Chlorbenzuron caused growth arrest through interference of glycolysis and energy metabolism in Hyphantria cunea (Lepidoptera: Erebidae) larvae.

Chlorbenzuron is a kind of benzoylphenylureas (BPUs), which plays a broad role in insect growth regulators (IGRs), with an inhibitory effect on chitin biosynthesis. However, BPUs how to regulate glycolysis and insect growth remains largely unclear. Here, we investigated the effects of chlorbenzuron on growth, nutritional indices, glycolysis, and carbohydrate homeostasis in Hyphantria cunea, a destructive and highly polyphagous forest pest, to elucidate the action mechanism of chlorbenzuron from the perspective of energy metabolism. The results showed that chlorbenzuron dramatically restrained the growth and nutritional indices of H. cunea larvae and resulted in lethality. Meanwhile, we confirmed that chlorbenzuron significantly decreased carbohydrate levels, adenosine triphosphate (ATP), and pyruvic acid (PA) in H. cunea larvae. Further studies indicated that chlorbenzuron caused a significant enhancement in the enzyme activities and mRNA expressions of hexokinase (HK), phosphofructokinase (PFK), and pyruvate kinase (PK), resulting in increased glycolytic flux. Expressions of genes involved in the AMP-activated protein kinase (AMPK) signaling pathway were also upregulated. Moreover, chlorbenzuron had remarkable impacts on H. cunea larvae from the perspective of metabolite enrichment, including the tricarboxylic acid (TCA) cycle and glycolysis, indicating an energy metabolism disorder in larvae. The findings provide a novel insight into the molecular mechanism by which chlorbenzuron abnormally promotes glycolysis and eventually interferes with insect growth and nutritional indices.

Knockdown of GFAT disrupts chitin synthesis in Hyphantria cunea larvae.

Glutamine-fructose-6-phosphate transaminase (GFAT) has been reported to regulate the hexosamine biosynthetic pathway as the first rate-limiting enzyme. As a key enzyme that catalyzes the substrate of glycosylation modification, which has a wide-ranging effect on cellular functions. However, there are few studies on the relationship between GFAT and chitin metabolism in insects. In the present study, the GFAT gene from Hyphantria cunea was identified based on transcriptome and bioinformatic analysis. The role of HcGFAT in regulating development and chitin synthesis was analyzed by RNA interference (RNAi) in H. cunea larvae. The full-length HcGFAT gene (2028 bp) encodes a 676 amino acid (aa) polypeptide had typical structural features of the SIS and Gn_AT_II superfamily. Phylogenetic analyses showed that GFAT of H. cunea shares the highest homology and identity with GFAT of Ostrinia furnacalis. Expression profiles indicated that HcGFAT was expressed throughout larval, pupal and three tissues (midgut, fat body, epidermis), and highly expressed in the last instar of larvae and strongly expressed in epidermis among three tissues. Bioassay results showed that knockdown of HcGFAT repressed larval growth and development, resulting in a significant loss of larval body weight. Meanwhile, HcGFAT knockdown also significantly caused larval developmental deformity. Knockdown of HcGFAT regulated the expression of four other critical genes in the chitin synthesis pathway (HcGNA, HcPAGM, HcUAP, HcCHSA), and ultimately resulted in decreased chitin content in the epidermis. In summary, these findings indicated that GFAT plays a critical role in larval growth and development, as well as chitin synthesis in H. cunea.

E74 knockdown represses larval development and chitin synthesis in Hyphantria cunea.

