In-depth transcriptome and physiological function analysis reveals the toxicology of sodium fluoride in the fall webworm Hyphantria cunea.

Fluoride is an environmental pollutant that severely injures various organisms in ecosystems. Herein, the non-target organism, fall webworm (Hyphantria cunea), was used to determine the toxicological mechanism of NaF exposure. In this study, H. cunea exposed to NaF showed significant declines in growth and reproduction. The authors conducted RNA sequencing on adipose bodies and midgut tissues from NaF-exposed H. cunea larvae to uncover the toxicological mechanisms. The results showed that extracellular matrix-receptor interaction, pentose and glucuronate interconversions, fatty acid biosynthesis, and ferroptosis might contribute to NaF stress. NaF significantly decreased the antioxidant level, nitrous oxide synthase activity, and NO content, while significantly increasing lipid peroxidation. NaF induced significant changes in the expression of energy metabolism genes. However, the triglyceride content was significantly decreased and the lipase enzyme activity was significantly increased. Moreover, the expression levels of light and heavy chains of ferritin were inhibited in NaF-exposed H. cunea. NaF caused ferritin Fe2+overload in FerHCH1 and FerLCH knockdown H. cunea larvae, activated reactive oxygen species, and reduced the total iron content, eventually increasing the mortality H. cunea larvae. This study identified the toxicological mechanisms of NaF in lipid synthesis and energy metabolism in H. cunea, providing a basis for understanding the molecular mechanisms of NaF toxicity and developing pollution control strategies.

UDP-glucuronosyltransferase is involved in susceptibility of Chironomus kiiensis Tokunaga, 1936 (Diptera: Chironomidae) to insecticides.

UDP-glucuronosyltransferases (UGTs) could transform various exogenous and endogenous compounds, which help detoxification of pesticides in insects. To investigate the role of UGTs in the detoxification metabolism of insecticides in Chironomus kiiensis, CkUGT302M1, CkUGT302N1, CkUGT308N1 and CkUGT36J1 genes were identified with 1449-1599 bp encoding 482-532 amino acids. Four UGT genes shared 40.86∼53.36% identity with other homologous insect species, and expressed in all developmental stages, notably in the larval and adult stages. Expression of CkUGTs was higher in the gastric caecum, midgut and head. Moreover, CkUGTs expression and activity were significantly increased in C. kiiensis larvae in exposure to sublethal concentrations of carbaryl, deltamethrin and phoxim, respectively. To further explore the functions of UGT genes, the CkUGT308N1 was effectively silenced in 4th instar C. kiiensis larvae by RNA interference, which resulted in the mortality of dsCkUGT308N1 treated larvae increased by 71.43%, 111.11% and 62.50% under sublethal doses of carbaryl, deltamethrin and phoxim at the 24-h time point, respectively. The study revealed that the CkUGT308N1 gene in C. kiiensis could contribute to the metabolism of pesticides and provide a scientific basis for evaluating the water pollution of pesticides.

Integration of miRNA and mRNA expression profiles in Asian spongy moth Lymantria dispar in response to cyantraniliprole.

The Asian spongy moth, Lymantria dispar, is a worldwide forest pest that damages >500 plant species. Nowadays, chemical control is the most widely used method because of its rapidity and effectiveness, but the insecticide resistance is a growing concern for spongy moth. As important post-transcriptional regulators of gene expression, whether microRNAs (miRNAs) are involved in insecticide tolerance is little known in spongy moth. Therefore, an integrated analysis of miRNA and mRNA was performed on L. dispar larvae treated with cyantraniliprole. Compared to the control group, a total of 432 differentially expressed genes (DEGs) and 23 differentially expressed miRNAs (DEMs) were identified in L. dispar larvae under cyantraniliprole exposure. Among them, twelve DEGs encoding detoxification enzymes/proteins were further analyzed. Twenty-one genes related to insecticide tolerance were predicted by 11 DEMs, of which 25 miRNA-mRNA interactions were identified. In the miRNA-mRNA network, novel-miR-4 and mmu-miR-3475-3p were involved in the response of L. dispar to cyantraniliprole stress by regulating five genes associated with detoxification, respectively. The P450 gene CYP4C1 (c34384.graph_c0) was the only DEG related to detoxification in the network, which was regulated by novel-miR-4. The expression levels of ten DEMs were confirmed by quantitative reverse transcription PCR (RT-qPCR) and the trends were consistent with miRNA-seq. This study identified some candidate miRNAs and mRNAs related to cyantraniliprole tolerance in L. dispar, which provides valuable transcriptomic information for revealing the molecular mechanisms of insect tolerance and developing novel insecticides.

