Analysis of gut microbiota of ladybug beetle (Harmonia axyridis) after feeding on different artificial diets.

BACKGROUND: Harmonia axyridis is an effective natural enemy insect to a variety of phloem-sucking pests and Lepidopteran larvae, such as aphids, scabies, and phylloxera, while its industrial production is limited due to unmature artificial diet. Insect intestinal microbiota affect host development and reproduction. The aim of this study is to understand intestinal microbiota composition of H. axyridis and screen effective probiotics on artificial diet. Considering the role of the components and composition of the diet on the structure and composition of the intestinal microbiome, four kinds of diets were set up: (1) aphid; (2) basic diet; (3) basic diet + glucose; (4) basic diet + trehalose. The gut microbiota of H. axyridis was detected after feeding on different diets. RESULTS: Results showed that the gut microbiota between artificial diet group and aphid groups were far apart, while the basic and glucose groups were clearly clustered. Besides, the glucose group and trehalose group had one unique phylum, Cryptophyta and Candidatus Saccharibacteria, respectively. The highest abundance of Proteobacteria was found in the aphid diet. The highest abundance of Firmicutes was found in the basic diet. However, the addition of glucose or trehalose alleviated the change. In addition, the relative abundance of Enterobacter, Klebsiella, Enterobacteriaceae_unclassified, Enterobacteriales_unclassified and Serratia in the aphid group was higher than other groups. Moreover, the function of gut genes in each group also showed clear differences. CONCLUSION: These results have offered a strong link between artificial diets and gut microbes, and also have provided a theoretical basis for the screening of synergistic probiotics in artificial diet.

Regulation of three novel pepper thiothiazolidinones on the fecundity of Spodoptera frugiperda.

Spodoptera frugiperda has emerged as a major invasive pest worldwide. The utilization of chemical pesticides not only poses numerous ecological concerns but also fosters resistance in S. frugiperda. In this study, we designed and synthesized three novel thiothiazolidinone compounds (6a, 7b, and 7e) and incorporated innovative thiothiazolidinone structural elements into the piperine skeleton. Treatment with compounds 6a and 7e resulted in the blackening and agglomeration of oviduct eggs within the ovaries of certain female moths, impeding the release of normal eggs. The levels of vitellogenin and vitellogenin receptor, along with three trehalase inhibitors, exhibited a dynamic equilibrium state, leading to no discernible change in egg production but a notable increase in the generation of low-hatching-rate egg fragments. Compared with the injection of 2%DMSO, the eclosion rate of 6a injection was significantly decreased, as followed the spawning time and longevity were prolonged or significantly prolonged in the trehalase inhibitors of 6a, 7b, and 7e. We aimed to investigate the regulatory impacts of three new pepper thiothiazolidinone compounds on the reproduction of S. frugiperda, and to authenticate the efficacy of novel alginase inhibitors in inhibiting the reproduction of S. frugiperda. This research endeavors to aid in the identification of efficient and steadfast trehalase inhibitors, thereby expediting the research and development of potent biological pesticides.

Comparative transcriptomic and metabolomics analysis of modified atmosphere responses in Tribolium castaneum (Coleoptera: Tenebrionidae).

Modified atmosphere is effective in controlling Tribolium castaneum Herbst, but it has adaptations. Comprehending the potential mechanism of resistance to T. castaneum in a modified atmosphere will help advance related management methods. This study conducted a comparative transcriptomic and metabolomic analysis to understand the physiological mechanism of T. castaneum in adapting to CO2 stress. Results showed that there were a large number of differentially expressed genes (DEGs) in T. castaneum treated with different concentrations of CO2. Gene ontology (GO) analysis revealed significant enrichment of DEGs mainly in binding, catalytic activity, cell, membrane, membrane part, protein-containing complex, biological regulation, and cellular and metabolic process. Kyoto Encyclopedia of Genes and Genomes analysis showed that different treatments had different effects on the metabolic pathways of T. castaneum. DEGs induced by 25% CO2 were involved in arginine and proline metabolism, and 50% air + 50% CO2 treatment affected most kinds of metabolic pathways, mainly the signal transduction pathway, including PI3K-Akt signaling pathway, AMPK signaling pathway, neurotrophin signaling pathway, insulin signaling pathway, and thyroid hormone signaling. Ribosome and DNA replication were enriched under high CO2 stress (75% and 95%). The metabolomics revealed that different concentrations of CO2 treatments might inhibit the growth of T. castaneum through acidosis, or they may adapt to anoxic conditions through histamine and N-acetylhistamine. Multiple analyses have shown significant changes in histamine and N-acetylhistamine levels, as well as their associated genes, with increasing CO2 concentration. In conclusion, this study comprehensively revealed the molecular mechanism of T. castaneum responding to CO2 stress and provided the basis for an effectively modified atmosphere in the T. castaneum.

