Spraying double-stranded RNA targets UDP-N-acetylglucosamine pyrophosphorylase in the control of Nilaparvata lugens.

The rice pest Nilaparvata lugens (the brown planthopper, BPH) has developed different levels of resistance to at least 11 chemical pesticides. RNAi technology has contributed to the development of environmentally friendly RNA biopesticides designed to reduce chemical use. Consequently, more precise targets need to be identified and characterized, and efficient dsRNA delivery methods are necessary for effective field pest control. In this study, a low off-target risk dsNlUAP fragment (166 bp) was designed in silico to minimize the potential adverse effects on non-target organisms. Knockdown of NlUAP via microinjection significantly decreased the content of UDP-N-acetylglucosamine and chitin, causing chitinous structural disorder and abnormal phenotypes in wing and body wall, reduced fertility, and resulted in pest mortality up to 100 %. Furthermore, dsNlUAP was loaded with ROPE@C, a chitosan-modified nanomaterial for spray application, which significantly downregulated the expression of NlUAP, led to 48.9 % pest mortality, and was confirmed to have no adverse effects on Cyrtorhinus lividipennis, an important natural enemy of BPH. These findings will contribute to the development of safer biopesticides for the control of N. lugens.

A dsRNA delivery system based on the rosin-modified polyethylene glycol and chitosan induces gene silencing and mortality in Nilaparvata lugens.

BACKGROUND: RNA interference (RNAi) technology has been considered as a promising pest control strategy due to its species selectivity. One of the popular RNAs is exogenous double strand RNA (dsRNA). However, dsRNA is easily degraded by nucleases and is difficult to pass through the insect body walls, and these factors restrict the application of RNAi technology in pest management. Here, the brown planthopper (BPH, Nilaparvata lugens), a major hemipteran pest of rice in Asia countries was used as a model insect, and a dsRNA topical delivery system was constructed. RESULTS: The carrier part of the delivery system was composed of rosin-modified polyethylene glycol and chitosan, termed ROPE@C. When the N/P ratio was greater than 1:1.25, the dsRNA/ROPE@C complex encouraged full binding of the dsRNA. The gel electrophoresis results showed that ROPE@C improved the stability of dsRNA in the presence of nucleases in gut and lumen contents for at least 6 h and in the temperature range from 4 °C to 37 °C. The dsNlCHSA/ROPE@C/alkyl polyglycoside (APG) nano-formulation directly penetrated the body walls reaching hemocoel within 6 h, and consequently, the relative expression of chitin synthetase A (CHSA) in BPH was reduced by 54.3% and the mortality rate was 65.8%. CONCLUSION: We developed an appropriate delivery method for dsRNA application in BPH, which is helpful for a large-scale application of RNAi pesticides. © 2022 Society of Chemical Industry.

RNA interference of eclosion hormone gene reveals its roles in the control of ecdysis behavior in Heortia vitessoides Moore.

Eclosion hormone (EH) is an important neuropeptide that regulates growth and development. This study predicted the EH gene (HvEH) of Heortia vitessoides Moore (Lepidoptera: Crambidae) from the transcriptome database and its expression patterns were determined using quantitative real-time polymerase chain reaction. HvEH was expressed in all developmental stages and especially in the head area. RNA interference-mediated silencing of HvEH (2 µg/individual) with double-stranded HvEH RNA (dsHvEH) was achieved within 48 hr. Abnormal phenotypes appeared in the pupa and adult stages. dsHvEH injection suppressed pupation and eclosion rates. HvEH expression increased upon treatment with 20-hydroxyecdysone but decreased at extreme temperatures. These results suggest that HvEH plays an essential role in ecdysis and wing formation in H. vitessoides.

Cloning, Expression Analysis, 20-Hydroxyecdysone Induction, and RNA Interference Study of Autophagy-Related Gene 8 from Heortia vitessoides Moore.