E74 is a key transcription factor induced by 20E, which plays a broad role in many physiological events during insect growth and development, including vitellogenesis, organ remodeling and new tissue formation, programmed cell death and metamorphosis. However, whether it is involved in regulating insect chitin biosynthesis remains largely unclear. Here, the E74 gene was identified for the first time from Hyphantria cunea, a notorious defoliator of forestry. Thereafter, the role of HcE74 in regulating growth, development and chitin synthesis in H. cunea larvae was evaluated. Bioinformatics analysis showed that HcE74 shared the highest identity (95.53%) with E74A of Spodoptera litura, which belonged to Ets superfamily. The results of RNAi bioassay showed that the larval mortality on 6 d after HcE74 knockdown was up to 51.11 ± 6.94%. Meanwhile, a distinct developmental deformity phenotype was found when HcE74 was silenced. These results indicated that HcE74 plays an important role in the development and molting of H. cunea larvae. Moreover, HcE74 knockdown also significantly decreased the expression of four key genes related to chitin synthesis, including glucose-6-phosphate isomerase (HcG6PI), UDP-N-acetylglucosamine pyrophosphorylase (HcUAP), chitin synthetase A (HcCHSA), and chitin synthetase B (HcCHSB). As a result, the content of chitin in midgut and epidermis decreased by 0.54- and 0.08-fold, respectively. Taken together, these results demonstrated that HcE74 not only plays a critical role in the growth and molting of H. cunea larvae, but also probably participates in the transcriptional regulation of genes involved in chitin biosynthesis.

Metformin-induced AMPK activation suppresses larval growth and molting probably by disrupting 20E synthesis and glycometabolism in fall webworm, Hyphantria cunea Drury.

Metformin, considered to be a potent AMPK activator, is widely used for clinical therapy of cancer and diabetes due to its distinct function in regulating cell energy balance and body metabolism. However, the effect of metformin-induced AMPK activation on the growth and development of insects remains largely unknown. In the present study, we focused on the role of metformin in regulating the growth and development of Hyphantria cunea, a notorious defoliator in the forestry. Firstly, we obtained the complete coding sequences of HcAMPKα2, HcAMPKß1, HcAMPKγ2 from H. cunea, which encoded a protein of 512, 281, and 680 amino acids respectively. Furthermore, the phylogenetic analysis revealed that these three subunits were highly homologous with the AMPK subunits from other lepidopteran species. According to the bioassay, we found metformin remarkably restrained the growth and development of H. cunea larvae, and caused molting delayed and body weight reduced. In addition, expressions of HcAMPKα2, HcAMPKß1, and HcAMPKγ2 were upregulated 3.30-, 5.93- and 5.92-folds at 24 h after treatment, confirming that metformin activated AMPK signaling at the transcriptional level in H. cunea larvae. Conversely, the expressions of two vital Halloween genes (HcCYP306A1 and HcCYP314A1) in the 20E synthesis pathway were remarkably suppressed by metformin. Thus, we presumed that metformin delayed larval molting probably by impeding 20E synthesis in the H. cunea larvae. Finally, we found that metformin accelerated glycogen breakdown, elevated in vivo trehalose level, promoted chitin synthesis, and upregulated transcriptions of the genes in chitin synthesis pathway. Taken together, the findings provide a new insight into the molecular mechanisms by which AMPK regulates carbohydrate metabolism and chitin synthesis in insects.

3-Bromopyruvate-induced glycolysis inhibition impacts larval growth and development and carbohydrate homeostasis in fall webworm, Hyphantria cunea Drury.

As a typical glycolytic inhibitor, 3-bromopyruvate (3-BrPA) has been extensively studied in cancer therapy in recent decades. However, few studies focused on 3-BrPA in regulating the growth and development of insects, and the relationship and regulatory mechanism between glycolysis and chitin biosynthesis remain largely unknown. The Hyphantria cunea, named fall webworm, is a notorious defoliator, which caused a huge economic loss to agriculture and forestry. Here, we investigated the effects of 3-BrPA on the growth and development, glycolysis, carbohydrate homeostasis, as well as chitin synthesis in H. cunea larvae. To elucidate the action mechanism of 3-BrPA on H. cunea will provide a new insight for the control of this pest. The results showed that 3-BrPA dramatically restrained the growth and development of H. cunea larvae and resulted in larval lethality. Meanwhile, we confirmed that 3-BrPA caused a significant decrease in carbohydrate, adenosine triphosphate (ATP), pyruvic acid (PA), and triglyceride (TG) levels by inhibiting glycolysis in H. cunea larvae. Further studies indicated that 3-BrPA significantly affected the activities of hexokinase (HK), phosphofructokinase (PFK), pyruvate kinase (PK), glucose 6-phosphate dehydrogenase (G6PDH) and trehalase, as well as expressions of the genes related to glycolysis, resulting in carbohydrate homeostasis disorder. Moreover, it was found that 3-BrPA enhanced 20-hydroxyecdysone (20E) signaling by upregulating HcCYP306A1 and HcCYP314A1, two critical genes in 20E synthesis pathway, and accelerated chitin synthesis by upregulating transcriptional levels of genes in the chitin synthesis pathway in H. cunea larvae. Taken together, our findings provide a novel insight into the mechanism of glycolytic inhibitor in regulating the growth and development of insects, and lay a foundation for the potential application of glycolytic inhibitors in pest control as well.

