Infection with 'Candidatus Liberibacter asiaticus' improves the fecundity of Diaphorina citri aiding its proliferation: A win-win strategy.

The evolution of insect vector-pathogen relationships has long been of interest in the field of molecular ecology. One system of special relevance, due to its economic impacts, is that between Diaphorina citri and 'Candidatus Liberibacter asiaticus' (CLas), the cause of the severe Asian form of huanglongbing. CLas-positive D. citri are more fecund than their CLas-negative counterparts, boosting opportunities for pathogens to acquire new vector hosts. The molecular mechanism behind this life-history shift remains unclear. Here, we found that CLas promoted ovarian development and increased the expression of the vitellogenin receptor (DcVgR) in ovaries. DcVgR RNAi significantly decreased fecundity and CLas titer in ovaries, extended the preoviposition period, shortened the oviposition period and blocked ovarian development. Given their importance in gene regulation, we explored the role of miRNAs in shaping these phenotypes and their molecular triggers. Our results showed that one miRNA, miR-275, suppressed DcVgR expression by binding to its 3' UTR. Overexpression of miR-275 knocked down DcVgR expression and CLas titer in ovaries, causing reproductive defects that mimicked DcVgR knockdown phenotypes. We focused, further, on roles of the Juvenile Hormone (JH) pathway in shaping the observed fecundity phenotype, given its known impacts on ovarian development. After CLas infection, this pathway was upregulated, thereby increasing DcVgR expression. From these combined results, we conclude that CLas hijacks the JH signalling pathway and miR-275, thereby targeting DcVgR to increase D. citri fecundity. These changes simultaneously increase CLas replication, suggesting a pathogen-vector host mutualism, or a seemingly helpful, but cryptically costly life-history manipulation.

Molecular identification of carboxylesterase genes and their potential roles in the insecticides susceptibility of Grapholita molesta.

Grapholita molesta is one of the most damaging pests worldwide in stone and pome fruits. Application of chemical pesticides is still the main method to control this pest, which results in resistance to several types of insecticides. Carboxylesterase (CarE) is one of the important enzymes involved in the detoxification metabolism and tolerance of xenobiotics and insecticides. However, the roles of CarEs in insecticides susceptibility of G. molesta are still unclear. In the present study, the enzyme activity of CarEs and the mRNA expression of six CarE genes were consistently elevated after treatment with three insecticides (emamectin benzoate, lambda-cyhalothrin, and chlorantraniliprole). According to spatio-temporal expression profiles, six CarE genes expressed differently in different developmental stages, and highly expressed in some detoxification metabolic organs. RNAi-mediated knockdown of these six CarE genes indicated that the susceptibility of G. molesta to all these three insecticides were obviously raised after GmCarE9, GmCarE14, GmCarE16, and GmCarE22 knockdown, respectively. Overall, these results demonstrated that GmCarE9, GmCarE14, GmCarE16, and GmCarE22 play a role in the susceptibility of G. molesta to emamectin benzoate, lambda-cyhalothrin, and chlorantraniliprole treatment. This study expands our understanding of CarEs in insects, that the same CarE gene could participate in the susceptibility to different insecticides.

Baculoviruses hijack the visual perception of their caterpillar hosts to induce climbing behaviour thus promoting virus dispersal.

Baculoviruses can induce climbing behaviour in their caterpillar hosts to ensure they die at elevated positions to enhance virus transmission, providing an excellent model to study parasitic manipulation of host behaviour. Here, we demonstrate that climbing behaviour occurred mostly during daylight hours, and that the height at death of Helicoverpa armigera single nucleopolyhedrovirus (HearNPV)-infected larvae increases with the height of the light source. Phototaxic and electroretinogram (ERG) responses were enhanced after HearNPV-infection in host larvae, and ablation of stemmata in infected larvae prevented both phototaxis and climbing behaviour. Through transcriptome and quantitative PCR, we confirmed that two opsin genes (a blue light-sensitive gene, HaBL; and a long wave-sensitive gene, HaLW) as well as the TRPL (transient receptor potential-like channel protein) gene, all integral to the host's visual perception pathway, were significantly upregulated after HearNPV infection. Knockout of HaBL, HaLW, or TRPL genes using the CRISPR/Cas9 system resulted in significantly reduced ERG responses, phototaxis, and climbing behaviour in HearNPV-infected larvae. These results reveal that HearNPV alters the expression of specific genes to hijack host visual perception at fundamental levels-photoreception and phototransduction-in order to induce climbing behaviour in host larvae.

