Integrated transcriptome and proteome unveiled distinct toxicological effects of long-term cadmium pollution on the silk glands of Pardosa pseudoannulata.

Cadmium (Cd) induces severe soil pollution worldwide and exerts adverse effects on paddy field arthropods. Spiders grant a novel perspective to assess the Cd-induced toxicity, yet the impacts of long-term Cd stress on spider silk glands and its underlying mechanism remain elusive. The study showed that Cd stress enervated the antioxidant system in the spider Pardosa pseudoannulata, manifested as the decreases of glutathione peroxidase and peroxidase, and the increase of malonaldehyde (p < 0.05). In addition, a total of 1459 differentially expressed genes (DEGs) and 404 differentially expressed proteins (DEPs) were obtained from the silk glands' transcriptome and proteome. DEGs and DEPs encoding spidroin (e.g., tubuliform spidroin and ampullate spidroin) and amino acids metabolism (e.g., alanine, proline, and glycine) were distinctively down-regulated. Further enrichment analysis verified that Cd stress could inhibit amino acid metabolism via the down-regulation of several key enzymes, including glutathione synthase, methylthioadenosine phosphorylase, S-adenosylmethionine synthetase, etc. In addition, the hedgehog signaling pathway regulating cellular growth and development was down-regulated under Cd stress. A protein-protein interaction network showed that long-term Cd stress could inhibit some key biological processes in the silk glands, including peptide biosynthetic process and cytoskeleton part. Collectively, this comprehensive study established an effective animal detection model for evaluating Cd-induced toxicity, presented key biomarkers for further validation, and provided novel insights to investigate the molecular mechanisms of spiders to Cd pollution.

Genome-wide and expression-profiling analyses of the cytochrome P450 genes in Tenebrionidea.

Cytochrome P450 monooxygenases (CYPs) are present in almost all areas of the tree of life. As one of the largest and most diverse superfamilies of multifunctional enzymes, they play important roles in the metabolism of xenobiotics and biosynthesis of endogenous compounds, shaping the success of insects. In this study, the CYPome (an omics term for all the CYP genes in a genome) diversification was examined in the four Tenebrionidea species through genome-wide analysis. A total of 483 CYP genes were identified, of which 103, 157, 122, and 101 were respectively deciphered from the genomes of Tebebrio molitor, Asbolus verucosus, Hycleus cichorii and Hycleus phaleratus. These CYPs were classified into four major clans (mitochondrial, CYP2, CYP3, and CYP4), and clans CYP3 and CYP4 are most diverse. Phylogenetic analysis showed that most CYPs of these Tenebrionidea beetles from each clan had a very close 1:1 orthology to each other, suggesting that they originate closely and have evolutionally conserved function. Expression analysis at different developmental stages and in various tissues showed the life stage-, gut-, salivary gland-, fat body-, Malpighian tubule-, antennae-, ovary- and testis-specific expression patterns of T. molitor CYP genes, implying their various potential roles in development, detoxification, immune response, digestion, olfaction, and reproduction. Our studies provide a platform to understand the evolution of Tenebrionidea CYP gene superfamily, and a basis for further functional investigation of the T. molitor CYPs involved in various biological processes.

Comparative genomic analysis of carboxylesterase genes in Tenebrio molitor and other four tenebrionids.

Carboxylesterases (COEs) have various functions in wide taxons of organisms. In insects, COEs are important enzymes involved in the hydrolysis of a variety of ester-containing xenobiotics, neural signal transmission, pheromone degradation, and reproductive development. Understanding the diversity of COEs is basic to illustrate their functions. In this study, we identified 53, 105, 37, and 39 COEs from the genomes of Tenebrio molitor, Asbolus verucosus, Hycleus cichorii, and H. phaleratus in the superfamily of Tenebrionidea, respectively. Phylogenetic analysis showed that 234 COEs from these four species and those reported in Tribolium castaneum (63) could be divided into 12 clades and three major classes. The α-esterases significantly expanded in T. molitor, A. verucosus, and T. castaneum compared to dipteran and hymenopteran insects. In T. molitor, most COEs showed tissue and stage-specific but not a sex-biased expression. Our results provide insights into the diversity and evolutionary characteristics of COEs in tenebrionids, and lay a foundation for the functional characterization of COEs in the yellow mealworm.

Identification and expression profiling of serine protease-related genes in Tenebrio molitor.

