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.

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 Wntl were selected as target genes to construct the co-CRISPR strategy using kmo marker.By coinjection 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 Wntl 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.