Host miRNAs are involved in hormonal regulation of HaSNPV-triggered climbing behaviour in Helicoverpa armigera.

Baculoviruses manipulate host climbing behaviour to ensure that the hosts die at elevated positions on host plants to facilitate virus proliferation and transmission, which is a process referred to as tree-top disease. However, the detailed molecular mechanism underlying tree-top disease has not been elucidated. Using transcriptome analysis, we showed that two hormone signals, juvenile hormone (JH) and 20-hydroxyecdysone (20E), are key components involved in HaSNPV-induced tree-top disease in Helicoverpa armigera larvae. RNAi-mediated knockdown and exogenous hormone treatment assays demonstrated that 20E inhibits virus-induced tree-top disease, while JH mediates tree-top disease behaviour. Knockdown of BrZ2, a downstream signal of JH and 20E, promoted HaSNPV-induced tree-top disease. We also found that two miRNAs target BrZ2 and are involved in the cross-talk regulation between 20E and JH manipulating HaSNPV replication, time to death and HaSNPV-induced tree-top disease.

Digital gene expression analysis of Helicoverpa armigera in the early stage of infection with Helicoverpa armigera nucleopolyhedrovirus.

Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus (HearNPV) is an obligatory and lethal parasite of the cotton bollworm and has been extensively used in China for the control of this notorious pest. Digital gene expression (DGE) analysis was adopted for an overall comparison of transcriptome profiling between HearNPV-infected and control healthy Helicoverpa armigera larvae during an early stage post-inoculation. A total of 908 differentially expressed genes (DEGs) were identified, of which 136 were up-regulated and 597 were down-regulated. GO category and KEGG pathway analysis demonstrated that the identified DEGs involved in ribosome biogenesis, aminoacyl-tRNA biosynthesis, protein processing in endoplasmic reticulum, biosynthesis of valine, leucine, isoleucine and the spliceosome were significantly down-regulated, whereas genes involved in pancreatic secretion, protein digestion and absorption and salivary secretion showed obviously up-regulated transcription. The DEGs were verified by quantitative real-time PCR, and genes that participated in defensive response, nutritional digestion and developmental regulation exhibited specific expression patterns in a continuous time-course assessment. These results provide basic data for future research on the molecular mechanism of HearNPV infection and the interactions between lepidopteran hosts and their specific NPV parasites.

Balancing metabolism and reproduction.

The bacterium responsible for a disease that infects citrus plants across Asia facilitates its own proliferation by increasing the fecundity of its host insect.

Supplementary sugars enhance the production efficiency and parasitism performance of the egg parasitoid Trichogramma dendrolimi (Hymenoptera: Trichogrammatidae).

Trichogramma spp. wasps are egg parasitoids with a long history of mass rearing for augmentation biocontrol programs in field crop and orchard landscapes. Supplementary nutrition can improve the longevity, fecundity, and biocontrol efficacy of parasitoids. To improve the production efficiency and parasitism performance of Trichogramma dendrolimi Matsumura (Hymenoptera: Trichogrammatidae), the present study screened and examined the potential supplementary nutrients for this biological control agent. Dietary supplementation with a 10% sucrose solution significantly increased wasp longevity and parasitism potential of T. dendrolimi on host eggs, but provision of pollen did not provide additional benefits. Laboratory and greenhouse cage tests demonstrated that wasp access to soybean aphid Aphis glycines Matsumura (Hemiptera: Aphididae) honeydew, comprised primarily of melezitose and trehalose, improved T. dendrolimi longevity and parasitism. In conclusion, provision of a 10% sucrose solution to adult wasps will enhance the mass-rearing efficiency of T. dendrolimi; furthermore, field release of T. dendrolimi by plant vectors bearing honeydew-producing aphids holds promise for improving the biocontrol efficacy of T. dendrolimi.

miR-2765 Modulates the Seasonal Polyphenism in Cacopsylla chinensis by Targeting a Novel Cold Rreceptor CcTRPC3.