In insects, serine proteases and serine protease homologs (SPs/SPHs) are involved in a variety of physiological processes including digestion, development, and immunity. Here, we identified 112 SP and 88 SPH genes in the genome of the yellow mealworm, Tenebrio molitor. Based on the features of domain structure, they were divided into "S" group containing single Tryp-SPc or Tryp-SPHc domain, "C" group containing 1-4 CLIP domain (CLIPA-D) and "M" group containing the CBD, CUB, EGF, Fz, Gd, LDLa, PAN, SEA, SR, Sushi, and TSP domains, and have 115, 48, and 37 gene members, respectively. According to the active sites in the catalytic triad, the putative trypsin, chymotrypsin, or elastase-like enzyme specificity of the identified SPs/SPHs were predicted. Phylogenetic and genomic location analyses revealed that gene duplication exists in the large amount of SPs/SPHs. Gene expression profiling using RNA-seq data along with real time reverse transcription-polymerase chain reaction analysis showed that most SP/SPH genes display life stage specific expression patterns, indicating their important roles in development. Many SP/SPH genes are specifically or highly expressed in the gut, salivary gland, fat body, hemocyte, ovary, and testis, suggesting that they participate in digestion, immunity, and reproduction. The findings lay the foundation for further functional characterization of SPs/SPHs in T. molitor.

Differences in pulmonary nodular consolidation and pulmonary cavity among drug-sensitive, rifampicin-resistant and multi-drug resistant tuberculosis patients: a computerized tomography study with history length matched cases.

Background: There have been concerns that literature described radiological feature differences between drug-sensitive pulmonary tuberculosis (DS-PTB) and multidrug-resistant (MDR)-PTB were confounded by that MDR-PTB cases tend to have a longer history. Using history length matched DS-PTB and MDR-PTB cases from a well-defined urban region in Dalian, we retrospectively analysed the CT feature differences of these paired cases with a focus on pulmonary nodular (PN) consolidation and pulmonary cavity (PC). Methods: There were 33 consecutive MDR-PTB cases [inclusive of rifampicin-resistant (RR) cases, 27 males and 6 females, mean age: 49.2 years], with 19 cases had a history of <1 month and 8 and 6 cases had a history of 1-6 and >6 months respectively. To pair the MDR-PTB cases with history length, matched 33 cases of DS-PTB patients (21 males and 12 females, mean age: 56.5 years) were included. All patients were new PTB without HIV infection. The first CT exams prior to treatment were analysed. Results: Compared with DS cases, MDR cases had a much higher prevalence of PN (75.76% vs. 45.45%) and a higher number of PN per positive case for PN (6.2 vs.1.53). For the cases >1 month history, MDR-PTB had a higher number of PC per positive case than that of DS-PTB cases (7.18 vs. 2.36). To differentiate DS-PTB from MDR-PTB, receiver operating characteristic (ROC) analysis showed a cutoff PN number of ≥3 had 48.5% sensitivity and 93.9% specificity, and a cutoff PC number of ≥4 had 39.4% sensitivity and 84.9% specificity. The lung field distribution of all lesions tended to be wider for MDR-PTB cases. MDR-PTB cases appeared to be associated with a faster progression in the absence of treatment. Conclusions: MDR-TB is likely intrinsically more invasive than DS-TB. Multiple PN and Multiple PC are promising signs for the suspicion of MDR-PTB on chest imaging.

Genomic and transcriptomic analyses of chitin metabolism enzymes in Tenebrio molitor.

Chitin is of great importance in the cuticle and inner cuticular linings of insects. Chitin synthases (CHSs), chitin deacetylases (CDAs), chitinases (CHTs), and ß-N-acetylhexosaminidases (HEXs) are important enzymes required for chitin metabolism, and play essential roles in development and metamorphosis. Although chitin metabolism genes have been well characterized in limited insects, the information in the yellow mealworm, Tenebrio molitor, a model insect, is presently still unavailable. With the help of bioinformatics, we identified 54 genes that encode putative chitin metabolism enzymes, including 2 CHSs, 10 CDAs, 32 CHTs, and 10 HEXs in the genome of T. molitor. All these genes have the conserved domains and motifs of their corresponding protein family. Phylogenetic analyses indicated that CHS genes were divided into two groups. CDA genes were clustered into five groups. CHT genes were phylogenetically grouped into 11 clades, among which 1 in the endo-ß-N-acetylglucosaminidases group and the others were classified in the glycoside hydrolase family 18 groups. HEX genes were assorted into six groups. Developmental and tissue-specific expression profiling indicated that the identified chitin metabolism genes showed dynamical expression patterns concurrent with specific instar during molting period, suggesting their significant roles in molting and development. They were predominantly expressed in different tissues or body parts, implying their functional specialization and diversity. The results provide important information for further clarifying their biological functions using the yellow mealworm as an ideal experimental insect.

Characterization and expression profiling of serine protease inhibitors in the yellow mealworm Tenebrio molitor.