Functional characterization of a diuretic hormone receptor associated with desiccation, starvation and temperature tolerance in gypsy moth, Lymantria dispar.

Variety of diuretic hormone neuropeptides is known to regulate water and ion balance in invertebrates. By activating their specific neuropeptide, diuretic hormone receptor (DHR) transmits extracellular signals into the cell, and then produces functional cell activity, which plays an important role in regulating physiology and behavior. However, little is known about the function of DHR gene in Lymantria dispar. DHR gene was firstly identified in L. dispar and its physiological functions were investigated using RNA interference (RNAi) technology. The results showed that except for the 6th instar larvae, the expression levels of DHR gene in the larval stages are higher than that in the egg, pupal and adult stages. The DHR gene is highly expressed in hindgut and midgut tissues. The L. dispar larvae significantly increased their water content and high temperature tolerance after the DHR was silenced, while decreasing excretion and feeding behavior. The physiological function of DHR is associated with desiccation, high temperature and starvation resistance. DHR could contribute to future development of novel insecticide to manage this global forest pest population.

Morphology and Distribution of Antennal Sensilla in an Egg Parasitoid Wasp, Anastatus disparis (Hymenoptera: Eupelmidae).

The wasp Anastatus disparis is an egg endoparasitoid of a number of Lepidopteran pest species. To better understand the A. disparis olfactory system, we observed the antennal sensilla of males and females under a scanning electron microscope and quantified their sizes and morphological characteristics. We identified the types of sensilla and counted the numbers and locations of the different types on the dorsal and ventral antennal surfaces. The antennae of A. disparis are geniculate, with flagella that comprise 11 subsegments in females and eight in males. The mean antenna length was 1324.10 ± 52.50 µm in females and 1323.93 ± 65.20 µm in males. Ten sensillum types were identified in both sexes: Böhm bristles (BBs), sensilla trichodea (ST, with subtypes STI and STII), sensilla chaetica (SCh), sensilla basiconica (SB, with subtypes SBI and SBII), sensilla placodea (SP), sensilla coeleoconica (SCo), sensilla grooved peg (SGP), sensilla auricillica (SAu), sensilla campaniformia (SCa), and glandular pores (GPs). The total numbers of BBs, STI, SBII, SCa, SCo, and GPs did not differ significantly between the sexes, whereas the total numbers of SCh, SBI, and SAu were significantly greater in females, and those of STII, SP, and SGP were significantly lower. The types, number, and density of antennal sensilla increased from the base to the end. The possible functions of these sensilla in host-detection behavior are discussed.

Functional identification and characterization of GST genes in the Asian gypsy moth in response to poplar secondary metabolites.

The Asian gypsy moth, Lymantria dispar, as one of the most important forest pests in the world, can feed on more than 500 species of host plants, causing serious damage to the forests. Poplar is one of the favorite host plants of L. dispar. The present study aimed to explore the effects of poplar secondary metabolites on the growth and detoxification function of L. dispar larvae. We also aimed to study the expression of glutathione S-transferase (GST) genes in different developmental stages and in response to treatment with secondary metabolites. Six kinds of main secondary metabolites and three groups of characteristic mixed secondary metabolites were selected as follows: Caffeic acid, salicin, rutin, quercetin, catechol, flavone, mixture 1 (salicin and flavone), mixture 2 (salicin, caffeic acid and catechol), and mixture 3 (flavone, caffeic acid and catechol) according to the content changes of secondary metabolites in poplar. The thirteen GST genes were selected as candidate genes to study the expression of GST genes in different developmental stages and after treatment with secondary metabolites using quantitative real-time reverse transcription PCR. The LdGSTe4 and LdGSTo1 genes could be induced by secondary metabolites and were screened to explore their detoxification function against secondary metabolites using RNA interference technology. The results showed that salicin and rutin significantly induced the expression of LdGSTe4 and LdGSTo1. Under the stress of secondary metabolites, LdGSTe4 silencing affected the adaptability of L. dispar larvae to salicin and rutin. LdGSTe4 silencing resulted in a significant decrease in the body weight of L. dispar, but had little effect on the relative growth rate, relative consumption rate, efficiency of conversion of ingested food, efficiency of conversion of digested food, and approximate digestibility, as well as the survival rate and development time. These results provide a deeper understanding of the adaptive mechanism of L. dispar to host plants, form the foundation for the further research into the host resistance mechanism, and identify target genes for breeding resistant transgenic poplar.