Polyphenism is a beneficial way in organisms to better cope with changing circumstances and is a hot topic in entomology, evolutionary biology, and ecology. Until now, this phenomenon has been proven to be season-, density-, and diet-dependent; however, there are very few reports on temperature regulation. Cacopsylla chinensis showed seasonal polyphenism, namely as summer- and winter-form, with obvious diversity in phenotypic characteristics in response to seasonal variation. Previous studies have found that low temperature in autumn is an extremely important element in inducing summer-form change to winter-form, but the underlying regulatory mechanism is still a mystery. Herein, we provided the initial evidence that the third instar of the summer-form is the critical period for developing to the winter-form, and 10 °C induces this transition by affecting the total pigment, chitin level, and thickness of the cuticle. Second, CcTPRC3 was proven to function as a novel cold receptor to control this seasonal polyphenism. Moreover, miR-2765 was found to mediate seasonal polyphenism by inhibiting CcTRPC3 expression. Last, we found that cuticle binding proteins CcCPR4 and CcCPR9 function as the downstream signals of CcTRPC3 to regulate the seasonal polyphenism in C. chinensis. In conclusion, our results displayed a novel signal pathway of miR-2765 and CcTRPC3 for the regulation of seasonal polyphenism in C. chinensis. These findings provide insights into the comprehensive analysis of insect polyphenism and are useful in developing potential strategies to block the phase transition for the pest control of C. chinensis.

Nanoparticle-mediated calmodulin dsRNA and cyantraniliprole co-delivery system: High-efficient control of two key pear pests while ensuring safety for natural enemy insects.

Currently, the predominant method for managing pests in orchards is chemical control. However, prolonged use of chemicals leads to resistance issues and raise ecological safety. A promising approach to tackle these challenges involves nanoparticles-mediated delivery system of dsRNA and pesticides. Despite its potential, this strategy has not been widely applied in controlling pests in pear orchards. In this study, we developed a nanoparticle-mediated ternary biopesticide to tackle resistance and safety concerns associated with calmodulin dsRNA and cyantraniliprole. Initially, we assessed the effectiveness of cyantraniliprole against two key pear pests, Grapholita molesta and Cacopsylla chinensis. Subsequently, we observed an upregualtion of genes CaM and CN following cyantraniliprole treatment. Furthermore, inhibiting or silencing GmCaM and CcGaM enhanced the sensitivity to cyantraniliprole more effectively. By introducing hairpin RNA into the pET30a-BL21 RNaseIII system to silence GmCaM and CcCaM, we developed a nanoparticle-mediated co-delivery system that exhibited improved control over these two pests. Importantly, our research demonstrated that using reduced cyantraniliprole dosages through ternary biopesticides could help mitigate risks to natural enemies. Overall, our research emphasizes the enhanced effectiveness of ternary biopesticides in boosting the performance of dsRNA and pesticide against pear pests, while fostering environmental sustainability-a novel advancement in this field.

miR-252 targeting temperature receptor CcTRPM to mediate the transition from summer-form to winter-form of Cacopsylla chinensis.

Temperature determines the geographical distribution of organisms and affects the outbreak and damage of pests. Insects seasonal polyphenism is a successful strategy adopted by some species to adapt the changeable external environment. Cacopsylla chinensis (Yang & Li) showed two seasonal morphotypes, summer-form and winter-form, with significant differences in morphological characteristics. Low temperature is the key environmental factor to induce its transition from summer-form to winter-form. However, the detailed molecular mechanism remains unknown. Here, we firstly confirmed that low temperature of 10 °C induced the transition from summer-form to winter-form by affecting the cuticle thickness and chitin content. Subsequently, we demonstrated that CcTRPM functions as a temperature receptor to regulate this transition. In addition, miR-252 was identified to mediate the expression of CcTRPM to involve in this morphological transition. Finally, we found CcTre1 and CcCHS1, two rate-limiting enzymes of insect chitin biosyntheis, act as the critical down-stream signal of CcTRPM in mediating this behavioral transition. Taken together, our results revealed that a signal transduction cascade mediates the seasonal polyphenism in C. chinensis. These findings not only lay a solid foundation for fully clarifying the ecological adaptation mechanism of C. chinensis outbreak, but also broaden our understanding about insect polymorphism.

