FDP-Na-induced enhancement of glycolysis impacts larval growth and development and chitin biosynthesis in fall webworm, Hyphantria cunea (Lepidoptera: Arctiidae).

Fructose 1, 6-diphosphate (FDP) is an endogenous intermediate in the glycolytic pathway, as well as an allosteric activator of phosphofructokinase (PFK). Based on the role in promoting glycolysis, FDP has been widely used as a therapeutic agent for mitigating the damage of endotoxemia and ischemia/reperfusion in clinical practice. However, the effect of exogenous FDP-induced glycolysis activation on insect carbohydrate metabolism and chitin synthesis remains largely unclear. Here, we investigated for the first time the effects of FDP-Na, an allosteric activator of PFK, on the growth and development of Hyphantria cunea larvae, a serious defoliator in agriculture and forestry, especially on glycolysis and chitin synthesis. The results showed that FDP-Na significantly restrained the growth and development of H. cunea larvae and resulted in larval lethality. After treatment with FDP-Na, hexokinase (HK), phosphofructokinase (PFK) and pyruvate kinase (PK) were significantly activated, and HcHK2, HcPFK, HcPK were dramatically upregulated, which suggested that FDP-Na enhanced glycolysis in H. cunea larvae. Meanwhile, FDP-Na also distinctly impacted chitin biosynthesis by disturbing transcriptions of genes in the chitin synthesis pathway, resulting in changes of chitin contents in the midgut and epidermis of H. cunea larvae. Therefore, we considered that FDP-Na caused the growth and development arrest, and impacted chitin biosynthesis, probably by disturbing in vivo glycolysis and carbohydrate metabolism in H. cunea larvae. The findings provide a new perspective on the mechanism by which glycolysis regulates insect growth and development, and lay the foundation for exploring the potential application of glycolysis activators in pest control as well.

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.

Steroid hormone 20-hydroxyecdysone disturbs fat body lipid metabolism and negatively regulates gluconeogenesis in Hyphantria cunea larvae.

The steroid hormone 20-hydroxyecdysone (20E) has been described to regulate fat body lipid metabolism in insects, but its accurate regulatory mechanism, especially the crosstalk between 20E-induced lipid metabolism and gluconeogenesis remains largely unclear. Here, we specially investigated the effect of 20E on lipid metabolism and gluconeogenesis in the fat body of Hyphantria cunea larvae, a notorious pest in forestry. Lipidomics analysis showed that a total of 1 907 lipid species were identified in the fat body of H. cunea larvae assigned to 6 groups and 48 lipid classes. The differentially abundant lipids analysis showed a significant difference between 20E-treated and control samples, indicating that 20E caused a remarkable alteration of lipidomics profiles in the fat body of H. cunea larvae. Further studies demonstrated that 20E accelerated fatty acid ß-oxidation, inhibited lipid synthesis, and promoted lipolysis. Meanwhile, the activities of pyruvate carboxylase, phosphoenolpyruvate carboxykinase, fructose-1,6-bisphosphatase, and glucose-6-phosphatase were dramatically suppressed by 20E in the fat body of H. cunea larvae. As well, the transcriptions of genes encoding these 4 rate-limiting gluconeogenic enzymes were significantly downregulated in the fat body of H. cunea larvae after treatment with 20E. Taken together, our results revealed that 20E disturbed fat body lipid homeostasis, accelerated fatty acid ß-oxidation and promoted lipolysis, but negatively regulated gluconeogenesis in H. cunea larvae. The findings might provide a new insight into hormonal regulation of glucose and lipid metabolism in insect fat body.

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.

Fenoxycarb and methoxyfenozide (RH-2485) affected development and chitin synthesis through disturbing glycometabolism in Lymantria dispar larvae.

