The intermediate process between mating and postmating behavioral changes in insects is still poorly known. Here, we studied mating-induced common and sex-specific behavioral and transcriptional changes in both sexes of Spodoptera frugiperda and tested whether the transcriptional changes are linked to postmating behavioral changes in each sex. A behavioral study showed that mating caused a temporary suppression of female calling and male courting behavior, and females did not lay eggs until the next day after the first mating. The significant differences on daily fecundity under the presence of males or not, and the same or novel males, suggest that females may intentionally retain eggs to be fertilized by novel males or to be fertilized competitively by different males. RNA sequencing in females revealed that there are more reproduction related GO (gene ontology) terms and KEGG (Kyoto encyclopedia of genes and genomes) pathways (mainly related to egg and zygote development) enriched to upregulated DEGs (differentially expressed genes) than to downregulated DEGs at 0 and 24 h postmating. In males, however, mating induced DEGs did not enrich any reproduction related terms/pathways, which may be because male reproductive bioinformatics is relatively limited in moths. Mating also induced upregulation on soma maintenance (such as immune activity and stress reaction) related processes in females at 0, 6 and 24 h postmating. In males, mating also induced upregulation on soma maintenance related processes at 0 h postmating, but induced downregulation on these processes at 6 and 24 h postmating. In conclusion, this study demonstrated that mating induced sex-specific postmating behavioral and transcriptional changes in both sexes of S. frugiperda and suggested that the transcriptional changes are correlated with postmating physiological and behavioral changes in each sex.

Mating promotes reproductive activity, which may impact immune performance. Paradoxically, mating frequently challenges females’ immunity (e.g., infections). Therefore, studies of postmating resource allocation between reproduction and survival are likely to shed new light on life-history trade-off and sexual selection. Here, we used RNAseq to test whether and how mating affected mRNA expression in genes related to reproduction and immunity in Spodoptera litura female moths. Results show a divergent change in the differentially expressed genes (DEGs) between reproduction and immunity: the immune response was largely downregulated shortly after mating (~6 h postmating), which has some recovery at 24 h postmating; reproductive response is trivial shortly after mating (~6 h postmating), but it largely upregulated at 24 h postmating (e.g., egg maturation related genes were highly upregulated). Considering the fact that most of the total DEGs downregulated from 0 to 6 h postmating (from 51/68 to 214/260) but most of the total DEGs upregulated at 24 h postmating (816/928), it is possible that trade-offs between reproduction and immunity occurred in mated females. For example, they may shut down immunity to favor sperm storage and save limited resources to support the increased energy required in reproduction (e.g., egg maturation and oviposition). Mating-induced infections should be trivial due to low polyandry in S. litura. A reduced immune defense may have no threat to S. litura survival but may benefit reproduction significantly. Furthermore, obvious expression changes were detected in genes related to hormone production, suggesting that endocrine changes could play important roles in postmating responses.

Mating may promote microbial diversity through sexual transmission, while mating-induced immune responses may decrease it. Therefore, the study of mating-induced microbiomes changes under different mating systems is informative to unravel its biological relevance and evolutionary significance. Here, we studied the microbiomes in a community context within the abdomen of Spodoptera frugiperda females using 16S rDNA sequences by setting virgin females, and females mated once, twice, or thrice with the same or different males. Alpha and beta diversities revealed that mating significantly affected the composition of microbiomes in S. frugiperda females, wherein virgin females have the highest diversity, followed by one-time mated females and females mated with multiple males, while females mated repeatedly with the same male showed the lowest diversity. The low diversity in females mated repeatedly with the same male may be due to lower sexual transmission as only mated with one mate and higher immune response from repeated matings. Functional prediction by FAPROTAX and literature searching found 17 possible pathogens and 12 beneficial microbiomes. Multiple mating turned over the abundance of pathogens and beneficial microbes, for example, Enterococcus and Lactobacillus spp. (beneficial) showed higher abundance in virgin females while Morganella and Serratia spp. (pathogens) showed higher abundance in females mated with multiple males. These results suggest that mating causes a decline in the diversity of symbiotic microbiomes and promiscuity incurs a higher pathogen abundance in S. frugiperda females, which may be the result of sexual transmission of bacterial strains and immune responses targeting members of the microbiomes. To our knowledge, we demonstrate microbiomes changes in female insects under virgin and different mating regimes for the first time.

Of all the things that can fly, the flight mechanisms of insects are possibly the least understood. By using RNAseq, we studied the aging-associated gene expression changes in the thorax of Spodoptera frugiperda females. Three possible flight energy metabolism pathways were constructed based on 32 key metabolic enzymes found in S. frugiperda. Differential expression analysis revealed up to 2000 DEGs within old females versus young ones. Expression and GO and KEGG enrichment analyses indicated that most genes and pathways related to energy metabolism and other biological processes, such as transport, redox, longevity and signaling pathway, were downregulated with aging. However, activity assay showed that the activities of all the five tested key enzymes increased with age. The age-associated transcriptional decrease and activity increase in these enzymes suggest that these enzymes are stable. S. frugiperda is a long-distance migrator, and a high activity of enzymes may be important to guarantee a high flight capacity. The activity ratio of GAPDH/HOAD ranged from 0.594 to 0.412, suggesting that lipid is the main fuel of this species, particularly in old individuals. Moreover, the expression of enzymes in the proline oxidation pathway increased with age, suggesting that this energy metabolic pathway also is important for this species or linked to some aging-specific processes. In addition, the expression of immunity- and repair-related genes also increased with age. This study established the overall transcriptome framework of the flight muscle and aging-associated expression change trajectories in an insect for the first time.