Mutation of the clock gene timeless disturbs diapause induction and adult emergence rhythm in Helicoverpa armigera.

BACKGROUND: Circadian rhythms are physical and behavioral changes that follow the 24-h cycle of Earth's light and temperature and are regulated by clock genes. Timeless (Tim) has been identified as a canonical clock gene in some insects, however, its functions have been little studied in lepidopteran pests. RESULTS: To investigate Tim (HaTim) gene function in Helicoverpa armigera, an important lepidopteran pest, we obtained the HaTim mutant using the CRISPR/Cas9 gene editing system. Our results showed that the transcript levels of HaTim rhythmically peaked at night in heads of the wild larvae and adult, and the diel expression of HaTim was sensitive to photoperiod and temperature. The expression rhythms of other clock genes, such as HaPer, HaCry1, HaCry2 and HaCwo, were disturbed in the HaTim mutant larvae, as that stage is a sensitivity period for diapause induction. Fifth-instar wild-type larvae could be induced to pupate in diapause under a short-day photoperiod and low temperature, however, fifth-instar HaTim mutant larvae could not be induced under the same conditions. In addition, the emergence of wild-type adults peaked early at night, but the rhythm was disturbed in the HaTim mutant with arrhythmic expression of some clock genes, such as HaPer, HaCry1 and HaCwo in adults. CONCLUSION: Our results suggest that the clock gene Tim is involved in diapause induction and adult emergence in H. armigera, and is a potential target gene for controlling pest. © 2023 Society of Chemical Industry.

Peroxiredoxin 1 transfer during mating protects eupyrene sperm against oxdative stress in Grapholita molesta.

BACKGROUND: Each Grapholita molesta female only copulates once during its lifetime and thus must maintain the viability of stored eupyrene sperm for male reproductive success. The male ejaculate comprises abundant accessory gland proteins produced by the male accessory gland (AG), and many of which are major effectors for sperm storage and maintenance. RESULTS: Here, we reported that an antioxidant protein, peroxiredoxin 1 (GmolPrx1), secreted by the male AG, is essential for protecting eupyrene sperm from oxidative stress and maintaining their quality during storage in the female bursa copulatrix (BC). Our data showed that GmolPrx1 is highly expressed in the AG of sexually mature males. The GmolPrx1 protein is localized to the cytoplasm of AG cells and delivered to the female BC during mating. Knockdown of GmolPrx1 strongly decreased the fertility of mated females. Additionally, we evaluated oxidative status in the spermatophore of females and found that the content of hydrogen peroxide increased significantly after mating with GmolPrx1 knockdown males. Finally, the quality assessment of eupyrene sperm demonstrated that the plasma membrane integrity, acrosome integrity, and DNA integrity were all severely impaired in the spermatophore of females after mating with GmolPrx1 knockdown males, which may contribute to the fertility decline in males. CONCLUSION: Our current data demonstrated that activities of eupyrene sperm stored in females can be significantly impaired by enhanced oxidative stress through knocking down of GmolPrx1 in males. Our finding thus may further lay new foundations for the control of G. molesta through suppressing their populations by manipulating male reproductive genes. © 2023 Society of Chemical Industry.

Safety of Bacillus thuringiensis Cry1Ah and Vip3Aa toxins for the predatory stink bug Arma custos (Hemiptera: Pentatomidae).

