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.

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.

Suppression of yolk formation, oviposition and egg quality of locust (Locusta migratoria manilensis) infected by Paranosema locustae.

Locusta migratoria manilensis is one of the most important agricultural pests in China. The locust has high fecundity and consumes large quantities of food, causing severe damage to diverse crops such as corn, sorghum, and rice. Immunity against pathogens and reproductive success are two important components of individual fitness, and many insects have a trade-off between reproduction and immunity when resources are limited, which may be an important target for pest control. In this study, adult females L. migratoria manilensis were treated with different concentrations (5 × 106 spores/mL or 2 × 107 spores/mL) of the entomopathogenic fungus Paranosema locustae. Effects of input to immunity on reproduction were studied by measuring feeding amount, enzyme activity, vitellogenin (Vg) and vitellogenin receptor (VgR) production, ovary development, and oviposition amount. When infected by P. locustae, feeding rate and phenol oxidase and lysozyme activities increased, mRNA expression of Vg and VgR genes decreased, and yolk deposition was blocked. Weight of ovaries decreased, with significant decreases in egg, length and weight.Thus, locusts used nutritive input required for reproduction to resist invasion by microsporidia. This leads to a decrease in expression of Vg and VgR genes inhibited ovarian development, and greatly decreased total fecundity. P. locustae at 2 × 107 spores/mL had a more obvious inhibitory effect on the ovarian development in migratory locusts. This study provides a detailed trade-off between reproduction and immune input of the female, which provides a reliable basis to find pest targets for biological control from those trade-off processes.

Regulatory Functions of Nilaparvata lugens GSK-3 in Energy and Chitin Metabolism.

Glucose metabolism is a biologically important metabolic process. Glycogen synthase kinase (GSK-3) is a key enzyme located in the middle of the sugar metabolism pathway that can regulate the energy metabolism process in the body through insulin signaling. This paper mainly explores the regulatory effect of glycogen synthase kinase on the metabolism of glycogen and trehalose in the brown planthopper (Nilaparvata lugens) by RNA interference. In this paper, microinjection of the target double-stranded GSK-3 (dsGSK-3) effectively inhibited the expression of target genes in N. lugens. GSK-3 gene silencing can effectively inhibit the expression of target genes (glycogen phosphorylase gene, glycogen synthase gene, trehalose-6-phosphate synthase 1 gene, and trehalose-6-phosphate synthase 2 gene) in N. lugens and trehalase activity, thereby reducing glycogen and glucose content, increasing trehalose content, and regulating insect trehalose balance. GSK-3 can regulate the genes chitin synthase gene and glucose-6-phosphate isomerase gene involved in the chitin biosynthetic pathway of N. lugens. GSK-3 gene silencing can inhibit the synthesis of chitin N. lugens, resulting in abnormal phenotypes and increased mortality. These results indicated that a low expression of GSK-3 in N. lugens can regulate the metabolism of glycogen and trehalose through the insulin signal pathway and energy metabolism pathway, and can regulate the biosynthesis of chitin, which affects molting and wing formation. The relevant research results will help us to more comprehensively explore the molecular mechanism of the regulation of energy and chitin metabolism of insect glycogen synthase kinases in species such as N. lugens.

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.