Diapause is a developmental arrest that allows an organism to survive unfavorable environmental conditions and is induced by environmental signals at a certain sensitive developmental stage. In Helicoverpa armigera, the larval brain receives the environmental signals for diapause induction and then regulates diapause entry at the pupal stage. Here, combined proteomic and metabolomic differential display analysis was performed on the H. armigera larval brain. Using two-dimensional electrophoresis, it was found that 22 proteins were increased and 27 proteins were decreased in the diapause-destined larval brain, 37 of which were successfully identified by MALDI-TOF/TOF mass spectrometry. RT-PCR and Western blot analyses showed that the expression levels of the differentially expressed proteins were consistent with the 2-DE results. Furthermore, a total of 49 metabolites were identified in the larval brain by GC–MS analysis, including 4 metabolites at high concentrations and 14 metabolites at low concentrations. The results gave us a clue to understand the governing molecular events of the prediapause phase. Those differences that exist in the induction phase of diapause-destined individuals are probably relevant to a special memory mechanism for photoperiodic information storage, and those differences that exist in the preparation phase are likely to regulate accumulation of specific energy reserves in diapause-destined individuals.Keywords: brain; Helicoverpa armigera; metabolome; prediapause phase; proteome;

•TGF-β signaling regulates insect diapause via the insulin pathway.•PP2A-B′ dephosphorylates Akt during insect development.•TGF-β signaling negatively regulates p-Akt levels by elevating PP2A activity.Akt, which is a key kinase in the insulin signaling pathway, plays important roles in glucose metabolism, cell proliferation, transcription and cell migration. Our previous studies indicated that low insulin levels and high p-Akt levels are present in diapause-destined individuals. Here, we show that PI3K, which is upstream of Akt, is low in diapause-destined pupal brains but high in p-Akt levels, implying that p-Akt is modified by factors other than the insulin signaling pathway. Protein phosphatase 2A (PP2A), which is a key regulator in the TGF-β signaling pathway, can directly bind to and dephosphorylate Akt. Low PP2A expression and activity in diapause-destined individuals suggest that a weak Akt dephosphorylation contributes to p-Akt accumulation. In addition, transforming growth factor-β receptor I (TβRI), which is upstream of PP2A, increases the activity of PP2A and decreases the p-Akt levels. These results show that TGF-β signaling decreases p-Akt levels by increasing the activity of PP2A. This is the first report showing that TGF-β signaling negatively regulates the insulin pathway in insect development or diapause.Download high-res image (178KB)Download full-size image

Developmental arrest, a critical component of the life cycle in animals as diverse as nematodes (dauer state), insects (diapause), and vertebrates (hibernation), results in dramatic depression of the metabolic rate and a profound extension in longevity. Although many details of the hormonal systems controlling developmental arrest are well-known, we know little about the interactions between metabolic events and the hormones controlling the arrested state. Here, we show that diapause is regulated by an interplay between blood-borne metabolites and regulatory centers within the brain. Gene expression in the fat body, the insect equivalent of the liver, is strongly suppressed during diapause, resulting in low levels of tricarboxylic acid (TCA) intermediates circulating within the blood, and at diapause termination, the fat body becomes activated, releasing an abundance of TCA intermediates that act on the brain to stimulate synthesis of regulatory peptides that prompt production of the insect growth hormone ecdysone. This model is supported by our success in breaking diapause by injecting a mixture of TCA intermediates and upstream metabolites. The results underscore the importance of cross-talk between the brain and fat body as a regulator of diapause and suggest that the TCA cycle may be a checkpoint for regulating different forms of animal dormancy.

Diapause is a period of developmental arrest that allows a species to adapt to unfavorable conditions. Many insect species reduce metabolic activity and then enter diapause at a certain stage in their life cycles. The cotton bollworm, Helicoverpa armigera, will be destined for pupal diapause when larvae are reared under short daylengths and low temperature. The brain is an important organ for diapause decision, and some signaling molecules from the brain of diapause-destined individuals are released into the hemolymph to regulate diapause. In this study, we performed 2-D gel-based comparative proteomic and phosphoproteomic analyses to search for differentially expressed proteins between nondiapause- and diapause-destined pupal brains. A total of 79 proteins and 23 phosphoproteins showed significant differences between these two groups, and 41 proteins and 10 phosphoproteins were identified by MALDI-TOF/TOF MS. Further, gene expression patterns in diapause- and nondiapause-destined pupal brains were confirmed by RT-PCR or Western blot analysis. These differentially expressed proteins act in the metabolic change, stress response, and signal transduction pathways at early pupal stage for diapause initiation. Thus, these identified proteins may depress metabolism in diapause-destined pupae to lead the insect to enter developmental arrest.

Prothoracicotropic hormone (PTTH), a neuropeptide hormone stimulating the prothoracic glands to synthesize ecdysone, plays an important role in regulating postembryonic development in insects. The cDNA encoding PTTH was isolated and sequenced from the beet armyworm, Spodoptera exigua (Spe). The deduced amino acid sequence is composed of a signal peptide, a peptide (65 amino acids) of unknown function, and a mature PTTH molecule (111 amino acids). The Spe-PTTH shows similarities (45.5%–70.3%) to other known PTTHs reported in Lepidoptera species, but 7 cysteine residues and the hydrophobic regions were conserved. Whole-mount immunocytochemistry by using an antiserum against recombinant Helicoverpa armigera PTTH showed that Spe-PTTH was synthesized in two pairs of neurosecretory cells in the S. exigua brain. Northern blot analysis demonstrates the presence of a 1.2-kb transcript in the brain. The Spe-PTTH mRNA is detectable at high levels at the wandering larval stage, early pupal stage, and pharate adult stage, suggesting that the Spe-PTTH gene might be correlated with molting, metamorphosis, and reproduction.