The widespread adoption of Bt crops expressing insecticidal proteins derived from Bacillus thuringiensis has created a need to assess the potential effects of these toxins on non-target organisms, especially species such as Arma custos, a generalist predator that provides important biological control services in many field crops in Asia. Direct dietary exposure of A. custos to Cry1Ah and Vip3Aa proteins produced no adverse effects on life history traits, despite continuous exposure throughout development and early adult life to concentrations significantly higher than the Bt protein concentration likely encountered by A.custos in the field, even when feeding directly on Bt plants. Enzyme-linked immunosorbent assay confirmed the presence of Bt proteins in A. custos midguts, but quantitative real-time PCR analysis of 12 genes associated with detoxification, antioxidative responses, immune responses, and metabolism revealed no significant changes in expression in adult bugs. Indirect exposure to these toxins via consumption of intoxicated prey, larvae of Helicoverpa armigera (Hübner), likewise produced no negative impacts on survival, development, adult weight, or female fecundity in either the F0 (exposed) or F1 (unexposed) generation, but female fresh weight was reduced in the F0 generation by the Cry1Ah (50 µg/g) treatment. Finally, a competitive binding assay with labelled protein and a ligand blotting assay both demonstrated that the Cry1Ah protein could not bind to receptors on the midgut brush border membrane vesicles (BBMVs) of A. custos adults. Therefore, we conclude that Cry1Ah and Vip3Aa proteins are unlikely to have significant negative effects on A. custos populations if employed as plant-incorporated protectants in field crops.

Laboratory evaluation of the compatibility of Beauveria bassiana with the egg parasitoid Trichogramma dendrolimi (Hymenoptera: Trichogrammatidae) for joint application against the oriental fruit moth Grapholita molesta (Lepidoptera: Tortricidae).

BACKGROUND: The entomopathogenic fungus Beauveria bassiana and the egg parasitoid Trichogramma dendrolimi can both contribute to biological control of the global fruit borer Grapholita molesta. To derive insights for optimizing their combined application in augmentation programs, we assayed fungal pathogenicity to both G. molesta (eggs, larvae and adults) and adult parasitoids, and assessed wasp acquisition and transmission of fungal spores following their emergence from B. bassiana-treated host eggs. RESULTS: Concentrations of 108 conidia mL-1 of B. bassiana and higher caused virtually complete mortality of G. molesta larvae, but less than 30% mortality of eggs, and female moths surviving treatments had reduced fecundity. More than 80% of adult T. dendrolimi survived B. bassiana treatments of 107 conidia mL-1 , and more than 60% survived 109 conidia mL-1 , with surviving females achieving reproductive success equivalent to controls. Parasitoid females preferred healthy eggs over B. bassiana-infected ones in choice tests; wasps emerging from treated eggs carried about 104 conidia each and contributed an additional 11% host mortality in trials combining both agents. CONCLUSION: Our results indicate that combined applications of B. bassiana and T. dendrolimi can have complimentary impacts on G. molesta, the wasps compensating for low fungal pathogenicity to eggs and their progeny potentially aiding in subsequent disease transmission. Although foraging wasps tended to avoid infected eggs, negative interactions between the two agents might be further mitigated by timing B. bassiana applications from late instar larva to early moth stage, and T. dendrolimi releases several days later to coincide with peak oviposition periods. © 2022 Society of Chemical Industry.

Functional analysis by RNAi of an glutaredoxin gene in Helicoverpa armigera.

Glutaredoxins play crucial roles in maintaining intracellular redox homeostasis via scavenging of excess reactive oxygen species. In this study, a glutaredoxin domain-containing cysteine-rich gene from Helicoverpa armigera, named HaGdccr, was characterized. Sequence analysis revealed that it contains a glutaredoxin domain and a conserved cysteine and shares high sequence identity with other insect genes. HaGdccr mRNA expression was highest in molting larvae of the 3rd instar and was mainly detected in the central nervous system of larvae and the wings of adults. Quantitative real-time PCR results revealed that the expression of HaGdccr was suppressed at 1 and 6 h and increased at 24 h after the larvae were treated with 4 °C and hydrogen peroxide. When the larvae were exposed to 20 °C, HaGdccr decreased at 1 h and was induced at 12 and 24 h. HaGdccr transcription level was downregulated at 2 and 12 h and upregulated at 24 h after the adults were exposed to 0 °C. However, transcript levels were increased by high temperature in both larvae and adults. After knockdown of HaGdccr by RNA interference, the expression of antioxidant genes, including thioredoxin-like (Trx-like), catalase (CAT), glutathione-S-transferase (GST), thioredoxin reductase (TrxR), and thioredoxin (Trx), was increased, whereas that of thioredoxin peroxidase (Tpx) was decreased. In addition, we found that HaGdccr knockdown enhanced the enzymatic activity of superoxide dismutase and the contents of hydrogen peroxide and ascorbate. Taken together, these results indicate that HaGdccr may play significant roles in protecting organisms against oxidative damage.