Characterisation of GST genes from the Hyphantria cunea and their response to the oxidative stress caused by the infection of Hyphantria cunea nucleopolyhedrovirus (HcNPV).

The fall webworm, Hyphantria cunea (Drury) (Lepidoptera: Noctuidae), is a major pest found in forests. In this study, the effects of Hyphantria cunea nucleopolyhedrovirus (HcNPV) infection on the transcription levels and activities of glutathione S-transferases (GSTs) in H. cunea were determined. In the present study, 18 GST family genes were identified from the H. cunea transcriptome dataset by using bioinformatic analyses. These GST genes were classified into cytosolic (15 genes) and microsomal (three genes) classes. The 15 cytosolic GST genes belonged to four different subclasses (epsilon, sigma and delta). The all GST genes, especially GSTe4, showed high expression levels in egg and 1st~4th instar larval stage while their low expression levels in 5th~7th instar larvae using real-time quantitative PCR analysis. However, the expression levels of the 18 GST genes were varied after exposure to sublethal doses of HcNPV. The expression levels of most GSTs were downregulated and upregulated at low and high concentrations of HcNPV, respectively. The corresponding total GST activities also showed similar patterns. In H. cunea, changes in the expression levels and enzymatic activities of GSTs after exposure to HcNPV indicated that they may have important functions in the defense against HcNPV, and the stress, which may be reflected by the high GST enzymatic activities.

Effects of phenol on glutathione S-transferase expression and enzyme activity in Chironomus kiiensis larvae.

Detoxifying enzyme mRNAs are potentially useful stress biomarkers. Glutathione S-transferase (GST) metabolises lipophilic organic contaminants and mitigates oxidative damage caused by environmental pollutants. Herein, 12 Chironomus kiiensis GSTs (CkGSTs1-6, CkGSTt1-2, CkGSTd1-2, CkGSTm1-2) were cloned and grouped into sigma, theta, delta and microsomal subclasses. Open reading frames (450-699 bp) encode 170-232 amino acid proteins with predicted molecular masses of 17.31-26.84 kDa and isoelectric points from 4.94 to 9.58. All 12 GSTs were expressed during all tested developmental stages, and 11 displayed higher expression in fourth-instar larvae than eggs. GST activity after 24 h of phenol exposure was used to estimate environmental phenol contamination. After exposure to sublethal concentrations of phenol for 48 h, expression and activity of CkGSTs were inhibited in C. kiiensis larvae. Expression of CkGSTd1-2 and CkGSTs1-2 varied with phenol concentration, indicating potential use as biomarkers for monitoring environmental phenol contamination.

Physiological functions of a methuselah-like G protein coupled receptor in Lymantria dispar Linnaeus.

Insect G protein coupled receptors (GPCRs) have been identified as a highly attractive target for new generation insecticides discovery due to their critical physiological functions. However, few insect GPCRs have been functionally characterized. Here, we cloned the full length of a methuselah-like GPCR gene (Ldmthl1) from the Asian gypsy moth, Lymantria dispar. We then characterized the secondary and tertiary structures of Ldmthl1. We also predicted the global structure of this insect GPCR protein which is composed of three major domains. RNA interference of Ldmthl1 resulted in a reduction of gypsy moths' resistance to deltamethrin and suppressed expression of downstream stress-associated genes, such as P450s, glutathione S transferases, and heat shock proteins. The function of Ldmthl1 was further investigated using transgenic lines of Drosophila melanogaster. Drosophila with overexpression of Ldmthl1 showed significantly longer lifespan than control flies. Taken together, our studies revealed that the physiological functions of Ldmthl1 in L. dispar are associated with longevity and resistance to insecticide stresses. Potentially, Ldmthl1 can be used as a target for new insecticide discovery in order to manage this notorious forest pest.