Silencing Br-C impairs larval development and chitin synthesis in Lymantria dispar larvae.

In insects, 20-hydroxyecdysone (20E) mediates developmental transitions and regulates molting processes through activation of a series of transcription factors. Broad-Complex (Br-C), a vital gene in the 20E signalling pathway, plays crucial roles during insect growth processes. However, whether Br-C affects chitin synthesis in insects remains unclear. In the present study, the Br-C gene from Lymantria dispar, a notorious defoliator of forestry, was identified based on transcriptome data, and subjected to bioinformatic analysis. The regulatory functions of LdBr-C in chitin synthesis and metabolism in L. dispar larvae were analysed by RNA interference (RNAi). The full-length LdBr-C gene (1431 bp) encodes a 477 amino acid (aa) polypeptide containing a common BRcore region (391 aa) at the N-terminus and a C-terminal Zinc finger domain (56 aa) harbouring two characteristic C2H2 motifs (CXXC and HXXXXH). Phylogenetic analyses showed that LdBr-C shares highest homology and identity with Br-C isoform 7 (83.12%) of Helicoverpa armigera. Expression profiles indicate that LdBr-C was expressed throughout larval and pupal stages, and highly expressed in prepupal and pupal stages. Furthermore, LdBr-C expression was strongly induced by exogenous 20E, and suppressed dramatically after application of dsLdBr-C. Bioassay results showed that knockdown of LdBr-C caused larval developmental deformity, significant weight loss, and a mortality rate of 67.18%. Knockdown of LdBr-C significantly down-regulated transcription levels of eight critical genes (LdTre1, LdTre2, LdG6PI, LdUAP, LdCHS1, LdCHS2, LdTPS and LdCHT) related to chitin synthesis and metabolism, thereby lowering the chitin content in the midgut and epidermis. Our findings demonstrate that Br-C knockdown impairs larval development and chitin synthesis in L. dispar.

Purification, characterization, and sensitivity to pesticides of carboxylesterase from Dendrolimus superans (Lepidoptera: Lasiocampidae).

Through a combination of steps including centrifugation, ammonium sulfate gradient precipitation, sephadex G-25 gel chromatography, diethylaminoethyl cellulose 52 ion-exchange chromatography and hydroxyapatite affinity chromatography, carboxylesterase (CarE, EC3.1.1.1) from sixth instar larch caterpillar moth, Dendrolimus superans (Lepidoptera: Lasiocampidae) larvae was purified and its biochemical properties were compared between crude homogenate and purified CarE. The final purified CarE after hydroxyapatite chromatography had a specific activity of 52.019 µmol/(min·mg protein), 138.348-fold of crude homogenate, and the yield of 2.782%. The molecular weight of the purified CarE was approximately 84.78 kDa by SDS-PAGE. Three pesticides (dichlorvos, lambda-cyhalothrin, and avermectins) showed different inhibition to crude CarE and purified CarE, respectively. In vitro median inhibitory concentration indicated that the sensitivity of CarE (both crude homogenate and final purified CarE) to pesticides was in decreasing order of dichlorvos > avermectins > lambda-cyhalothrin. By the kinetic analysis, the substrates alpha-naphthyl acetate (α-NA) and beta-naphthyl acetate (ß-NA) showed lesser affinity to crude extract than purified CarE. The results also indicated that both crude homogenate and purified CarE had more affinity to α-NA than to ß-NA, and the Kcat and Vmax values of crude extract were lower than purified CarE using α-NA or ß-NA as substrate.