The vast majority of lepidopterans, about 90%, are moths. Some moths, particularly their caterpillars, are major agricultural and forestry pests in many parts of the world. However, some other members of moths, such as the silkworm Bombyx mori, are famous for their economic value. Fire et al. in 1998 initially found that exogenous double-stranded RNA (dsRNA) can silence the homolog endogenous mRNA in organisms, which is called RNA interference (RNAi). Soon after, the RNAi technique proved to be very promising not only in gene function determination but also in pest control. However, later studies demonstrate that performing RNAi in moths is not as straightforward as shown in other insect taxa. Nevertheless, since 2007, especially after 2010, an increasing number of reports have been published that describe successful RNAi experiments in different moth species either on gene function analysis or on pest management exploration. So far, more than 100 peer-reviewed papers have reported successful RNAi experiments in moths, covering 10 families and 25 species. By using classic and novel dsRNA delivery methods, these studies effectively silence the expression of various target genes and determine their function in larval development, reproduction, immunology, resistance against chemicals, and other biological processes. In addition, a number of laboratory and field trials have demonstrated that RNAi is also a potential strategy for moth pest management. In this review, therefore, we summarize and discuss the mechanisms and applications of the RNAi technique in moths by focusing on recent progresses.

The neuropeptide natalisin (NTL) has been determined to play essential roles in reproduction in two Diptera and one Coleoptera species. Whether NTL has similar or even different functions in Lepidoptera remains to be determined. Here, we cloned the NTL transcript in the common cutworm moth Spodoptera litura. This transcript encodes a 438-amino acid protein. Twelve putative Sl-NTL neuropeptides were defined by cleavage sites. These NTL peptides share a DDPFWxxRamide C-terminal motif. The expressions of Sl-NTL is low during the egg and larval stages, which increased to a higher level during the pupal stage, and then reached the maximum during the adult stage. Moreover, the expression pattern during the pupal stage is similar between sexes while during the adult stage, it is dimorphic. To explore the function of Sl-NTL and assess its potential as a target for pest control, we knocked down the expression of Sl-NTL in both sexes by using bacteria-mediated RNAi. This technique significantly down regulated (reduced up to 83%) the expression of Sl-NTL in both sexes. Knocking down Sl-NTL expression did not significantly affect its development, survival and morphology but significantly reduced adults’ reproductive behavior (including female calling, male courtship, mating and remating patterns and rates) and reproductive output (offspring gain reduced more than 70%).

Investigation of mating-induced trade-offs between reproduction and survival is conducive to provide evolutionary insights into reproductive strategies and aging. Here, we used RNAseq and bioinformatics to reveal mating-induced changes of genes and pathways related to reproduction and survival in female Cephalcia chuxiongica, a pine defoliator with facultative parthenogenesis and long larval dormancy. Results showed that mating induced substantial downregulation on genes and pathways associated to immunity, stress response, and longevity. However, mating induced divergent reproductive response, with downregulation on genes and pathways related to egg production while upregulation on genes and pathways related to egg fertilization. Considering the nature of limited resources in adults, low fecundity, and egg protection behavior in C. chuxiongica, we suggest that mating triggers trade-offs between reproduction and survival in this insect and females of this species may have evolved specific strategies to adapt to the environmental and hosts’ conditions, e.g., restrict whole fecundity to ensure higher fertilization and offspring’s survival. Moreover, mating induced significant responses on genes and pathways that play important roles in vertebrate reproduction while their function in insects are unclear, such as the progesterone-mediated oocyte maturation pathway; the significant regulation after mating suggests that their function may be evolutionarily conserved in animal kingdom.

It is recognized that the behavioral rhythms of organisms are controlled by the circadian clock, while the reverse direction, i.e., whether changes in physiology and behavior react to the internal rhythms, is unclear. Cryptochromes (CRYs) are photolyase-like flavoproteins with blue-light receptor function and other functions on circadian clock and migration in animals. Here, we cloned the full-length cDNA of CRY1 and CRY2 in Spodoptera litura (Fabricius, 1775) (Lepidoptera: Noctuidae). Sl-CRYs show high similarity to orthologs from other insects, and their conserved regions contain a DNA photolyase domain and a FAD-binding seven domain. The expression levels of both genes were relatively low during the larval stage, which increased during the pupal stage and then peaked at the adult stage. The expression of Sl-CRY1 and Sl-CRY2 showed differences between males and females and between scotophase and photophase. Further, our study demonstrated that copulation has a significant effect on the expression of Sl-CRYs. More interestingly, the changes in the expression of Sl-CRY1 and Sl-CRY2 due to copulation showed the same trend in both sexes, in which the expression levels of both genes in copulated males and females decreased in the subsequent scotophase after copulation and then increased significantly in the following photophase. Considering the nature of the dramatic changes in reproductive behavior and physiology after copulation in S. litura, we propose that the changes in the expression of Sl-CRYs after copulation could have some function in the reproductive process.

Recent studies on polyandry evolution have revolutionized our understanding of sexual selection. Therefore, study on traits in females, such as sensory perception and remating strategies, is expected to disclose a greater role of sexual selection in female evolution. Here, we tested how bodyweights of both sexes affected female and male remating behavior in Spodoptera litura (Fabricius, 1775) (Lepidoptera: Noctuidae). Results show most (74.4%; unmated virgin females may be sexually immature) females and males mated the first time in the second scotophase after eclosion. In the subsequent scotophase, 41.9% once-mated females and 94.1% once-mated males mated the second time with a novel virgin mate. Analyses indicate that female remating probability significantly increased with the increase of her own and the second male’s bodyweight but decreased with the increase of the first male’s bodyweight, while bodyweights of both sexes have no significant effect on male remating probability. Considering the nature of the low level of polyandry (two times on average) and last male precedence in S. litura, we propose that 1) females are more choosy when remating, from which females will benefit indirectly by sequential mate comparison and remate to replace sperm from the previous mates with those from higher quality mates, and 2) males will generally like to remate with any possible females, from which males will gain direct benefit by siring more offspring. The outcome of polyandry rate is likely to be the result of male investment × female choice interactions.

Moths are the most widely studied example of pheromones in animals. However, little is known about the mechanism of intrasexual and mating-related intersexual regulation of pheromone production and release in female moths. Our previous studies inSpodoptera liturafound that mating induced a higher sex pheromone titre in the pheromone gland (PG) and mating or male accessory gland (MAG) extract suppressed female calling behavior. We therefore hypothesize that the inhibition of female calling behavior by mating or MAG factors likely suppresses the release of sex pheromones and thus results in a higher pheromone titre in the PG. To test this hypothesis, in the present study, we introduced an artificial calling behavior suppression treatment by gently knocking on and shaking the testing boxes contained moths once every 10 minutes. Results show that this treatment significantly increased pheromone titres in virgin or saline injected virgin females, and the increase rates are similar to those of mating and MAG extract treated ones. These results have suggested that the increase of sex pheromone titer in the female PG after mating inS. liturais due to the inhibition of female calling behavior by MAG factors. Moreover, results of this study also suggest that female calling behavior is positively correlated to pheromone release and likewise, the calling behavior and sex pheromone release inS. liturafemales are directly under the neural control, and modulated by molecular and environmental factors.