Diapause is a state of arrested growth, which allows insects to adapt to diverse environments. Serine protease inhibitors (serpins) play an important role in various physiological processes, including blood coagulation, fibrinolysis, development, complement activation and extracellular matrix remodeling. We hypothesized that serpin may affect energy metabolism and thereby control diapause of migratory locust (Locusta migratoria) embryos by regulating protease cascades. A total of seven nonredundant serpin genes (named serpin1-serpin7) of L. migratoria were obtained through transcriptomic analysis. We further performed label-free proteomic sequencing and analysis of diapause and nondiapause eggs of L. migratoria, revealing significant differences in serpin7 expression. A significant reduction in diapause rate under the short photoperiod was observed in insects treated with serpin7 double-stranded RNA. Furthermore, knockdown of the serpin7 gene resulted in significant upregulation of the activity of polyphenol oxidase. We therefore propose that the observed serpin7 gene plays a crucial role in diapause, suggesting that control of energy metabolism may have potential as a future strategy for the reproductive control of insect pests.

Diapause hormone (DH) neuropeptides in insects are produced by the genes belonging to pban/capa family. Previous studies show that DH contains a conserved sequence of WFGPRXa that plays vital role in diapause regulation of some Lepidopteran species. However, the function of DH in other species is still unknown. In order to expand our understanding of DH function in diapause induction, Lom-pban, Lom-capa, and five candidates DH precursor genes (Lom-dh1, Lom-dh2, Lom-dh3, Lom-dh4, Lom-dh5) of Locusta migratoria L. were subsequently cloned. We identified Lom-dh1 to Lom-dh5 as novel genes that encoded five types (type I-V) of 44 tandem repeats of DH-like neuropeptides, which might promote egg diapause of L. migratoria. To test this hypothesis, we identified four types of eight new neuropeptides encoded by Lom-dh using liquid chromatography-tandem mass spectrometry from the central neuron system of L. migratoria under both short (10:14 L:D) and long (16:8 L:D) photoperiods. Later on, we synthesized four type I DH-like neuropeptides, LDH1, SDH1, LDH2, and SDH2, encoded by Lom-dh2/Lom-dh3 and injected them into fifth instar female locusts. Egg diapause incidences were observed after female oviposition. The four DH-like neuropeptides significantly increased the incidence of egg diapause under the short photoperiod, but the response was absent under the long photoperiod. Injection of dsLom-dh into female adults of L. migratoria under the short photoperiod could inhibit egg diapause, with no response under the long photoperiod. This study identified a new member of pban/capa family being the second example beside Bombyx mori, where the DH showed significant role on maternal induction of diapause.

Photoperiod is one of the most important maternal factors with an impact on the offspring diapause induction of Locusta migratoria. Previous studies have shown that forkhead box protein O (FOXO) plays an important role in regulating insect diapause, but how photoperiod stimulates maternal migratory locusts to regulate the next generation of egg diapause through the FOXO signaling pathway still needs to be addressed. In this study, the transcriptomes of ovaries and fat bodies of adult locusts under a long and short photoperiod were obtained. Among the total of 137 differentially expressed genes (DEGs) in both ovaries and fat bodies, 71 DEGs involved in FOXO signaling pathways might be closely related to diapause induction. 24 key DEGs were selected and their expression profiles were confirmed to be consistent with the transcriptome results using qRT-PCR. RNA interference was then performed to verify the function of retinoic acid induced protein gene (rai1) and foxo. Egg diapause rates were significantly increased by RNAi maternal locusts rai1 gene under short photoperiods. However, the egg diapause rates were significantly decreased by knock down of the foxo gene in the maternal locusts under a short photoperiod. In addition, reactive oxygen species (ROS) and superoxide dismutase (SOD) activities were promoted by RNAi rai1. We identified the candidate genes related to the FOXO pathway, and verified the diapause regulation function of rai1 and foxo under a short photoperiod only. In the future, the researchers can work in the area to explore other factors and genes that can promote diapause induction under a long photoperiod.