Biosynthesis and secretion of prothoracicotropic hormone (PTTH) of diapause- and nondiapause-destined individuals in Helicoverpa armigera were studied using whole-mount immunocytochemistry and enzyme-linked immunosorbent assay (ELISA). The immunocytochemistry revealed that PTTH is expressed in two pairs of lateral neurosecretory cells of the brain. The presence of immunoreactivity has not significant difference between the brains of the diapause- and nondiapause-destined 6th instar larvae. However, the obvious differences of expressional pattern from day 4 pupae were observed between the two types. PTTH titers in hemolymph from the 6th instar larvae to pharate adults were measured by the ELISA. Although there were similar titer changes between the two types of individuals at the larval stage, a significant difference from developmental expression was detected at the pupal stage, suggesting that the expression and secretion of PTTH does play a crucial role in regulation of pupal diapause of H. armigera.

The transcription factor fork head (FoxA) plays important roles in development and metabolism. Here, we cloned a fork head gene in Helicoverpa armigera, and found that the fork head protein is mainly located in the nucleus. This fork head gene belongs to the FoxA subfamily of the Fox transcription factors. The diapause hormone and pheromone biosynthesis-activating neuropeptide (DH-PBAN), which are two well-documented insect neuropeptides that regulate insect development and pheromone biosynthesis, are encoded by a single mRNA. In the present study, fork head was shown to bind strongly to the promoter of H. armigera DH-PBAN gene, and regulate its promoter activity. Furthermore, the effect of SUMOylation of the FH protein on the regulation of Har-DH-PBAN gene was investigated, and we show that the SUMO can modify Har-FH protein and cause down-regulation of DH-PBAN gene expression. These results suggest that SUMOylated FH plays a key role in insect diapause in H. armigera.Download high-res image (81KB)Download full-size imageHighlights► We cloned the fork head gene in Helicoverpa armigera. ► Fork head could bind to the promoter of DH-PBAN. ► Fork head promotes the transcription of DH-PBAN. ► Fork head could be modified by SUMO on 289 Lysine. ► SUMOylation of fork head represses its transcriptional activity.

•Wnt-β-catenin signals are activated in non-diapause-destined pupal brains.•The blockage of Wnt/β-catenin signaling delays pupal development.•Har-c-Myc binds to the promoter of Har-Ap-4 to enhance its expression.•Ecdysone can regulate the Wnt/β-catenin pathway.Seasonally changing environmental conditions perceived by insect brains can be converted into hormonal signals that prompt insects to make a decision to develop or enter developmental arrest (diapause). Diapause is a complex physiological response, and many signaling pathways may participate in its regulation. However, little is known about these regulatory pathways. In this study, we cloned four genes related to the Wnt/β-catenin signaling pathway from Helicoverpa armigera, a pupal diapause species. Western blotting shows that expression of Har-Wnt1, Har-β-catenin, and Har-c-Myc are higher in non-diapause pupal brains than in diapause-destined brains. Har-Wnt1 can promote the accumulation of Har-β-catenin in the nucleus, and Har-β-catenin in turn increases the expression of Har-c-Myc. The blockage of Wnt/β-catenin signaling by the inhibitor XAV939 significantly down-regulates Har-β-catenin and Har-c-Myc expression and delays pupal development, suggesting that the Wnt/β-catenin pathway functions in insect development. Furthermore, Har-c-Myc binds to the promoter of Har-AP-4 and regulates its expression. It has been reported that Har-AP-4 activates diapause hormone (DH) expression and that DH up-regulates the growth hormone ecdysteroid for pupal development. Thus, pupal development is regulated by Wnt/β-catenin signaling through the pathway Wnt-β-catenin-c-Myc-AP-4-DH-ecdysteroid. In contrast, the down-regulation of Wnt/β-catenin signaling is likely to induce insects to enter diapause.Download high-res image (147KB)Download full-size image

•Cathepsins play key roles in the remodeling of the midgut.•Cathepsin L functions in the dissociation of the midgut.•Caspase-1 is activated by cathepsin L to induce apoptosis in the midgut.•Ecdysone is an upstream signal to induce cathepsin L expression.The larval midgut in holometabolous insects must undergo a remodeling process during metamorphosis to form the pupal-adult midgut. However, the molecular mechanism of larval midgut cell dissociation remains unknown. Here, we show that the expression and activity of Helicoverpa armigera cathepsin L (Har-CatL) are high in the midgut at the mid-late stage of the 6th-instar larvae and are responsive to the upstream hormone ecdysone. Immunocytochemistry shows that signals for Har-CatL-like are localized in midgut cells, and an inhibitor experiment demonstrates that Har-CatL functions in the dissociation of midgut epithelial cells. Mechanistically, Har-CatL can cleave pro-caspase-1 into the mature peptide, thereby increasing the activity of caspase-1, which plays a key role in apoptosis, indicating that Har-CatL is also involved in the apoptosis of midgut cells by activating caspase-1. We believe that this is the first report that Har-CatL regulates the dissociation and apoptosis of the larval midgut epithelium for midgut remodeling.Download high-res image (207KB)Download full-size image