Regulation of Vicia faba L. Response and Its Effect on Megoura crassicauda Reproduction under Zinc Stress.

The heavy metal zinc (Zn) is known to be transmitted in the food chain; however, the effect of Zn stress on beans and herbivorous insects is largely unclear. This study aimed to investigate the resistance of broad bean plants to Zn stress and the consequent changes in their physiological and biochemical metabolism by simulating heavy metal pollution in soil. Simultaneously, the effects of aphid progeny treated with different Zn concentrations on the expression of carbohydrate and related genes were analyzed. The results showed that Zn had no effect on the germination rate of broad beans, but other effects mainly manifested as follows. (1) Chlorophyll content decreased. (2) The total soluble sugar and Zn content in stems and leaves increased with increasing Zn content. (3) The proline content first increased and then decreased with increasing Zn content. (4) The height of the seedlings indicates that low concentrations promote growth and high concentrations inhibit growth. In addition, only the first-generation fecundity decreased significantly when aphids fed on heavy metal broad beans. Continuous high Zn levels increase the trehalose content of aphid F1 and F2, while F3 decreases. These results can not only provide a theoretical basis for exploring the impact of soil heavy metal pollution on ecosystems but also preliminarily evaluate the possibility of broad beans as a means of pollution remediation.

Effects of long-term cadmium exposure on trehalose metabolism, growth, and development of Aedes albopictus (Diptera: Culicidae).

Trehalose is the major blood sugar in insects; it not only serves as an energy source but also plays important roles in physiological responses to adverse conditions. However, only a few studies have explored the effects of heavy metal exposure stress on trehalose metabolism in insects. Therefore, in this study, we examined the effects of cadmium stress on changes in trehalose metabolism in Aedes albopictus. Three concentrations of cadmium (0.005, 0.01, and 0.1 mg/L) were selected for evaluation of long-term stress in Ae. albopictus (from eggs to adults); Ae. albopictus in double-distilled water was used as the control group. The trehalose and glucose contents, trehalase activity, and trehalose metabolism-related gene expression were determined. The effects of long-term cadmium exposure on growth, development, and reproduction were also assessed. Trehalose contents were increased, whereas glucose contents and trehalase activity were decreased in Ae. albopictus following long-term exposure to low concentrations of cadmium compared with those in untreated individuals. Moreover, the expression of trehalose-6-phosphate synthase was upregulated, and that of trehalase was downregulated, indicating that Ae. albopictus may enhance trehalose synthesis to resist cadmium stress. Cadmium exposure also caused Ae. albopictus individuals to become smaller with a longer developmental duration, whereas both reproduction and hatching rates of the offspring were decreased compared with those in the control group. Our findings demonstrated that cadmium exposure affected the morphology, physiology, and biochemistry of Ae. albopictus. These findings also confirmed the role of trehalose in the response of Ae. albopictus to cadmium stress, providing insights into the effects of heavy metal stress on trehalose metabolism in an insect model.

Suppressing the activity of trehalase with validamycin disrupts the trehalose and chitin biosynthesis pathways in the rice brown planthopper, Nilaparvata lugens.