Autophagy is a highly conserved and regulated process in eukaryotic cells and remodels cytoplasm, recovers essential nutrients, and disposes of unwanted cytoplasmic components. Autophagy-related gene (ATG) 8, identified in Heortia vitessoides Moore, which is an oligophagous pest of Aquilaria sinensis (Lour.), was characterized (HvATG8). Multiple sequence alignment showed that HvATG8 possesses highly conserved domain structures. Stage- and tissue-specific expressions indicated that HvATG8 is highly expressed in prepupal, pupal, and adult stages and in the midgut of larvae and abdomen of adults. Lack of function of HvATG8 by RNA interference resulted in a significant decrease in survival rate and an increase in abnormal or nonviable phenotypes in H. vitessoides. Transition rate from larval to pupal stages was 33.0% and from pupal to adult stages was 15.0% after injection. Reduction of ATG8 expression reduced survival of H. vitessoides. Therefore, HvATG8 possibly plays a key role in normal growth stage of H. vitessoides. HvATG8 suppression downregulates HvATG3 expression, suggesting that the two genes are interconnected. Further, HvATG8 expression increased by 20-hydroxyecdysone treatment, starvation, and extreme temperature exposure. Starvation also altered expression of other ATGs in H. vitessoide. This study may be used to guide research on molecular mechanisms of autophagy in insects.

RNA interference of a trehalose-6-phosphate synthase gene reveals its roles in the biosynthesis of chitin and lipids in Heortia vitessoides (Lepidoptera: Crambidae).

Trehalose-6-phosphate synthase (TPS), an enzyme that hydrolyzes two glucose molecules to yield trehalose, plays a pivotal role in various physiological processes. In this study, we cloned the trehalose-6-phosphate synthase gene (HvTPS) and investigated its expression patterns in various tissues and developmental stages in Heortia vitessoides Moore (Lepidoptera: Crambidae). HvTPS was highly expressed in the fat body and after pupation or before molting. We knocked down TPS in H. vitessoides by RNA interference and found that 3.0 µg of dsHvTPS resulted in optimal interference at 24 h and 36 h post-injection and caused a sharp decline in the survival rate during the 5th instar larval-pupal stage and obviously abnormal or lethal phenotypes. Additionally, compared to the controls, TPS activity and trehalose contents were significantly lower and the glucose content was significantly higher 24 h or 36 h after injection with 3.0 µg of dsHvTPS. Furthermore, the silencing of HvTPS suppressed the expression of six key genes in the chitin biosynthesis pathway and one key gene related to lipid catabolism. The expression levels of two genes associated with lipid biosynthesis were upregulated. These results strongly suggest that HvTPS is essential for the normal growth and development of H. vitessoides and provide a reference for further studies of the utility of key genes involved in chitin and lipid biosynthesis for controlling insect development.

Suppression of Gene Juvenile Hormone Diol Kinase Delays Pupation in Heortia vitessoides Moore.

Juvenile hormone diol kinase (JHDK) is a critical enzyme involved in juvenile hormone degradation in insects. In this study, HvJHDK in the Heortia vitessoides Moore (Lepidoptera: Crambidae) transcriptional library was cloned. Stage-specific expression patterns of HvJHDK, HvJHEH, and HvJHE as well as juvenile hormone titers were determined. The three tested enzymes participated in juvenile hormone degradation. Moreover, juvenile hormone titers peaked after larval-larval molts, consistent with a role for juvenile hormone in inhibition of metamorphosis. HvJHDK was subsequently suppressed using RNA interference (RNAi) to reveal its functions. Different concentrations of dsJHDK elicited the optimal interference efficiency at different life stages of H. vitessoides. Suppression of HvJHDK decreased HvJHDK content and increased the juvenile hormone titer, thereby resulting in reduced triglyceride content, sharply declined survival rate, clearly lethal phenotypes, and extended larval growth. Moreover, suppression of HvJHDK upregulated HvJHEH and HvJHE expression levels, suggesting that there is feedback regulation in the juvenile hormone metabolic pathway. Taken together, our findings provide molecular references for the selection of novel insecticidal targets.

Chitin deacetylase 1 and 2 are indispensable for larval-pupal and pupal-adult molts in Heortia vitessoides (Lepidoptera: Crambidae).

