Wei-Hua Xu

Identification of a novel gene encoding the trehalose phosphate synthase in the cotton bollworm, Helicoverpa armigera

Trehalose and trehalose metabolism are crucial for insect development. We measured the content of polyhydric compounds in the hemolymph of diapause- and nondiapause-destined individuals of Helicoverpa armigera. We found that the trehalose content is much higher in diapause-destined individuals than that in nondiapause individuals. The activity of trehalose-6-phosphate synthase (TPS) during H. armigera larval-pupal development is significantly higher in diapause-type individuals and is closely correlated with the changes in the trehalose content. The cDNA encoding TPS, which converts uridine-5-diphosphoglucose and glucose-6-phosphate to trehalose-6-phosphate, was cloned from the fat body of H. armigera using rapid amplification of cDNA ends (RACE). The molecular characterization of the cDNA revealed that the mRNA encodes a precursor polypeptide of 826-amino-acid residues, containing Har-TPS at residues 6-507 and a putative trehalose-6-phosphate phosphatase, which converts trehalose-6-phosphate into free trehalose, at residues 512-783. The Har-TPS precursor polypeptide shows 73% identity with that of Drosophila melanogaster. The presence of a 2.8 kb transcript in the fat body and ovary was detected with a northern blot. The Har-TPS mRNA was detected at high levels in the late stage of sixth larval instar and the early middle stage of diapause-destined pupae, which are most likely to respond the changes in TPS activity and trehalose in the hemolymph. The Har-TPS protein was successfully overexpressed in the Bombyx mori baculovirus expression system, and the catalytic activity of Har-TPS was found to be approximately 5-fold higher in B. mori blood infected by the recombined-baculovirus than the control. When diapause is broken, the trehalose content drops significantly and glucose increases rapidly. These results suggest that trehalose is involved in regulating H. armigera pupal diapause.

Wnt/β-catenin signaling regulates Helicoverpa armigera pupal development by up-regulating c-Myc and AP-4

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.

Transcription factor fork head regulates the promoter of diapause hormone gene in the cotton bollworm, Helicoverpa armigera, and the modification of SUMOylation.

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.xa0armigera 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.xa0armigera.

HIF-1 regulates insect lifespan extension by inhibiting c-Myc-TFAM signaling and mitochondrial biogenesis.

Diapause (developmental arrest) is characterized by dramatic depression of metabolic activity and profoundly extends insect lifespan, similar to the Caenorhabditis elegans dauer stage and Drosophila longevity; however, the molecular mechanism of low metabolism in insect diapause is unclear. Here, we show that HIF-1α expression is significantly increased in diapause-destined pupal brains compared to nondiapause-destined pupal brains and that HIF-1α negatively regulates mitochondrial biogenesis. HIF-1α mediates this effect by inhibiting c-Myc activity via proteasome-dependent degradation of c-Myc. The mitochondrial transcription factor A (TFAM), which encodes a key factor involved in mitochondrial transcription and mitochondrial DNA replication, is activated by the binding of c-Myc to the TFAM promoter, thereby inducing transcription. Loss of TFAM expression is a major factor contributing to reducing the mitochondrial activity. Thus, the HIF-1α-c-Myc-TFAM signaling pathway participates in the regulation of mitochondrial activity for insect diapause or lifespan extension.

Analysis of four achaete-scute homologs in Bombyx mori reveals new viewpoints of the evolution and functions of this gene family

Backgroundachaete-scute complexe (AS-C) has been widely studied at genetic, developmental and evolutional levels. Genes of this family encode proteins containing a highly conserved bHLH domain, which take part in the regulation of the development of central nervous system and peripheral nervous system. Many AS-C homologs have been isolated from various vertebrates and invertebrates. Also, AS-C genes are duplicated during the evolution of Diptera. Functions besides neural development controlling have also been found in Drosophila AS-C genes.ResultsWe cloned four achaete-scute homologs (ASH) from the lepidopteran model organism Bombyx mori, including three proneural genes and one neural precursor gene. Proteins encoded by them contained the characteristic bHLH domain and the three proneural ones were also found to have the C-terminal conserved motif. These genes regulated promoter activity through the Class A E-boxes in vitro. Though both Bm-ASH and Drosophila AS-C have four members, they are not in one by one corresponding relationships. Results of RT-PCR and real-time PCR showed that Bm-ASH genes were expressed in different larval tissues, and had well-regulated expressional profiles during the development of embryo and wing/wing disc.ConclusionThere are four achaete-scute homologs in Bombyx mori, the second insect having four AS-C genes so far, and these genes have multiple functions in silkworm life cycle. AS-C gene duplication in insects occurs after or parallel to, but not before the taxonomic order formation during evolution.