Trehalase (TRE) is a key enzyme in trehalose degradation and has important functions in insect growth and chitin synthesis. Though validamycin has the potential for pest control by suppressing TRE activities, it is not known whether validamycin acts on both trehalose and chitin metabolism. TRE1 and TRE2 activities and glucose and glycogen contents decreased significantly after the injection of different doses of validamycin solution compared with the control group, while the trehalose content increased significantly. Overall, it showed that about 13 to 38% insects was appeared abnormal phenotypes, and 10 to 57% of insects died 48h after injection of solutions with different concentrations of validamycin; the chitin content also decreased significantly. Validamycin altered the relative expression levels of trehalose, glycogen and chitin metabolism-related genes by suppressing the activities of two TREs. We showed that the expression levels of three TRE and two trehalose-6-phosphate synthase (TPS) genes increased, while the expression levels of GP; CHS1 and its two transcripts, CHS1a, CHS1b; six chitinases, including Cht3, Cht4, Cht5, Cht6, Cht7, Cht9; and the HK, G6PI2, GFAT, GNPNA, PAGM1, UAP, VVL, CI and AP genes decreased significantly 48h after the injection of any validamycin concentration compared with the control group. These results demonstrate that by inhibiting the activities of two TREs, validamycin alters N. lugens chitin synthesis and degradation and affects trehalose and chitin metabolism-related gene expression. The development of TRE inhibitors may provide effective pest control in the future.

Glycogen Phosphorylase and Glycogen Synthase: Gene Cloning and Expression Analysis Reveal Their Role in Trehalose Metabolism in the Brown Planthopper, Nilaparvata lugens Stål (Hemiptera: Delphacidae).

RNA interference has been used to study insects' gene function and regulation. Glycogen synthase (GS) and glycogen phosphorylase (GP) are two key enzymes in carbohydrates' conversion in insects. Glycogen content and GP and GS gene expression in several tissues and developmental stages of the Brown planthopper Nilaparvata lugens Stål (Hemiptera: Delphacidae) were analyzed in the present study, using quantitative reverse-transcription polymerase chain reaction to determine their response to double-stranded trehalases (dsTREs), trehalose-6-phosphate synthases (dsTPSs), and validamycin injection. The highest expression of both genes was detected in the wing bud, followed by leg and head tissues, and different expression patterns were shown across the developmental stages analyzed. Glycogen content significantly decreased 48 and 72 h after dsTPSs injection and 48 h after dsTREs injection. GP expression increased 48 h after dsTREs and dsTPSs injection and significantly decreased 72 h after dsTPSs, dsTRE1-1, and dsTRE1-2 injection. GS expression significantly decreased 48 h after dsTPS2 and dsTRE2 injection and 72 h after dsTRE1-1 and dsTRE1-2 injection. GP and GS expression and glycogen content significantly decreased 48 h after validamycin injection. The GP activity significantly decreased 48 h after validamycin injection, while GS activities of dsTPS1 and dsTRE2 injection groups were significantly higher than that of double-stranded GFP (dsGFP) 48 h after injection, respectively. Thus, glycogen is synthesized, released, and degraded across several insect tissues according to the need to maintain stable trehalose levels.

Knockdown of five trehalase genes using RNA interference regulates the gene expression of the chitin biosynthesis pathway in Tribolium castaneum.

BACKGROUND: RNA interference is a very effective approach for studies on gene function and may be an efficient method for controlling pests. Trehalase is a key gene in the chitin biosynthesis pathway in insects. Five trehalase genes have been cloned in Tribolium castaneum, though it is not known whether the detailed functions of these trehalases can be targeted for pest control. RESULTS: The functions of all five trehalase genes were studied using RNAi, and the most important results showed that the expression of all 12 genes decreased significantly from 12 to 72 h compared with the control groups, except GP1 at 72 h, when the expression of the TcTre2 gene was suppressed. The results also revealed different abnormal phenotypes, and the observed mortality rates ranged from 17 to 42 %. The qRT-PCR results showed that the expression of TPS, GS, two GP, CHS1a and CHS1b genes decreased significantly, while that of the CHS2 gene decreased or increased after RNAi after the five trehalases were silenced at 48 h. In addition, TPS gene expression decreased from 12 to 72 h after dsTcTre injection. CONCLUSIONS: These results demonstrate that silencing of any individual trehalase gene, especially Tre1-4 and Tre2 gene can lead to moulting deformities and a high mortality rate through the regulation of gene expression in the chitin biosynthesis pathway and may be a potential approach for pest control in the future.

Suppressing the expression of a forkhead transcription factor disrupts the chitin biosynthesis pathway in Spodoptera exigua.

