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.

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.

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 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.

Involvement of GOBP2 in the perception of a sex pheromone component in both larval and adult Spodoptera litura revealed using CRISPR/Cas9 mutagenesis.

General odorant-binding proteins (GOBPs) are long considered responsible for the perception of plant odorants. In this study with the important noctuid pest Spodoptera litura, we functionally characterized that GOBP2 is also involved in the perception of sex pheromone components using in vivo CRISPR/Cas9 technique. First, the GOBP2 sgRNA and Cas9 protein were injected into the newly laid insect eggs, resulting in a 35.6% target mutagenesis in G0 moths. Then, the homozygous GOBP2 knockout strain (GOBP2-/-) was obtained after the screening of three generations. The knockout male and female moths displayed a significant reduction in EAG responses to the sex pheromone components, and the knockout females also displayed a significant reduction to plant odorants. In the behavioral assay of food choice, GOBP2-/- larvae lost the preference to artificial diet added with the major sex pheromone component Z9, E11-tetradecadienyl acetate (Z9, E11-14:Ac), whereas the WT larvae highly preferred the pheromone diet. Y-tube olfactometer assay and direct pheromone stimulation assay showed that GOBP2-/- male adults reduced significantly than WT males in percentages of choice, hair pencil displaying and mating attempt to Z9, E11-14:Ac. In the oviposition test, GOBP2-/- females showed significantly reduced preference for the soybean plants compared to the WT females. Our study demonstrated that GOBP2 plays an important role in perceiving sex pheromones in adult and larval stages, providing new insight into sex pheromone perception and a potential target for sex pheromone-based behavioral regulation in the pest.

The Molecular Basis of Host Selection in a Crucifer-Specialized Moth.

Glucosinolates (GSs) are sulfur-containing secondary metabolites characteristic of cruciferous plants [1, 2]. Their breakdown products, isothiocyanates (ITCs), are released following tissue disruption by insect feeding or other mechanical damages [3, 4]. ITCs repel and are toxic to generalist herbivores, while specialist herbivores utilize the volatile ITCs as key signals for localizing host plants [5, 6]. However, the molecular mechanisms underlying detection of ITCs remain open. Here, we report that in the diamondback moth Plutella xylostella, a crucifer specialist, ITCs indeed drive the host preference for Arabidopsis thaliana, and the two olfactory receptors Or35 and Or49 are essential for this behavior. By performing gene expression analyses, we identified 12 (out of 59 in total) female-biased Ors, suggesting their possible involvement in oviposition choice. By ectopically expressing these Ors in Xenopus oocytes and screening their responses with 49 odors (including 13 ITCs, 25 general plant volatiles, and 11 sex pheromone components), we found that Or35 and Or49 responded specifically to three ITCs (iberverin, 4-pentenyl ITC, and phenylethyl ITC). The same ITCs also exhibited highest activity in electroantennogram recordings with female antennae and were the strongest oviposition stimulants. Knocking out either Or35 or Or49 via CRISPR-Cas9 resulted in a reduced oviposition preference for the ITCs, while double Or knockout females lost their ITC preference completely and were unable to choose between wild-type A. thaliana and a conspecific ITC knockout plant. We hence conclude that the ITC-based oviposition preference of the diamondback moth for its host A. thaliana is governed by the cooperation of two highly specific olfactory receptors.

Functional Characterization of Sex Pheromone Receptors in the Fall Armyworm (Spodoptera frugiperda).

Pheromone receptors (PRs) found in the antennae of male moths play a vital role in the recognition of sex pheromones released by females. The fall armyworm (FAW), Spodoptera frugiperda, is a notorious invasive pest, but its PRs have not been reported. In this report, six candidate PRs (SfruOR6, 11, 13, 16, 56 and 62) suggested by phylogenetic analysis were cloned, and their tissue-sex expression profiles were determined by quantitative real-time PCR (qPCR). All six genes except for SfruOR6 were highly and specifically expressed in the antennae, with SfruOR6, 13 and 62 being male-specific, while the other three (SfruOR11, 16 and 56) were male biased, suggesting their roles in sex pheromone perception. A functional analysis by the Xenopus oocyte system further demonstrated that SfruOR13 was highly sensitive to the major sex pheromone component Z9-14:OAc and the pheromone analog Z9,E12-14:OAc, but less sensitive to the minor pheromone component Z9-12:OAc; SfruOR16 responded weakly to pheromone component Z9-14:OAc, but strongly to pheromone analog Z9-14:OH; the other four candidate PRs did not respond to any of the four pheromone components and four pheromone analogs. This study contributes to clarifying the pheromone perception in the FAW, and provides potential gene targets for developing OR-based pest control techniques.

Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-Associated Protein 9 Mediated Knockout Reveals Functions of the yellow-y Gene in Spodoptera litura.

Yellow genes are thought to be involved in the melanin biosynthetic pathway and play a crucial role in pigmentation reactions in insects. However, little research has been done on yellow genes in lepidopteran pests. To clarify the function of one of the yellow genes (yellow-y) in Spodoptera litura, we cloned the full-length of yellow-y, and investigated its spatial and temporal expression profiles by quantitative real-time PCR (qPCR). It revealed that yellow-y was highly expressed in larva of fourth, fifth, and sixth instars, as well as in epidermis (Ep), fat bodies (FB), Malpighian tubes (MT), and midguts (MG) of the larvae; whereas it was expressed in very low levels in different tissues of adults, and was almost undetected in pupa. This expression profile suggests an important role of yellow-y in larvae, minor role in adults, and no role in pupae. To confirm this, we disrupted yellow-y using the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system, and obtained G0 insects with mutation in yellow-y. The mutation in yellow-y clearly rendered the larvae body, a color yellower than that of wide type insects, and in addition, the mutation resulted in abnormal segmentation and molting for older larvae. The mutation of yellow-y also made various adult tissues (antennae, proboscis, legs, and wings) yellowish. However, the mutation had no effect on pigmentation of the pupal cuticle. Taken together, our study clearly demonstrated the role of yellow-y not only in the body pigmentation of larvae and adults, and but also in segmentation and molting of larvae, providing new insights into the physiology of larval development, as well as a useful marker gene for genome editing based studies.