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.

Cytochrome P450 CYP6EV11 in Chironomus kiiensis Larvae Involved in Phenol Stress.

Phenol is one of the organic pollutants which can cause water environment pollution. It is not only enriched in aquatic organisms but is also a serious threat to human health. Chironomus kiiensis is very sensitive to the contaminants in water and its cytochrome P450s are usually chosen as biomarkers for water pollution. To examine whether CYP6EV11 plays a role in the oxidative metabolism of phenol, we measured the silencing efficiency of CYP6EV11 and evaluated larval susceptibility to sublethal phenol levels by RNA interference (RNAi) technology. The results showed that the transcription of CYP6EV11 was found significantly up-regulated when the 4th instar C.kiiensis larvae were exposed to three doses of phenol. However, the transcriptional levels of CYP6EV11 were significantly suppressed by 92.7% in the 4th instar C. kiiensis larvae soaked in dsCYP6EV11 compared with those soaked in dsGFP for 6 h. The CYP6EV11 expression and mortality of the 4th instar C. kiiensis larvae with CYP6EV11 silencing were mostly decreased under phenol stress. Therefore, the CYP6EV11 gene may be used as a molecular biomarker for earlier warning and monitoring for water pollution.

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.

Characterization of the transcriptome of the Asian gypsy moth Lymantria dispar identifies numerous transcripts associated with insecticide resistance.

Although the Asian gypsy moth Lymantria dispar causes extensive forest damage worldwide, little is known regarding the genes involved in its development or response to insecticides. Accordingly, characterization of the transcriptome of L. dispar larvae would promote the development of toxicological methods for its control. RNA-seq analysis of L. dispar larvae messenger RNA (mRNA) generated 62,063 unigenes with N50 of 993 bp, from which 23,975 unique sequences (E-value < 10(-5)) were identified using a BLASTx search of the NCBI non-redundant (nr) database. Using functional classification in the Gene Ontology (GO) and Clusters of Orthologous Groups (COG) databases, 7,309 indentified sequences were categorized into 51 functional groups and 8,079 sequences were categorized into 25 functional groups, respectively. Moreover, we identified a large number of transcripts encoding known insecticide targets, or proteins involved in the metabolism of insecticides. Reads per kilobase of unigene length per million mapped reads (RPKM) analysis identified 39 high abundance transcripts, of which 27 exhibited significantly altered expression patterns across the egg, larvae, pupae, male and female adult stages. Our study provides the most comprehensive transcriptomic sequence resource for L. dispar, which will form the basis for future identification of candidate insecticide resistance genes in L. dispar.

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.

Extensive Ace2 duplication and multiple mutations on Ace1 and Ace2 are related with high level of organophosphates resistance in Aphis gossypii.

Aphis gossypii (Glover) has been found to possess multiple mutations in the acetylcholinesterase (AChE) gene (Ace) that might involve target site insensitivity. In vitro functional expression of AChEs reveals that the resistant Ace1 (Ace1R) and Ace2 (Ace2R) were significantly less inhibited by eserine, omethoate, and malaoxon than the susceptible Ace1 (Ace1S) and Ace2 (Ace2S). Furthermore, in both the mutant and susceptible AChEs, Ace2 was significantly less sensitive to eserine, omethoate, and malaoxon than Ace1. These results suggested that both the mutant Ace1 and Ace2 were responsible for omethoate resistance, while the mutant Ace2 played a major role in insecticide resistance. The DNA copy number and transcription level of Ace2 were 1.52- and 1.88-fold higher in the ORR strain than in the OSS strain. Furthermore, the DNA copy number and transcription level of Ace2 were significantly higher than that of Ace1 in either OSS or ORR strains, demonstrating the involvement of Ace2 gene duplication in resistance. Thus, the authors conclude that omethoate resistance in cotton aphids appears to have evolved through a combination of multiple mutations and extensive Ace2R gene duplication.

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.

Hemolin increases the immune response of a caterpillar to NPV infection.