Forkhead (Fox) transcription factors display functional diversity and are involved in various metabolic and developmental processes. The Spodoptera exigua Fox (SeFox) encodes a protein of 353 amino acids with a theoretical molecular mass of approximately 38.99 kDa and an isoelectric point of 8.86. qPCR results revealed that SeFox was expressed mainly in the brain, fat body, epidermis, midgut, Malpighian tubules, and testis. SeFox was expressed, with some changes, throughout development in the fat body and whole body. Injection of dsSeFox (SeFox dsRNA) into larvae resulted in incidences of albino plus molting deformity (4.8%), molting deformity (26.2%), and albino phenotypes (69.1%). dsSeFox injection resulted in approximately 50% knockdown of transcript levels at 36 h. Compared with control groups, hexokinase (HK) expression was reduced to approximately 40% at 48 h postinjection. Chitin synthase A (CHSA) expression was reduced to two-thirds at 24 h, but increased at 72 h. Compared with untreated control and green fluorescent protein-treated groups, Chitin synthase B (CHSB) expression decreased to 33% following dsSeFox injection by 36 h. We infer from our results that forkhead transcription factors act in chitin synthesis in S. exigua.

The Role of TcCYP6K1 and TcCYP9F2 Influences Trehalose Metabolism under High-CO2 Stress in Tribolium castaneum (Coleoptera).

Cytochrome P450 monooxygenases (CYP), crucial detoxification enzymes in insects, are involved in the metabolism of endogenous substances as well as the activation and degradation of exogenous compounds. In this study, T. castaneum was utilized to investigate the roles of TcCYP6K1 and TcCYP9F2 genes influencing in the trehalose metabolism pathway under high-CO2 stress. By predicting the functional sequences of TcCYP6K1 and TcCYP9F2 genes and analyzing their spatiotemporal expression patterns, it was discovered that both genes belong to the CYP3 group and exhibit high expression levels during the larval stage, decreasing during the pupal stage, while showing high expression in the fatty body, intestine, and malpighian tubules. Furthermore, following the knockdown of TcCYP6K1 and TcCYP9F2 genes in combination with treating larvae with 75% CO2, it was observed that larval mortality increased, and glycogen content significantly decreased, while trehalose content increased significantly. Additionally, membrane-bound trehalase enzyme activity declined, TPS gene expression was significantly upregulated, GS gene expression was significantly downregulated, and ATP content showed a marked decrease. In conclusion, CYP genes are critical responsive genes of T. castaneum to high CO2 levels, potentially impacting the insect's resistance to carbon dioxide through their involvement in the synthesis or breakdown of the carbohydrate metabolism pathway. These findings could serve as a theoretical basis for the utilization of novel pesticides in low-oxygen grain storage techniques and offer new insights for environmentally friendly pest control strategies in grain storage.

Residues of chlorpyrifos in the environment induce resistance in Aedes albopictus by affecting its olfactory system and neurotoxicity.

Aedes albopictus, a virus-vector pest, is primarily controlled through the use of insecticides. In this study, we investigated the mechanisms of resistance in Ae. albopictus in terms of chlorpyrifos neurotoxicity to Ae. albopictus and its effects on the olfactory system. We assessed Ca2+-Mg2+-ATP levels, choline acetyltransferase (ChAT), Monoamine oxidase (MAO), odorant-binding proteins (OBPs), and olfactory receptor (OR7) gene expression in Ae. albopictus using various assays including Y-shaped tube experiments and DanioVision analysis to evaluate macromotor behavior. Our findings revealed that cumulative exposure to chlorpyrifos reduced the activity of neurotoxic Ca2+-Mg2+-ATPase and ChAT enzymes in Ae. albopictus to varying degrees, suppressed MAO-B enzyme expression, altered OBPs and OR7 expression patterns, as well as affected evasive response, physical mobility, and cumulative locomotor time under chlorpyrifos stress conditions for Ae. albopictus individuals. Consequently, these changes led to decreased feeding ability, reproductive capacity, and avoidance behavior towards natural enemies in Ae. albopictus populations exposed to chlorpyrifos stressors over time. To adapt to unfavorable living environments, Ae. albopictus may develop certain tolerance mechanisms against organophosphorus pesticides. This study provides valuable insights for guiding rational insecticide usage or dosage adjustments targeting the nervous system of Ae. albopictus.

Gene cloning and expression patterns of two prophenoloxidases from Catantops pinguis (Orthoptera: Catantopidae).