Homology of dipteran bristles and lepidopteran scales: requirement for the Bombyx mori achaete-scute homologue ASH2.

Lepidopteran wing scales and Drosophila bristles are considered homologous structures on the basis of the similarities in their cell lineages. However, the molecular mechanisms underlying scale development are essentially unknown as analysis of gene function in Lepidoptera is sorely limited. In this study, we used the Bombyx mori mutant scaleless (sl), which displays a nearly complete loss of wing scales, to explore the mechanism of lepidopteran wing-scale formation. We found that Bm-ASH2, one of four Bombyx achaete-scute homologs, is highly expressed in early pupal wings of wild-type silkworms, but its expression is severely reduced in sl pupal wings. Through molecular characterization of the mutant locus using luciferase and gel shift assays, genetic analysis of recombining populations, and in vivo rescue experiments, we provide evidence that a 26-bp deletion within the Bm-ASH2 promoter is closely linked to the sl locus and leads to loss of Bm-ASH2 expression and the scaleless-wings phenotype. Thus, the Bm-ASH2 appears to play a critical role in scale formation in B. mori. This finding supports the proposed homology of lepidopteran scales and dipteran bristles and provides evidence for conservation of the genetic pathway in scale/bristle development at the level of gene function.

Cathepsin L participates in the remodeling of the midgut through dissociation of midgut cells and activation of apoptosis via caspase-1

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.

TGF-β signaling regulates p-Akt levels via PP2A during diapause entry in the cotton bollworm, Helicoverpa armigera

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.

PTEN expression responds to transcription factor POU and regulates p-AKT levels during diapause initiation in the cotton bollworm, Helicoverpa armigera

Diapause is a complex physiological response accompanied by many signaling pathways participating in the process. Previous studies have shown that p-AKT levels in brains of diapause-destined pupae are elevated by ROS, and the activated AKT promotes Glut expression for glucose uptake during diapause entry in Helicoverpa armigera. However, the mechanism by which ROS activate AKT is still unclear. Here, we show that PTEN, a PI3K/p-AKT signaling inhibitor, was significantly lower in the brains of diapause-destined pupae and that p-AKT levels were elevated by a lack of PTEN dephosphorylating PIP3. In addition, POU was identified as a transcription factor that binds to the PTEN promoter and regulates its expression. POU expression was enhanced by ecdysone but suppressed by ROS, suggesting that POU/PTEN plays a central role in responding to ROS signaling and regulating p-AKT levels. These results suggest that ecdysone and ROS participate together in the regulation of insect diapause through downregulation of POU/PTEN, which elevates p-AKT levels.

TGF-β and BMP signals regulate insect diapause through Smad1-POU-TFAM pathway

The transforming growth factor-β (TGF-β) superfamily signaling pathway contains two general branches, known as TGF-β and bone morphogenetic protein (BMP), that regulate development in animals. It is well known that TGF-β superfamily signaling participates in the regulation of dauer (lifespan extension) in Caenorhabditis elegans, but little is known about the molecular mechanisms of lifespan extension in the pathway. Diapause, a programmed developmental arrest in insects, is similar to dauer in C. elegans. In this study, we find that TGF-β superfamily signaling regulates Helicoverpa armigera diapause via a novel mechanism. Both TGF-β and BMP signals are weaker in the brains of diapause-destined pupae than in nondiapause-destined pupae, and the levels of p-Smad1, POU, TFAM, and mitochondrial activity are decreased in diapause pupae. Development in nondiapause pupae is delayed by an injection of TGF-β or BMP receptor inhibitors. Both TGF-β and BMP signals can activate a common target, Smad1. ChIP and EMSA assays indicate that Smad1 can bind to the POU promoter to regulate its expression. POU can improve the transcription of TFAM, which regulates mitochondrial activity. This is the first report showing that both TGF-β and BMP signals regulate development or diapause through the Smad1-POU-TFAM-mitochondrial activity in insects.