Heortia vitessoides Moore is a notorious defoliator of Aquilaria sinensis (Lour.) Gilg trees. Chitin deacetylases (CDAs) catalyze the N-deacetylation of chitin, which is a crucial process for chitin modification. Here, we identified and characterized HvCDA1 and HvCDA2 from H. vitessoides. HvCDA1 and HvCDA2 possess typical domain structures of CDAs and belong to the Group I CDAs. HvCDA1 and HvCDA2 were highly expressed before and after the larval-larval molt. In addition, both exhibited relatively high mRNA expression levels during the larval-pupal molt, the pupal stage, and the pupal-adult molt. HvCDA1 and HvCDA2 transcript expression levels were highest in the body wall and relatively high in the larval head. Significant increases in the HvCDA1 and HvCDA2 transcript expression levels were observed in the larvae upon exposure to 20-hydroxyecdysone. RNA interference-mediated HvCDA1 and HvCDA2 silencing significantly inhibited HvCDA1 and HvCDA2 expression, with abnormal or nonviable phenotypes being observed. Post injection survival rates of the larvae injected with dsHvCDA1 and dsHvCDA2 were 66.7% and 46.7% (larval-pupal) during development and 23.0% and 6.7% (pupal-adult), respectively. These rates were significantly lower than those of the control group insects. Our results suggest that HvCDA1 and HvCDA2 play important roles in the larval-pupal and pupal-adult transitions and represent potential targets for the management of H. vitessoides.

Knockdown of ß-N-acetylglucosaminidase gene disrupts molting process in Heortia vitessoides Moore.

ß-N-acetylglucosaminidase (NAG) is a key enzyme in insect chitin metabolism and plays an important role in many physiological activities of insects. The HvNAG1 gene was identified from the Heortia vitessoides Moore (Lepidoptera: Crambidae) cDNA library and its expression patterns were determined using quantitative real-time polymerase chain reaction. The results indicated that HvNAG1 mRNA levels were high in the midgut and before molting, and 20E could induce its expression. Subsequently, the HvNAG1 gene was knocked down via RNA interference to identify its functions. We found that 3 µg of dsNAG1 resulted in optimal interference at 48 and 72 hr after injection, causing a decrease in NAG1 protein content, which resulted in abnormal or lethal phenotypes, and a sharp decrease in the survival rate. These results indicate that HvNAG1 plays a key role in the molting process of H. vitessoides. However, the silencing of HvNAG1 had no significant effect on the chitin metabolism-related genes tested in this study. Our present study provides a reference for further research on the utility of key genes involved in the chitin metabolic pathway in the insect molting process.

Candidate olfactory genes identified in Heortia vitessoides (Lepidoptera: Crambidae) by antennal transcriptome analysis.

Heortia vitessoides Moore is the most severe defoliating pest of Aquilaria sinensis (Lour.) Gilg (Thymelaeaceae) forests. Olfaction in insects is essential for host identification, mating, and oviposition, in which olfactory proteins, including odorant-binding proteins (OBPs), chemosensory proteins (CSPs), olfactory receptors (ORs), ionotropic receptors (IRs), and sensory neuron membrane proteins (SNMPs), are responsible for chemical signaling. Here, we determined the transcriptomes of male and female adult antennae of H. vitessoides. We assembled 52,383 unigenes and annotated their putative gene functions based on the gene ontology (GO), eukaryotic ortholog groups (KOG), and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Overall, 61 olfactory-related transcripts, including nine OBPs, 10 CSPs, 28 ORs, 12 IRs, and two SNMPs, were identified. Expression patterns of OBPs and CSPs in the female antennae, male antennae, and legs were performed using reverse transcription quantitative PCR (RT-qPCR). The results revealed that HvitOBP1, HvitOBP6, and HvitGOBP1 were enriched in the female antennae, while HvitOBP2, HvitOBP3, HvitOBP5, HvitGOBP2, and HvitPBP1 were enriched in the male antennae. HvitOBP4 was expressed at nearly the same level in the antennae of both males and females. Four CSPs (HvitCSP3, HvitCSP5, HvitCSP7, and HvitCSP10) and two CSPs (HvitCSP1 and HvitCSP4) were expressed at higher levels in the female and male antennae, respectively. HvitCSP6 was expressed at higher levels both in the female antennae and legs. Three CSP genes (HvitCSP2, HvitCSP8, and HvitCSP9) were expressed at higher levels in the legs. These results provide a basis for further studies on the molecular olfactory mechanisms of H. vitessoides.