In insect, fat body plays major roles in insect innate immunity. Phenoloxidase (PO) is an important component in insect innate immunity and is necessary for acclimatization. In our study, two prophenoloxidase (PPO) subunits were obtained from fat body of Catantops pinguis (Stål). The full-length cDNA sequence of one PPO (CpPPO1) consisted of 2347 bp with an open reading frame (ORF) of 2187 bp encoding 728 amino acids, while the other subunit (CpPPO2) had a full length of 2445 bp, encoding 691 amino acids. Both the PPO gene products are predicted to possess all the structural features of other PPO members, including two putative tyrosinase copper-binding motifs with six highly conserved histidine residues and a thiolester-like motif. Tissue distribution analysis showed that both PPO mRNAs were abundantly expressed in the fat body among 11 tissues examined, and they were transiently up-regulated after Escherichia coli infection, consistent with them being immune-responsive genes. Total levels of CpPPO1 and CpPPO2 mRNA transcripts were much higher in first instar larvae and adults. A much higher transcript level of CpPPO1 was detected in several months, while there were extremely high mRNA expression levels of CpPPO2 in January, July, October, and December. The above results suggested that PPO from fat body might also bring significant function during the processes of development and acclimatization for C. pinguis.

RNAi-mediated CHS-2 silencing affects the synthesis of chitin and the formation of the peritrophic membrane in the midgut of Aedes albopictus larvae.

BACKGROUND: Mosquitoes are an important vector of viral transmission, and due to the complexity of the pathogens they transmit, vector control may be the most effective strategy to control mosquito-borne diseases. Chitin is required for insect growth and development and is absent in higher animals and plants, so regulating the chitin synthesis pathway can serve as a potentially effective means to control vector insects. Most of the current research on the chitin synthase (CHS) gene is focused on chitin synthase-1 (CHS-1), while relatively little is known about chitin synthase-2 (CHS-2). RESULTS: The CHS-2 gene of Ae. albopictus is highly conserved and closely related to that of Aedes aegypti. The expression of CHS-2 in the third-instar larvae and pupal stage of Ae. albopictus was relatively high, and CHS-2 expression in adult mosquitoes reached the highest value 24 h after blood-feeding. In the fourth-instar larvae of Ae. albopictus, CHS-2 expression was significantly higher in the midgut than in the epidermis. Silencing CHS-2 in Ae. albopictus larvae had no effect on larval survival and emergence. The expression of four genes related to chitin synthesis enzymes was significantly upregulated, the expression level of three genes was unchanged, and only the expression level of GFAT was significantly downregulated. The expression of chitin metabolism-related genes was also upregulated after silencing. The level of chitin in the midgut of Ae. albopictus larvae was significantly decreased, while the chitinase activity was unchanged. The epithelium of the midgut showed vacuolization, cell invagination and partial cell rupture, and the structure of the peritrophic membrane was destroyed or even absent. METHODS: The expression of CHS-2 in different developmental stages and tissues of Aedes albopictus was detected by real-time fluorescence quantitative PCR (qPCR). After silencing CHS-2 of the fourth-instar larvae of Ae. albopictus by RNA interference (RNAi), the expression levels of genes related to chitin metabolism, chitin content and chitinase activity in the larvae were detected. The structure of peritrophic membrane in the midgut of the fourth-instar larvae after silencing was observed by paraffin section and hematoxylin-eosin (HE) staining. CONCLUSION: CHS-2 can affect midgut chitin synthesis and breakdown by regulating chitin metabolic pathway-related genes and is involved in the formation of the midgut peritrophic membrane in Ae. albopictus, playing an important role in growth and development. It may be a potential target for enhancing other control methods.

Novel compounds ZK-PI-5 and ZK-PI-9 regulate the reproduction of Spodoptera frugiperda (Lepidoptera: Noctuidae), with insecticide potential.

