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.

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.

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.

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.

Preparation of Trichoderma asperellum Microcapsules and Biocontrol of Cucumber Powdery Mildew.

Microencapsulation is an important technique for protecting the viability and activity of microorganisms under adverse environmental conditions. To improve biological control, controlled-release microcapsules of Trichoderma asperellum were prepared and embedded in combinations of the biodegradable wall materials sodium alginate (SA). The microcapsules were evaluated for their ability to control cucumber powdery mildew in the greenhouse. The results showed that the highest encapsulation efficiency of 95% was obtained by applying 1% SA and 4% calcium chloride. The microcapsules provided good, controlled release and UV resistance, and could be stored for a long time. The greenhouse experiment revealed that the T. asperellum microcapsules had a maximal biocontrol efficiency of 76% against cucumber powdery mildew. In summary, embedding T. asperellum in microcapsules is a promising technique to improve the survivability of T. asperellum conidia. The T. asperellum microcapsules exerted significant biocontrol efficiency against cucumber powdery mildew. IMPORTANCE Trichoderma asperellum is widely found in plant roots and soil and has been used for the biocontrol of various plant pathogens; however, the control efficiency of T. asperellum is usually unstable in field trials. To improve the control efficiency of T. asperellum, in the present study, T. asperellum microcapsules were prepared using sodium alginate as wall material to reduce the effects of temperature, UV irradiation, and other environmental factors on its activity, and to significantly improve its biocontrol efficiency on cucumber powdery mildew. Microcapsules can prolong the shelf life of microbial pesticides. This study provides a new way to prepare a biocontrol agent against cucumber powdery mildew with high efficiency.

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.

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.

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 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.

RNAi-based functional analysis of bursicon genes related to wing expansion in gypsy moths.

Bursicon is a heterodimeric neuropeptide composed of Burs-α and Burs-ß subunits that plays an important role in cuticle tanning and wing expansion in insects. In this study, full-length cDNAs of Burs-α (LdBurs-α) and Burs-ß (LdBurs-ß) genes were identified in gypsy moth (Lymantria dispar) and cloned. The 480 bp and 420 bp open reading frames (ORFs) encode 159 and 129 amino acid polypeptides, respectively. LdBurs-α and LdBurs-ß have 11 conserved cysteine residues, and LdBurs-α and LdBurs-ß genes were expressed during all developmental stages according to quantitative reverse transcription PCR (qRT-PCR), with highest expression in the egg stage. High expression levels were also detected in the haemolymph, cuticle and head. To explore the physiological functions of LdBurs-α and LdBurs-ß, the genes were knocked down in larvae and pupae using RNA interference (RNAi), and expression levels of LdBurs-α and LdBurs-ß were decreased by 42.26-80.09%. Wing defects were observed in L. dispar pupae following Ldbursion silencing, with a phenotypic percentage ranging from 10.17% to 15.00%. RNAi-mediated knockdown of Ldbursicon prevented the expansion of male and female L. dispar adult wings, with malformation rates ranging from 6.38% and 30.00% to 57.69% and 69.23%, but no cuticle tanning defects were observed in pupae or adults. The results indicate that bursicon plays a key role in wing expansion in L. dispar adults, making it a potentially novel molecular target for insecticide-based control of this pest species.

Bursicon receptor gene HLGR2 as a potential RNA interference target for control of the fall webworm Hyphantria cunea.

BACKGROUND: Insect G protein-coupled receptors (GPCRs) have been identified as a new generation of attractive targets for RNA interference (RNAi)-based pest control. A functional study of the leucine-rich repeat-containing (LGR2) gene in Hyphantria cunea (HLGR2) was performed to examine whether it can be used in the molecular control of this notorious pest. RESULTS: The complementary DNA (cDNA) sequence and deduced amino acids of HLGR2 were obtained and analyzed in the present study. HLGR2 is a typical GPCR and shows high structural and sequence similarity with other insect LGR2 proteins. The spatiotemporal expression profiles of HLGR2 showed that HLGR2 was highly expressed at the egg stage and tissues of head and silk gland. After RNAi of HLGR2, distinct phenotypes were observed when HLGR2 expression was suppressed, indicating that HLGR2 is essential in pupation and eclosion. HLGR2 RNAi led to a low pupation rate (45.00%), body malformation, abnormal wing expansion, failed cuticle melanization (63.33%), and high mortality rate (48.33%). Furthermore, we identified eight genes that are regulated by HLGR2. The expression of these eight genes was induced by the HLGR2 signaling pathway and correlated well with cuticle sclerotization. Unlike LGR2 in other insect species, HLGR2 was found to play a crucial role in the control of H. cunea during ecdysis and postecdysial stages. CONCLUSION: HLGR2 is essential for the growth and development and wing expansion and maturation in H. cunea, suggesting HLGR2 is a promising candidate for application in RNAi-based control of this notorious agriculture-forest pest. © 2021 Society of Chemical Industry.

