects coordinate a variety of mechanisms to overcome the feeding challenges, including gene transcriptional plasticity and stable symbioses in the gut. Here, Spodoptera frugiperda larvae were reared on corn and rice plants for successive generations to obtain two specific strains. The rice strain displayed a longer developmental period, lower female fecundity, and intrinsic growth rate at G1 and G5 but not at G10. KEGG analysis of the G1, G5, and G11 gut transcriptome indicated that detoxification enzymes might play vital roles in host adaptation. RNAi-mediated knockdown of CYP12A2 and UGT41B8, which were highly expressed in the gut of the rice strain, significantly reduced the larval adaptability to rice. Besides, the dsCYP12A2-treated larvae displayed an increased sensitivity to luteolin, a flavonoid phytochemical. The KEGG function prediction of gut microbiota indicated that the high enrichment level of metabolism in the rice strain would play essential roles in rice adaptation.

Co-CRISPR: A valuable toolkit for mutation enrichment in the gene editing of Spodoptera frugiperda.

The CRISPR/Cas9 system has been successfully applied in dozens of diverse species; although the screening of successful CRISPR/Cas9 editing events remains particularly laborious, especially for those that occur at relatively low frequency. Recently, a co-CRISPR strategy was proved to enrich the desired CRISPR events. Here, the co-CRISPR strategy was developed in the Fall armyworm, Spodoptera frugiperda, with kynurenine 3-monooxygenase gene (kmo) as a marker. The kmo mosaics induced by single-guide RNAs (sgRNAs)/Cas9 displayed the darker green color phenotype in larvae, compared with wild type (brown), and mosaic-eye adults were significantly acquired from the mosaic larvae group. In the kmo knockout strain, no significant difference was observed in larval development and adult reproduction. Acetylcholinesterase 2 (ace2) and Wnt1 were selected as target genes to construct the co-CRISPR strategy using kmo marker. By co-injection of kmo and ace2 sgRNAs, the mutant efficiency of ace2 was significantly increased in the kmo mosaic (larvae or adults) groups. Similarly, more malformed pupae with Wnt1 mutations were observed in the darker green larvae group. Taken together, these results demonstrated that kmo was a suitable visible marker gene for the application and extension of co-CRISPR strategy in Fall armyworm. Using darker green color in larvae or mosaic-eye in adults from kmo knockout as a marker, the mutant efficiency of a target gene could be enriched in a Fall armyworm group consisting of marked individuals. The co-CRISPR strategy is helpful for gene function studies by the knockout technique with no or lethal phenotypes.