Trehalase inhibitors prevent trehalase from breaking down trehalose to provide energy. Chitinase inhibitors inhibit chitinase activity affecting insect growth and development. This is an important tool for the investigation of regulation of trehalose metabolism and chitin metabolism in insect reproduction. There are few studies on trehalase or chitinase inhibitors' regulation of insect reproduction. In this study, ZK-PI-5 and ZK-PI-9 were shown to have a significant inhibitory effect on the trehalase, and ZK-PI-9 significantly inhibited chitinase activity in female pupae. We investigated the reproduction regulation of Spodoptera frugiperda using these new inhibitors and evaluated their potential as new insecticides. Compounds ZK-PI-5 and ZK-PI-9 were injected into the female pupae, and the control group was injected with solvent (2% DMSO). The results showed that the emergence failure rate for pupae treated with inhibitors increased dramatically and aberrant phenotypes such as difficulty in wings spreading occurred. The oviposition period and longevity of female adults in the treated group were significantly shorter than those in the control group, and the ovaries developed more slowly and shrank earlier. The egg hatching rate was significantly reduced by treatment with the inhibitor. These results showed that the two new compounds had a significant impact on the physiological indicators related to reproduction of S. frugiperda, and have pest control potential. This study investigated the effect of trehalase and chitin inhibitors on insect reproduction and should promote the development of green and efficient insecticides.

Polyketides as Secondary Metabolites from the Genus Aspergillus.

Polyketides are an important class of structurally diverse natural products derived from a precursor molecule consisting of a chain of alternating ketone and methylene groups. These compounds have attracted the worldwide attention of pharmaceutical researchers since they are endowed with a wide array of biological properties. As one of the most common filamentous fungi in nature, Aspergillus spp. is well known as an excellent producer of polyketide compounds with therapeutic potential. By extensive literature search and data analysis, this review comprehensively summarizes Aspergillus-derived polyketides for the first time, regarding their occurrences, chemical structures and bioactivities as well as biosynthetic logics.

RNAi-mediated CrebA silencing inhibits reproduction and immunity in Locusta migratoria manilensis.

Locusta migratoria manilensis is a major agricultural pest that causes severe direct and indirect damage to several crops. Thus, to provide a theoretical foundation for pest control, the role of CrebA in the reproduction and immune regulation of L. migratoria was investigated. CrebA is a bZIP transcription factor that critically regulates intracellular protein secretion. In this study, CrebA was widely expressed in the brain, fat body, integument, midgut, and reproductive tissues of different maturity stages of adult locusts, especially in the female fat body. RNA interfering (RNAi)-mediated silencing of CrebA inhibited locusts ovarian development, and key reproduction gene expressions, Vgs, VgRs, Chico, and JHAMT were downregulated. After the locusts were injected with Micrococcus luteus or Escherichia coli, M. luteus activated lysozyme expression, while the E. coli activated both phenol oxidase cascade and lysozyme expression. Furthermore, both bacteria stimulated the upregulation of the antimicrobial peptide genes DEF3 and DEF4. However, CrebA silencing is fatal to locusts infection with E. coli, with a mortality rate of up to 96.3%, and resulted in a significant decrease in the expression of DEF3 and DEF4 and changes in the activities of phenol oxidase and lysozyme of locusts infected by bacteria. Collectively, CrebA may be involved in diverse biological processes, including reproduction and immunity. CrebA inhibited locusts reproduction by regulating JH signaling pathway and inhibits the expression of immune genes TLR6, IMD, and AMPs. These results demonstrate CrebA seems to play a crucial role in reproduction and innate immunity.

Potential inhibitory effects of compounds ZK-PI-5 and ZK-PI-9 on trehalose and chitin metabolism in Spodoptera frugiperda (J. E. Smith).

Introduction: Spodoptera frugiperda is an omnivorous agricultural pest which is great dangerous for grain output. Methods: In order to investigate the effects of potential trehalase inhibitors, ZK-PI-5 and ZK-PI-9, on the growth and development of S. frugiperda, and to identify new avenues for S. frugiperda control, we measured the content of the trehalose, glucose, glycogen and chitin, enzyme activity, and gene expression levels in trehalose and chitin metabolism of S. frugiperda. Besides, their growth and development were also observed. Results: The results showed that ZK-PI-9 significantly reduced trehalase activity and ZK-PI-5 significantly reduced membraned-bound trehalase activity. Moreover, ZK-PI-5 inhibited the expression of SfTRE2, SfCHS2, and SfCHT, thus affecting the chitin metabolism. In addition, the mortality of S. frugiperda in pupal stage and eclosion stage increased significantly after treatment with ZK-PI-5 and ZK-PI-9, which affected their development stage and caused death phenotype (abnormal pupation and difficulty in breaking pupa). Discussion: These results have provided a theoretical basis for the application of trehalase inhibitors in the control of agricultural pests to promote future global grain yield.