Molecular Identification of Two Thioredoxin Genes From Grapholita molesta and Their Function in Resistance to Emamectin Benzoate.

Thioredoxins (Trxs), a member of the thioredoxin system, play crucial roles in maintaining intracellular redox homeostasis and protecting organisms against oxidative stress. In this study, we cloned and characterized two genes, GmTrx2 and GmTrx-like1, from Grapholita molesta. Sequence analysis showed that GmTrx2 and GmTrx-like1 had highly conserved active sites CGPC and CXXC motif, respectively, and shared high sequence identity with selected insect species. The quantitative real-time polymerase chain reaction results revealed that GmTrx2 was mainly detected at first instar, whereas GmTrx-like1 was highly concentrated at prepupa day. The transcripts of GmTrx2 and GmTrx-like1 were both highly expressed in the head and salivary glands. The expression levels of GmTrx2 and GmTrx-like1 were induced by low or high temperature, E. coli, M. anisopliae, H2O2, and pesticides (emamectin benzoate). We further detected interference efficiency of GmTrx2 and GmTrx-like1 in G. molesta larvae and found that peroxidase capacity, hydrogen peroxide content, and ascorbate content all increased after knockdown of GmTrx2 or GmTrx-like1. Furthermore, the hydrogen peroxide concentration was increased by emamectin benzoate and the sensitivity for larvae to emamectin benzoate was improved after GmTrx2 or GmTrx-like1 was silenced. Our results indicated that GmTrx2 and GmTrx-like1 played vital roles in protecting G. molesta against oxidative damage and also provided the theoretical basis for understanding the antioxidant defense mechanisms of the Trx system in insects.

Molecular identification of three novel glutaredoxin genes that play important roles in antioxidant defense in Helicoverpa armigera.

Glutaredoxins (Grxs), also known as thioltransferases, play key roles in maintaining intracellular redox balance and protecting cells from oxidative damage in plants and mammals. We tested whether Grxs play important roles in antioxidant defense in insects using the moth, Helicoverpa armigera. We obtained the full-length cDNA sequences of three novel Grx genes, named HaGrx, HaGrx3, and HaGrx5. Sequence analysis indicated that HaGrx shared a high amino acid identity (58%-78%) and a CPYC motif of conserved redox activity with homologues from other selected insect species. In contrast, HaGrx3 and HaGrx5 both shared a CGF(S/G) motif, a conserved catalytic domain, with other orthologous genes. Quantitative real-time PCR results revealed that HaGrx, HaGrx3, and HaGrx5 exhibited temporally- and spatially-dependent patterns of expression. The mRNA expression of HaGrx, HaGrx3, and HaGrx5 was induced by various temperature stresses and H2O2 treatments. We further investigated the knockdown of HaGrx, HaGrx3, and HaGrx5 in H. armigera larvae and found that most of the selected antioxidant genes were up regulated. However, Tpx was down regulated, and further interpretation of the complementary functions of these antioxidant genes is still required. We also determined the effect of HaGrx, HaGrx3, and HaGrx5 knockdown on antioxidant enzymatic activity and metabolite content. The enzymatic activities of SOD, CAT, and POD, and the metabolite contents of hydrogen peroxide, ascorbate, protein carbonyl, and total GSH increased after RNAi mediated knockdown of HaGrx, HaGrx3, and HaGrx5. These results supported our hypothesis that HaGrx, HaGrx3, and HaGrx5 play important roles in antioxidant defense of Helicoverpa armigera and provided a theoretical basis for further in-depth study of physiological function in the insect glutaredoxin family genes.

Sequence analysis, expression profiles and function of thioredoxin 2 and thioredoxin reductase 1 in resistance to nucleopolyhedrovirus in Helicoverpa armigera.