Egg diapause in Locusta migratoria L. (Orthoptera: Acridoidea) is believed to be influenced by maternal photoperiod. However, the molecular mechanism regulating the phenomenon of maternal diapause induction is unclear. Here we performed transcriptomic analyses from the central nervous system (CNS) of migratory locusts under long and short photoperiods to identify differentially expressed genes (DEGs) related to diapause induction. There were total of 165750 unigenes from 569491 transcripts, and 610 DEGs were obtained in S_CNS (CNS of short photoperiod treated locusts) vs. L_CNS (CNS of long photoperiod treated locusts). Of these, 360 were up-regulated, 250 were down-regulated, and 84 DEGs were found to be related to FOXO signaling pathways, including citrate cycle/TCA cycle, glycolysis/ gluconeogenesis, oxidative phosphorylation, and PI3K-Akt. The qRT-PCR validation of mRNA expression of 12 randomly selected DEGs showed consistency with transcriptome analysis. Furthermore, the takeout gene thought to be involved in circadian rhythm was cloned and used for RNAi to observe its function in maternal diapause induction. We found that the mRNA level of Lm-takeout was significantly lower in dstakeout treatments as compared to the control under both long and short photoperiods. Similarly, the offspring diapause rate was significantly higher in dstakeout treatment as compared to the control only in short photoperiod. This shows that the Lm-takeout gene might be involved in the inhibition of maternal diapause induction of L. migratoria under short photoperiods. The present study provides extensive data of the CNS transcriptome and particular insights into the molecular mechanisms of maternal effects on egg diapause of L. migratoria As well for the future, the researchers can explore other factors and genes that may promote diapause in insect species.

FMRFamide-related peptides (FaRPs) are a type of neuropeptide, which participate in a variety of physiological processes in insects. Previous study showed that myosuppressin, being a member of FaRPs, initiated pupal diapause in Mamestra brassicae. We presumed that FaRPs genes might play a critical role in photoperiodic diapause induction of L. migratoria. To verify our hypothesis, flrf, a precursor gene of FaRP from L. migratoria, was initially cloned under long and short photoperiods that encoded by flrf gene identified from central nervous system (CNS). Phylogenetic analysis showed that the protein encoded by L. migratoria flrf gene, clustered together with Nilaparvata lugens (Hemiptera: Delphacidae) with 100% bootstrap support, was basically an FMRFamide precursor homologue. We noticed the availability of -RFamide peptides (GSERNFLRFa, DRNFIRFa) under short photoperiod only, which suggested their functions related to photoperiodic diapause induction. RNAi and quantitative real-time PCR (qRT-PCR) results further confirmed that the flrf gene promoted locust's diapause.

Low temperature induces diapause in locusts. However, the physiological processes and initiation mechanism of diapause are not well understood. To understand the molecular basis of diapause, 'omics' analyses were performed to examine the differences between diapause and non-diapause eggs at both transcriptional and translational levels. Results indicated that a total of 62,241 mRNAs and 212 proteins were differentially expressed. Among them, 116 transcripts had concurrent transcription and translation profiles. Up-regulated genes related to diapause included glutathiones-S-transferase et al., and down-regulated genes including juvenile hormone esterase-like protein et al. KEGG analysis mapped 7,243 and 99 differentially expressed genes and proteins, to 83 and 25 pathways, respectively. Correlation enriched pathways indicated that there were nine identical pathways related to diapause. Gene Ontology analysis placed these genes and proteins into three categories, and a higher proportion of genes related to metabolism was up-regulated than down-regulated. Furthermore, three up-regulated pathways were linked to cryoprotection. This study demonstrates the applicability of high-throughput omics tools to identify molecules linked to diapause in the locust. In addition, it reveals cellular metabolism in diapause eggs is more active than in non-diapause eggs, and up-regulated enzymes may play roles in cryoprotection and storing energy for diapause and post-diapause stages.