Sequencing and characterization of glycogen synthase and glycogen phosphorylase genes from Spodoptera exigua and analysis of their function in starvation and excessive sugar intake.

Glycogen and trehalose are important energy source and key regulation factors in the development of many organisms' pass through energy metabolism, including bacteria, fungi, and insects. To study glycogen metabolism pathway in Spodoptera exigua, first cDNAs for glycogen synthase (SpoexGS) and glycogen phosphorylase (SpoexGP) were cloned from S. exigua. SpoexGS cDNA contains an open reading frame of 2,010 nucleotides encoding a protein of 669 amino acids with a predicted molecular mass of 76.19 kDa and a pI of 5.84. SpoexGP contains an open reading frame of 2,946 nucleotides, which encodes a protein of 841 amino acids with a predicted molecular mass of approximately 96.63 kDa and a pI of 6.03. Second, Northern blotting revealed that SpoexGS and SpoexGP mRNAs were expressed in brain, fat body, mid-gut, Malpighian tubules, spermary, and tracheae of S. exigua. Expression patterns for SpoexGS and SpoexGP mRNAs were similar in fat body, but differed in whole body at different developmental stages. The last, under starvation conditions, SpoexGS and SpoexGP transcript expression rapidly decreased with increasing starvation time. When the starvation stress was removed, SpoexGS and SpoexGP mRNA levels were lower in the groups starved for 6 and 12 h than in the 24-h starvation and control groups. Treatment with excessive sugar intake led to higher levels of SpoexGS and SpoexGP transcripts after 12 h compared to the control group. These findings provide new data on the tissue distribution, expression patterns, and potential function of glycogen synthase and glycogen phosphorylase proteins.

Elongation factor 1ß' gene from Spodoptera exigua: characterization and function identification through RNA interference.

Elongation factor (EF) is a key regulation factor for translation in many organisms, including plants, bacteria, fungi, animals and insects. To investigate the nature and function of elongation factor 1ß' from Spodoptera exigua (SeEF-1ß'), its cDNA was cloned. This contained an open reading frame of 672 nucleotides encoding a protein of 223 amino acids with a predicted molecular weight of 24.04 kDa and pI of 4.53. Northern blotting revealed that SeEF-1ß' mRNA is expressed in brain, epidermis, fat body, midgut, Malpighian tubules, ovary and tracheae. RT-PCR revealed that SeEF-1ß' mRNA is expressed at different levels in fat body and whole body during different developmental stages. In RNAi experiments, the survival rate of insects injected with SeEF-1ß' dsRNA was 58.7% at 36 h after injection, which was significantly lower than three control groups. Other elongation factors and transcription factors were also influenced when EF-1ß' was suppressed. The results demonstrate that SeEF-1ß' is a key gene in transcription in S. exigua.

Involvement of glucose transporter 4 in ovarian development and reproductive maturation of Harmonia axyridis (Coleoptera: Coccinellidae).

Glucose is vital to embryogenesis, as are glucose transporters. Glucose transporter 4 (Glut4) is one of the glucose transporters, which is involved in rapid uptake of glucose by various cells and promotes glucose homeostasis. Although energy metabolism in insect reproduction is well known, the molecular mechanism of Glut4 in insect reproduction is poorly understood. We suspect that Glut4 is involved in maintaining glucose concentrations in the ovaries and affecting vitellogenesis, which is critical for subsequent oocyte maturation and insect fertility. Harmonia axyridis (Pallas) is a model organism for genetic research and a natural enemy of insect pests. We studied the influence of the Glut4 gene on the reproduction and development of H. axyridis using RNA interference technology. Reverse transcription quantitative polymerase chain reaction analysis revealed that HaGlut4 was most highly expressed in adults. Knockdown of the HaGlut4 gene reduced the transcript levels of HaGlut4, and the weight and number of eggs produced significantly decreased. In addition, the transcript levels of vitellogenin receptor and vitellogenin in the fat bodies and the ovaries of H. axyridis decreased after the interference of Glut4, and decreased the triglyceride, fatty acid, total amino acid and adenosine triphosphate content of H. axyridis. This resulted in severe blockage of ovary development and reduction of yolk formation; there was no development of ovarioles in the developing oocytes. These changes indicate that a lack of HaGlut4 can impair ovarian development and oocyte maturation and result in decreased fecundity.