Functional Identification and Characterization of Leucokinin and Its Receptor in the Fall Webworm, Hyphantria cunea.

Neuropeptides function as central neuromodulators and circulating hormones that modulate insect behavior and physiology. Leucokinin (LK) is an intercellular signaling molecule that mediates many physiological and behavioral processes. However, the functions of LK associated with environmental stress and feeding behavior in the fall webworm, Hyphantria cunea, is little known. Our primary objective is to understand the function of LK and LK receptor (LKR) neuroendocrine system in H. cunea. In the present study, the results showed that LK/LKR are expressed at different developmental stages and in various tissues of H. cunea. A candidate receptor-ligand pairing for LK was identified in the larval transcriptome of H. cunea. In a heterologous expression system, the calcium assay was used to demonstrate that LKR is activated by HcLKs in a dose-dependent manner, with 50% effective concentration (EC50) values of 8.44-90.44nM. Knockdown of HcLK and HcLKR by microinjecting target-specific dsRNA leads to several effects in H. cunea, including feeding promotion, increase in resistance to desiccation and starvation stress, and regulation of water homeostasis. The transcript levels of HILP2 (except in the LK knockdown group), HILP5, and HILP8 increased, whereas those of HILP3, HILP4, and HILP6 decreased; HILP1, HILP2 (in the LK knockdown group), and HILP7 gene expression was not influenced after LK and LKR knockdown. Variations in mRNA expression levels in insulin-like peptide genes in the knockdown larvae suggest an essential role of these genes in survival in H. cunea. To our knowledge, the present study is the first comprehensive study of LK and LKR - from gene to behavior - in H. cunea.

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.

Effects of elevated CO2 treatment of Populus davidiana × P. bolleana on growth and detoxifying enzymes in gypsy moth, Lymantria dispar.

To date, elevated CO2 concentrations in the environment caused by various human activities influence diverse areas of life, including the interactions between insects and plants. The Lymantria dispar is one of the most severely destructive pests, which further could inflict ecological and economical damage. In this experiment, one-year-old Populus davidiana × P. bolleana plants were grown in CO2-enhanced environments for one month at three different CO2 concentrations: 397 ppm (atmospheric CO2 concentration), 550 ppm and 750 ppm (two predicted elevated CO2 concentrations). The 3rd instar L. dispar larvae then fed on the treated poplar seedlings covered in a nylon bag. The L. dispar larvae fed on poplar seedling treated for 96 h showed the highest growth rate at all CO2 concentrations. Enzymatic activity of treated larvae showed the highest GST and P450 activity at 750 ppm CO2. The relative expressions of seven CYP and ten GST genes in L. dispar larvae were analyzed quantitatively using real-time RT-PCR, which the results were expressed variably. Compared to 397 ppm CO2, the expression of CYP4L23 was down-regulated, while the expressions of other CYP genes were up-regulated. Meanwhile, only GSTo1 gene showed down-regulated at 48 h and 96 h in 750 ppm CO2 treatment, while GST expression level for the other nine GST genes showed up-regulated at 48 h and 72 h. These results offer the insight into plant-insect interactions under global climate change and furthermore will provide essential information for strategic pest control based on biochemical and molecular levels changes in gypsy moths.

Screening and evaluation of the stability of expression of reference genes in Lymantria dispar (Lepidoptera: Erebidae) using qRT-PCR.