The thioredoxin system, including NADPH, thioredoxin (Trx), and thioredoxin reductase (TrxR), plays significant roles in maintaining intracellular redox homeostasis and protecting organisms against oxidative damage. In this study, the characteristics and functions of H. armigera HaTrx2 and HaTrxR1 were identified. Sequence analysis showed that HaTrx2 and HaTrxR1 were both highly conserved and shared high sequence identity with other insect counterparts. The mRNA of HaTrx2 was expressed the highest in 5th instar 96 h and was mainly detected in heads and epidermis. The expression of HaTrxR1 was highly concentrated in 5th instar 72 h and 96 h, and higher in malpighian tube, midgut and hemocyte than other examined tissues. HaTrx2 and HaTrxR1 were markedly induced by various types of stress. HaTrx2- or HaTrxR1-knockdown increased ROS production in hemocytes and also increased the lipid damage in NPV infected H. armigera larvae. Furthermore, interference with expression of HaTrx2 or HaTrxR1 transcripts in H. armigera larvae resulted in increased sensitivity to NPV infection and shortened LT50 values. Our findings indicated that HaTrx2 and HaTrxR1 contribute to the susceptibility of H. armigera to NPV and also provided the theoretical basis for the in-depth study of insect thioredoxin system.

Identification of a thioredoxin peroxidase gene involved in resistance to nucleopolyhedrovirus infection in Helicoverpa armigera with RNA interference.

Thioredoxin peroxidases (Tpxs) play a crucial role in protection against oxidative damage in several insect species. However, studies on the characteristics and functions of Tpxs in Helicoverpa armigera are lacking. In this study, a novel 2-Cys Tpx gene from H. armigera (HaTpx) was identified. Sequence analysis revealed that HaTpx is highly conserved and shares two catalysis regions (VCP) with other insect species. HaTpx mRNA was found to be expressed in an age-dependent manner and was ubiquitous in all tissues examined. Hormone treatment showed that the expression of HaTpx is clearly induced by 20-hydroxyecdysone but repressed by Juvenile hormone. Additionally, extreme temperature, ultraviolet light, mechanical injury, Escherichia coli, Metarhizium anisopliae, nucleopolyhedrovirus (NPV) infection, and H2O2 treatment markedly induced HaTpx gene expression. Reactive oxygen species (ROS) levels in hemocytes and MDA concentrations in the hemolymph after NPV infection were evaluated, and the results indicated that NPV infection causes excessive ROS generation. After knockdown of HaTpx by RNA interference, the expression of three antioxidant genes (Cu/ZnSOD, Trx, and TrxR) was increased, whereas two antioxidant genes (CAT and GPX) showed decreased expression. Moreover, the susceptibility of H. armigera to NPV infection increased after HaTpx knockdown. These results indicated that HaTpx contributes to the susceptibility of H. armigera to NPV, and the results also provide a theoretical basis for a novel strategy for developing new chemicals and microbial pesticides that target HaTpx gene for controlling H. armigera.

Identification and validation of reference genes for normalization of gene expression analysis using qRT-PCR in Helicoverpa armigera (Lepidoptera: Noctuidae).

BACKGROUND: Recent studies have focused on determining functional genes and microRNAs in the pest Helicoverpa armigera (Lepidoptera: Noctuidae). Most of these studies used quantitative real-time PCR (qRT-PCR). Suitable reference genes are necessary to normalize gene expression data of qRT-PCR. However, a comprehensive study on the reference genes in H. armigera remains lacking. RESULTS: Twelve candidate reference genes of H. armigera were selected and evaluated for their expression stability under different biotic and abiotic conditions. The comprehensive stability ranking of candidate reference genes was recommended by RefFinder and the optimal number of reference genes was calculated by geNorm. Two target genes, thioredoxin (TRX) and Cu/Zn superoxide dismutase (SOD), were used to validate the selection of reference genes. Results showed that the most suitable candidate combinations of reference genes were as follows: 28S and RPS15 for developmental stages; RPS15 and RPL13 for larvae tissues; EF and RPL27 for adult tissues; GAPDH, RPL27, and ß-TUB for nuclear polyhedrosis virus infection; RPS15 and RPL32 for insecticide treatment; RPS15 and RPL27 for temperature treatment; and RPL32, RPS15, and RPL27 for all samples. CONCLUSION: This study not only establishes an accurate method for normalizing qRT-PCR data in H. armigera but also serve as a reference for further study on gene transcription in H. armigera and other insects.