Fenoxycarb as a juvenile hormone analogue and methoxyfenozide (RH-2485) as a 20-hydroxyecdysone (20E) agonist are two main insect growth regulators (IGRs) used for pest control, whose insecticidal mechanisms had been widely reported in past decades. However, there were few studies focused on their effects on the carbohydrate metabolism of insects. Here, we reported that two IGRs (fenoxycarb and RH-2485) significantly affected growth and development of L. dispar larvae and caused larval lethality. Furthermore, both contens of three sugars (glycogen, threhalose, glucose) in four tissues (fat body, midgut, hemolymph and epidermis) and trehalase activity in three tissues (fat body, midgut and hemolymph) of L. dispar larvae were markedly affected by these two IGRs. Moreover, we found that mRNA expression levels of LdTPS, LdTre1 and LdTre2 in L. dispar larvae were dramatically suppressed by two IGRs. Additionally, chitin content in both midgut and epidermis decreased significantly after L. dispar larvae treated with fenoxycarb or RH-2485. Summarily, these results indicated that these two IGRs disturbed glycometabolism in L. dispar larvae, resulting in impeding chitin synthesis, generating new epidermis failure, disrupting molting and larval lethality in the end.

Sanguinarine in Chelidonium majus induced antifeeding and larval lethality by suppressing food intake and digestive enzymes in Lymantria dispar.

Our previous studies had identified that both crude extracts and total alkaloid from Chelidonium majus exerted a significant antifeeding and larval lethality on Lymantria dispar. Moreover, sanguinarine, chelidonine, berberine hydrochloride and coptisine were the main alkaloid in C. majus exerting toxicity to L. dispar. In this paper, we evaluated the insecticidal and antifeeding activities of each alkaloid on the 3rd instar L. dispar larvae by bioassay. Meanwhile, the effects of alkaloids from C. majus on the activities and mRNA levels of three main digestive enzymes in L. dispar larvae were investigated. The results indicated that sanguinarine possessed the strongest insecticidal activity with a LD50 value of 4.963 µg/larva, and the coptisine showed little lethality to 3 rd instar L. dispar larvae among four alkaloids from C. majus. The insecticidal capacity of four alkaloids on 3rd instar L. dispar larvae was in the following decreasing order of sanguinarine > chelidonine > berberine hydrochloride > coptisine. Similarly, except coptisine, the other three alkaloids significantly reduced food intakes of third instar L. dispar larvae and suppressed activities of three digestive enzymes (α-amylase, lipase and total protease) simultaneously. Finally, qRT-PCR analysis revealed that the transcriptions of α-amylase, lipase and serine protease were affected by sanguinarine. Especially, at 48 h after treatment, the mRNA expressions of those digestive enzymes were significantly suppressed by sanguinarine. In conclusion, we suggested that alkaloids from C. majus induced antifeeding and larval lethality on L. dispar larvae by suppressing food intake and digestive enzymes in L. dispar. Our findings provide a novel insight into evaluating the antifeeding and insecticidal properties of C. majus, which afford a new strategy for integrated pest management programs as well.

Cucurbitacin B acts a potential insect growth regulator by antagonizing 20-hydroxyecdysone activity.

BACKGROUND: 20-Hydroxyecdysone (20E), a crucial insect steroid hormone, can bind to its cognate nuclear receptor composed of ecdysone receptor (EcR) and ultraspiracle (USP) to activate expression of 20E-response genes, enabling subsequent metamorphosis. In this study, we tried to find out which steroid-like compounds can block insect metamorphosis effectively and provide useful information for biopesticide study. For this purpose, we screened 126 steroid-like compounds for possible 20E antagonists using a dual-luciferase reporter assay with Drosophila melanogaster Kc and Bombyx mori Bm12 cells. RESULTS: Among 126 steroid-like compounds, three cucurbitacins (CucB, D and E) were identified as 20E antagonists in both Kc and Bm12 cells. Notably, CucB caused significant molting defects and mortality in both B. mori and D. melanogaster larvae, and dramatically hindered larval growth of Helicoverpa armigera by its anti-feeding activity. CONCLUSION: In vivo and in vitro experiments demonstrate that CucB acts as a potential insect growth regulator by antagonizing 20E activity and thus blocking molting and metamorphosis induced by 20E signaling. © 2017 Society of Chemical Industry.