The quantitative real-time polymerase chain reaction (qRT-PCR) has rapidly become the most sensitive and accurate method for the quantitative analysis of gene expression. Normalization of gene expression to that of relatively stably expressed housekeeping genes is required to facilitate the study of gene expression and to obtain more accurate RT-PCR data. However, no studies of the stability of expression of housekeeping genes in Lymantria dispar have been reported. In the present study, BestKeeper, GeNorm and NormFinder statistical software was used to evaluate the expression of thirteen candidate reference genes in L. dispar under different conditions. The expression levels of candidate reference genes were determined for two biological factors (developmental stages and tissues) and four abiotic treatments (temperature, insecticide, CO2 and starvation). The results showed that the best candidate reference genes in L. dispar were TUB, AK, RPS15 for developmental stages, RPL32 and GAPDH for tissues, ACTB and EF1-α for CO2 stress, GAPDH and RPL32 for temperature stress, RPS3 and GAPDH for insecticide stress, and GAPDH and RPS3 for starvation stress. In summary, EF1-α and TUB are preferential housekeeping genes in L. dispar under various conditions. These results provide a basis for the further study of functional genes of L. dispar.

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.

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.

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.

Effects of poplar secondary metabolites on performance and detoxification enzyme activity of Lymantria dispar.

To identify the effects of poplar secondary metabolites on Lymantria dispar, six poplar secondary metabolites (i.e., caffeic acid, salicin, rutin, quercetin, flavone, and catechol) and three mixtures containing characteristic secondary metabolites in poplar were selected. Mixture 1 contained flavone and salicin, mixture 2 contained salicin, caffeic acid, and catechol, and mixture 3 contained flavone, catechol, and caffeic acid. Mixtures were added to artificial diets used to feed 2nd instar L. dispar larvae. The effects of different secondary metabolites on larval growth and development, antifeedant activity, nutrient utilization, and detoxifying enzymatic activity were investigated. Results revealed that there were different influences on L. dispar larvae. The maximum antifeedant rate of flavone was 87.58%. Larvae treated with mixture 2 had a significantly longer development time of 5.61 d with a survival rate of 38.75% for 15 d, which is lower than a single secondary metabolite. No L. dispar larvae survived on feeding diets containing flavone for 7 d. An increase in GST and P450 activities in larvae was significantly induced during the 72 h feeding on artificial diets containing experimental secondary metabolites. After treatment containing salicin and flavone for 24-72 h, P450 activity increased at first then decreased. These results provide a foundation for further investigation on the host selection and underlying adaptation mechanisms in L. dispar.

Ocular Albinism Type 1 Regulates Deltamethrin Tolerance in Lymantria dispar and Drosophila melanogaster.

The ocular albinism type 1 (OA1), a pigment cell-specific integral membrane glycoprotein, is a member of the G-protein-coupled receptor (GPCR) superfamily that binds to heterotrimeric G proteins in mammalian cells. We aimed to characterize the physiological functions an insect OA1 from Lymantria dispar (LdOA1) employs in the regulation of insecticide tolerance. In the present study, we investigated the roles of LdOA1 in response to deltamethrin exposure in both L. dispar and Drosophila melanogaster. LdOA1 was expressed at the lowest level during the 4th instar stage, while LdOA1 was significantly upregulated in the 5th instar and male stages. Knockdown of LdOA1 by injecting dsRNA of LdOA1 into gypsy moth larvae caused a 4.80-fold higher mortality than in control larvae microinjected with dsRNA of GFP under deltamethrin stress. Nine out of 11 L. dispar CYP genes were significantly downregulated under deltamethrin stress in LdOA1 silenced larvae as compared to control larvae. Moreover, the LdOA1 gene was successfully overexpressed in D. melanogaster using transgenic technique. The deltamethrin contact assay showed that the LdOA1 overexpression in flies significantly enhanced the tolerance to deltamethrin compared to the control flies. Furthermore, the downstream Drosophila CYP genes were upregulated in the LdOA1 overexpression flies, suggesting LdOA1 may play a master switch role in P450-mediated metabolic detoxification. This study is the first report of an insect OA1 gene regulating insecticide tolerance and potentially playing a role in the regulation of downstream cytochrome P450 expression. These results contribute to the future development of novel insecticides targeting insect GPCRs.