Hemolin, a member of the immunoglobulin superfamily, plays a crucial role in the immune responses of insects against pathogens. However, the innate immune response of Hemolin to baculovirus infection varies among different insects, and the antiviral effects of Hemolin in Hyphantria cunea (HcHemolin) remain poorly understood. Our results showed that HcHemolin was expressed throughout all developmental stages, with higher expressions observed during pupal and adult stages of H. cunea. Additionally, HcHemolin was expressed in reproductive and digestive organs. The expression levels of the HcHemolin were induced significantly following H. cunea nucleopolyhedrovirus (HcNPV) infection. The susceptibility of H. cunea larvae to HcNPV decreased upon silencing of HcHemolin, resulting in a 40% reduction in median lifespan compared to the control group. The relative growth rate (RGR), the relative efficiency of consumption rate (RCR), the efficiency of the conversion of ingested food (ECI), and efficiency of the conversion of digested food (ECD) of silenced H. cunea larvae were significantly lower than those of the control group. Immune challenge assays showed that the median lifespan of treated H. cunea larvae was two-fold longer than the control group after HcNPV and HcHemolin protein co-injection. Therefore, we propose that HcHemolin plays a crucial role in regulating the growth, development, and food utilization of H. cunea, as well as in the antiviral immune response against HcNPV. These findings provide implications for the development of targeted nucleic acid pesticides and novel strategies for pollution-free biological control synergists for HcNPV.

The ethylene response factor gene, ThDRE1A, is involved in abscisic acid- and ethylene-mediated cadmium accumulation in Tamarix hispida.

Tamarix hispida is highly tolerant to salt, drought and heavy metal stress and is a potential material for the remediation of cadmium (Cd)-contaminated soil under harsh conditions. In this study, T. hispida growth and chlorophyll content decreased, whereas flavonoid and carotenoid contents increased under long-term Cd stress (25 d). The aboveground components of T. hispida were collected for RNA-seq to investigate the mechanism of Cd accumulation. GO and KEGG enrichment analyses revealed that the differentially expressed genes (DEGs) were significantly enriched in plant hormone-related pathways. Exogenous hormone treatment and determination of Cd2+ levels showed that ethylene (ETH) and abscisic acid (ABA) antagonists regulate Cd accumulation in T. hispida. Twenty-five transcription factors were identified as upstream regulators of hormone-related pathways. ThDRE1A, which was previously identified as an important regulatory factor, was selected for further analysis. The results indicated that ThABAH2.5 and ThACCO3.1 were direct target genes of ThDRE1A. The determination of Cd2+, ABA, and ETH levels indicated that ThDRE1A plays an important role in Cd accumulation through the antagonistic regulation of ABA and ETH. In conclusion, these results reveal the molecular mechanism underlying Cd accumulation in plants and identify candidate genes for further research.

Identification and characterization of key genes in insulin signaling pathway as molecular targets for controlling the fall webworm, Hyphantria cunea.

BACKGROUND: The insulin signaling pathway is closely related to metabolism, growth, reproductive capacity and lifespan of insects. However, the physiological function of the insulin signaling pathway is little known in Hyphantria cunea. RESULTS: Five insulin signaling pathway genes (HcInR, HcPI3K, HcAKT, HcFOXO and HcTOR) in H. cunea were identified and characterized in this study. The spatiotemporal expression profiles of the genes showed that HcInR, HcAKT, HcPI3K and HcTOR expressions were higher at the egg stage than those in other development stages, whereas HcFOXO was highly expressed in the adult stage; all of these genes were highly expressed in the larval digestive system, especially in the midgut and hindgut. After RNA interference (RNAi) of the five genes in 5th instar H. cunea larvae, weight gain and survival rate (except in the siHcAKT-injected group) were significantly decreased, and the developmental duration of larval and pupal stages were prolonged. In addition, knockdown of five genes in 7th instar larvae decreased the pupation rate, survival rate and oviposition capacity, and resulted in abnormal development during larval-pupal transition. CONCLUSION: Our findings indicate that the insulin signaling pathway plays essential roles in growth and development and the molting process in H. cunea, providing an important basis for developing new potentially molecular targets for RNAi-based pest control and understanding the mechanism of H. cunea outbreak. © 2022 Society of Chemical Industry.