Identification and characterization of the catalase gene involved in resistance to thermal stress in Heortia vitessoides using RNA interference.

To elucidate the role of catalase (CAT) in Heortia vitessoides Moore, which is one of the most destructive defoliating pests in Aquilaria sinensis (Loureiro) Sprenger forests, a CAT gene (HvCAT) was identified in the transcriptome of adult H. vitessoides. Sequence analyses indicated that HvCAT encodes a protein containing 507 amino acids, including a proximal active site sequence (FXRERIPERVVHAKGXGA), heme-ligand sequence (RLFSYNDTX), heme-binding residues (H73, S112, N146, F151, F159, R352, and Y356), and NADPH-binding residues (P149, H192, Y196, G199, R201, N211, H233, K235, I300, W301, P302, H303, Q442, and L445). A phylogenetic analysis indicated that CAT from lepidopteran species could be assigned to one well-supported cluster. Regarding its stage- and tissue-specific expression profiles, HvCAT was expressed at high levels in fifth-instar larvae, fat body of larvae, and abdomen of adults. Furthermore, when fifth-instar larvae were exposed to thermal stress at 35, 37, and 39 °C, hydrogen peroxide and malondialdehyde content significantly increased. HvCAT mRNA was upregulated when the larvae were exposed to temperatures of 31, 33, 35, 37, and 39 °C. The enzymatic activity of HvCAT was significantly elevated following thermal stress (35 and 37 °C). After the knockdown of HvCAT by double-stranded RNA interference, the expression of thioredoxin peroxidase (Tpx) increased, whereas that of copper zinc superoxide dismutase (Cu/ZnSOD) decreased. Additionally, knocking down HvCAT transcripts in fifth-instar larvae resulted in accelerated death following thermal stress at 35 °C. In summary, the results suggest that HvCAT plays a major role in the thermotolerance of H. vitessoides.

Multiple Glutathione S-Transferase Genes in Heortia vitessoides (Lepidoptera: Crambidae): Identification and Expression Patterns.

To elucidate the role of glutathione S-transferases (GSTs) in Heortia vitessoides Moore (Lepidoptera: Crambidae), one of the most destructive defoliating pests in Aquilaria sinensis (Lour.) Gilg (Thymelaeaceae) forests, 16 GST cDNAs were identified in the transcriptome of adult H. vitessoides. All cDNAs included a complete open reading frame and were designated HvGSTd1-HvGSTu2. A phylogenetic analysis showed that the 16 HvGSTs were classified into seven different cytosolic classes; three in delta, two in epsilon, three in omega, three in sigma, one in theta, two in zeta, and two in unclassified. The expression patterns of these HvGSTs in various larval and adult tissues, following exposure to half the lethal concentrations (LC50s) of chlorantraniliprole and beta-cypermethrin, were determined using real-time quantitative polymerase chain reaction (RT-qPCR). The expression levels of the 16 HvGSTs were found to differ among various larval and adult tissues. Furthermore, the RT-qPCR confirmed that the transcription levels of nine (HvGSTd1, HvGSTd3, HvGSTe2, HvGSTe3, HvGSTo3, HvGSTs1, HvGSTs3, HvGSTu1, and HvGSTu2) and six (HvGSTd1, HvGSTd3, HvGSTe2, HvGSTo2, HvGSTs1, and HvGSTu1) HvGST genes were significantly higher in the fourth-instar larvae following exposure to the insecticides chlorantraniliprole and beta-cypermethrin, respectively. These genes are potential candidates involved in the detoxification of these two insecticides. Further studies utilizing the RNA interference approach are required to enhance our understanding of the functions of these genes in this forest pest.