Diapause, a programmed developmental arrest primarily induced by seasonal environmental changes, is very common in the animal kingdom, and found in vertebrates and invertebrates alike. Diapause provides an adaptive advantage to animals, as it increases the odds of surviving adverse conditions. In insects, individuals perceive photoperiodic cues and modify endocrine signaling to direct reproductive diapause traits, such as ovary arrest and increased fat accumulation. However, it remains unclear as to which endocrine factors are involved in this process and how they regulate the onset of reproductive diapause. Here, we found that the long day-mediated drop in the concentration of the steroid hormone ecdysone is essential for the preparation of photoperiodic reproductive diapause in Colaphellus bowringi, an economically important cabbage beetle. The diapause-inducing long-day condition reduced the expression of ecdysone biosynthetic genes, explaining the drop in the titer of 20-hydroxyecdysone (20E, the active form of ecdysone) in female adults. Application of exogenous 20E induced vitellogenesis and ovarian development but reduced fat accumulation in the diapause-destined females. Knocking down the ecdysone receptor (EcR) in females destined for reproduction blocked reproductive development and induced diapause traits. RNA-seq and hormone measurements indicated that 20E stimulates the production of juvenile hormone (JH), a key endocrine factor in reproductive diapause. To verify this, we depleted three ecdysone biosynthetic enzymes via RNAi, which confirmed that 20E is critical for JH biosynthesis and reproductive diapause. Importantly, impairing Met function, a component of the JH intracellular receptor, partially blocked the 20E-regulated reproductive diapause preparation, indicating that 20E regulates reproductive diapause in both JH-dependent and -independent manners. Finally, we found that 20E deficiency decreased ecdysis-triggering hormone signaling and reduced JH production, thereby inducing diapause. Together, these results suggest that 20E signaling is a pivotal regulator that coordinates reproductive plasticity in response to environmental inputs.

Diapause, also known as dormancy, is a state of arrested development that allows insects to survive unfavorable environmental conditions. Diapause-destined insects store large amounts of fat when preparing for diapause. However, the extent to which these accumulated fat reserves influence diapause remains unclear. To address this question, we investigated the function of fatty acid synthase (FAS), which plays a central role in lipid synthesis, in stress tolerance, the duration of diapause preparation, and whether insects enter diapause or not. In diapause-destined adult female cabbage beetles, Colaphellus bowringi, FAS2 was more highly expressed than FAS1 at the peak stage of diapause preparation. FAS2 knockdown suppressed lipid accumulation and subsequently affected stress tolerance genes expression and water content. However, silencing FAS2 had no significant effects on the duration of diapause preparation or the incidence of diapause. FAS2 transcription was suppressed by juvenile hormone (JH) and the JH receptor methoprene-tolerant (Met). These results suggest that the absence of JH-Met induces FAS2 expression, thereby promoting lipid storage in diapause-destined female beetles. These results demonstrate that fat reserves regulate stress tolerance genes expression and water content, but have no significant effect on the duration of diapause preparation or the incidence of diapause.

In addition to the classical nuclear receptor pathway, there is a nongenomic pathway in the cell membrane that regulates gene expression in animal steroid hormone signaling; however, this mechanism is unclear. Here, we report that the insect steroid hormone 20-hydroxyecdysone (20E) regulates calcium influx via phospholipase Cγ1 (PLCG1) to modulate the protein kinase C phosphorylation of the transcription factor ultraspiracle (USP1) in the lepidopteran insect Helicoverpa armigera. The PLCG1 mRNA levels are increased during the molting and metamorphic stages. The depletion of PLCG1 by RNA interference can block 20E-enhanced pupation, cause larvae death and pupation defects, and repress 20E-induced gene expression. 20E may induce the tyrosine phosphorylation of PLCG1 at the cytosolic tyrosine kinase (Src) homology 2 domains and then determine the migration of PLCG1 toward the plasma membrane. The G-protein-coupled receptor (GPCR) inhibitor suramin, Src family kinase inhibitor PP2, and the depletions of ecdysone-responsible GPCR (ErGPCR) and Gαq restrain the 20E-induced tyrosine phosphorylation of PLCG1. PLCG1 participates in the 20E-induced Ca(2+) influx. The inhibition of GPCR, PLC, inositol 1,4,5-trisphosphate receptor, and calcium channels represses the 20E-induced Ca(2+) influx. Through calcium signaling, PLCG1 mediates the transcriptional activation driven by the ecdysone-response element. Through PLCG1 and calcium signaling, 20E regulates PKC phosphorylation of USP1 at Ser-21 to determine its ecdysone-response element binding activity. These results suggest that 20E activates PLCG1 via the ErGPCR and Src family kinases to regulate Ca(2+) influx and PKC phosphorylation of USP1 to subsequently modulate gene transcription for metamorphosis.

The cabbage beetle Colaphellus bowringi Baly is a serious insect pest of crucifers and undergoes reproductive diapause in soil. An understanding of the molecular mechanisms of diapause regulation, insecticide resistance, and other physiological processes is helpful for developing new management strategies for this beetle. However, the lack of genomic information and valid reference genes limits knowledge on the molecular bases of these physiological processes in this species. Using Illumina sequencing, we obtained more than 57 million sequence reads derived from C. bowringi, which were assembled into 39,390 unique sequences. A Clusters of Orthologous Groups classification was obtained for 9,048 of these sequences, covering 25 categories, and 16,951 were assigned to 255 Kyoto Encyclopedia of Genes and Genomes pathways. Eleven candidate reference gene sequences from the transcriptome were then identified through reverse transcriptase polymerase chain reaction. Among these candidate genes, EF1α, ACT1, and RPL19 proved to be the most stable reference genes for different reverse transcriptase quantitative polymerase chain reaction experiments in C. bowringi. Conversely, aTUB and GAPDH were the least stable reference genes. The abundant putative C. bowringi transcript sequences reported enrich the genomic resources of this beetle. Importantly, the larger number of gene sequences and valid reference genes provide a valuable platform for future gene expression studies, especially with regard to exploring the molecular mechanisms of different physiological processes in this species.