Larvicidal activity and insecticidal mechanism of Chelidonium majus on Lymantria dispar.

Based on the broad spectrum of its biological activities, Chelidonium majus has been studied extensively in the medical field. However, few studies have focused on the insecticidal activity of C. majus, and the precise mechanism of its insecticidal activity. In the present study, larvicidal activity and insecticidal mechanism of C. majus on Lymantria dispar were investigated using bioassays, in vitro and in vivo enzyme activity assays, determination of the nutritional index, and gene transcription analysis. The results showed that alkaloids are the main insecticidal ingredients in C. majus. Among the five isoquinoline alkaloids, coptisine was present at the highest concentration (1624.23mg/L), while tetrahydrocoptisine showed the lowest concentration (0.47mg/L). Both the crude extract of C. majus (CECm) and the total alkaloids of C. majus (TACm) possessed a potent insecticidal activity toward L. dispar larvae. TACm had significant effects on the relative consumption rate, efficiency of conversion of digested food into growth, approximate digestibility, and approximate digestibility of L. dispar larvae. Enzyme activity assays suggested that both CECm and TACm displayed their strongest inhibitory activity to in vitro glutathione S-transferase (GST) and acetylcholinesterase (AChE), and showed the weakest inhibition of in vitro carboxylesterase (CarE). Moreover, CECm and TACm affected the in vivo activities of five enzymes. The in vivo activities of AChE and CarE in L. dispar larvae were inhibited significantly by CECm and TACm. Additionally, qRT-PCR analysis revealed that the transcription of the five enzymes was also affected by TACm. In conclusion, alkaloids in C. majus showed a prominent toxicity to L. dispar by reducing food intake, influencing nutritional indices, and affecting the activity and mRNA transcription of detoxifying and protective enzymes. This study provides novel insights into the insecticidal mechanism of C. majus.

Role of ocular albinism type 1 (OA1) GPCR in Asian gypsy moth development and transcriptional expression of heat-shock protein genes.

The ocular albinism type 1 gene, named OA1, is a coding pigment cell-specific G protein-coupled receptor exclusively localized in intracellular organelles. However, the function of OA1 in insects remains generally unknown. In the present study, we explore for the first time the function of LdOA1 in the Asian gypsy moth, Lymantria dispar. To identify the function of LdOA1 gene in the development and growth of the Asian gypsy moth, the LdOA1 gene in third instar larvae was knocked down by RNAi. Compared with the controls, the knockdown of LdOA1 increased larval mortality but did not significantly affect their utilization of nutrition. Moreover, LdOA1 was stably transformed into the third chromosome of Drosophila melanogaster. The LdOA1 gene in the transformed D. melanogaster modulated the expression of heat-shock protein (hsp) and increased the expression of hsp genes under deltamethrin stress, which indicates that LdOA1 is involved in the regulation of hsp gene expression. These results deepen our understanding of the molecular function of OA1 in insects.

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.

Transcription profiling of 12 asian gypsy moth (Lymantria dispar) cytochrome P450 genes in response to insecticides.

As the main group of detoxification enzymes, cytochrome P450 monoxygenases (P450s) catalyse an extremely diverse range of reactions that play an important role in the detoxification of foreign compounds. Transcription profiling of 12 Lymantria dispar P450 genes from the CYP6 subfamily believed to be involved in insecticide metabolism was performed in this study. Life-stage transcription profiling of CYP6 genes revealed significant variations between eggs, larvae, pupae, and adult males and females. Exposure of larvae to sublethal doses of deltamethrin, omethoate, and carbaryl enhanced the transcription of most of the CYP6 P450 genes, with induction peaking between 24 and 72 h after exposure. Transcription profiles were dependent on the levels of insecticide exposure and the various developmental stages.