Serine protease inhibitors (SPIs) act in diverse biological processes in insects such as immunity, development, and digestion by preventing the unwanted proteolysis. So far, the repertoire of genes encoding SPIs has been identified from few insect species. In this study, 62 SPI genes were identified from the genome of the yellow mealworm, Tenebrio molitor. According to their modes of action, they were classified into three families, serpin (26), canonical SPI (31), and α-macroglobulins (A2M) (5). These SPIs feature eight domains including serpin, Kazal, TIL, Kunitz, WAP, Antistasin, pacifastin, and A2M. In total, 39 SPIs contain a single SPI domain, while the others encode at least two inhibitor units. Based on the amino acids in the cleaved reactive sites, the abilities of these SPIs to inhibit trypsin, chymotrypsin, or elastase-like enzymes are predicted. The expression profiling based on the RNA-seq data showed that these genes displayed stage-specific expression patterns during development, suggesting to us their significance in development. Some of the SPI genes were exclusively expressed in particular tissues such as hemocyte, fat body, gut, ovary, and testis, which may be involved in biological processes specific to the indicated tissues. These findings provide necessary information for further investigation of insect SPIs.

Comparative genomic analysis of ABC transporter genes in Tenebrio molitor and four other tenebrionid beetles (Coleoptera: Tenebrionidea).

ATP-binding cassette (ABC) transporters, one of the largest transmembrane protein families, transport a diverse number of substate across membranes. Details of their diverse physiological functions have not been established. Here, we identified 87 ABC transporter genes in the genomes of Tenebrio molitor along with those from Asbolus verrucosus (104), Hycleus cichorii (65), and Hycleus phaleratus (80). Combining these genes (336 in total) with genes reported in Tribolium castaneum (73), we analyzed the phylogeny of ABC transporter genes in all five Tenebrionids. They are assigned into eight subfamilies (ABCA-H). In comparison to other species, the ABCC subfamily in this group of beetles appears expanded. The expression profiles of the T. molitor genes at different life stages and in various tissues were also investigated using transcriptomic analysis. Most of them display developmental specific expression patterns, suggesting to us their possible roles in development. Most of them are highly expressed in detoxification-related tissues including gut and Malpighian tubule, from which we infer their roles in insecticide resistance. We detected specific or abundant expressions of many ABC transporter genes in various tissues such as salivary gland, ovary, testis, and antenna. This new information helps generate new hypotheses on their biological significance within tissues.

The Wnt gene family in Tenebrio molitor and other coleopterans.

The Wnt gene family is involved in a wide range of developmental processes. Despite its significance, the evolution and function of Wnt genes remain largely unclear. Here, an exhaustive survey of Wnt genes was conducted in Tenebrio molitor and 17 other beetle genomes. A total of 146 Wnt genes were identified, creating a comprehensive coleopteran Wnt gene catalog. Comparative genomics indicates that dynamic evolutionary patterns of Wnt gene loss and duplication occurred in Coleoptera, leading to the diverse Wnt gene repertoire in various beetles. A striking loss of particular Wnt gene subfamilies occurs in Coleoptera. Remarkably, Wnt gene duplication was discovered for the first time in insects. Further analysis of Wnt gene expression in T. molitor indicates that each Wnt gene, including the duplicated ones, has a unique spatial or temporal expression pattern. The current study provides valuable insight into the evolution and functional validation of Wnt genes in Coleoptera.

DNA methylation-mediated expression of zinc finger protein 615 affects embryonic development in Bombyx mori.

Cell division and differentiation after egg fertilization are critical steps in the development of embryos from single cells to multicellular individuals and are regulated by DNA methylation via its effects on gene expression. However, the mechanisms by which DNA methylation regulates these processes in insects remain unclear. Here, we studied the impacts of DNA methylation on early embryonic development in Bombyx mori. Genome methylation and transcriptome analysis of early embryos showed that DNA methylation events mainly occurred in the 5' region of protein metabolism-related genes. The transcription factor gene zinc finger protein 615 ( ZnF615) was methylated by DNA methyltransferase 1 (Dnmt1) to be up-regulated and bind to protein metabolism-related genes. Dnmt1 RNA interference (RNAi) revealed that DNA methylation mainly regulated the expression of nonmethylated nutrient metabolism-related genes through ZnF615. The same sites in the ZnF615 gene were methylated in ovaries and embryos. Knockout of ZnF615 using CRISPR/Cas9 gene editing decreased the hatching rate and egg number to levels similar to that of Dnmt1 knockout. Analysis of the ZnF615 methylation rate revealed that the DNA methylation pattern in the parent ovary was maintained and doubled in the offspring embryo. Thus, Dnmt1-mediated intragenic DNA methylation of the transcription factor ZnF615 enhances its expression to ensure ovarian and embryonic development.

Integrated transcriptomics and proteomics provide new insights into the cadmium-induced ovarian toxicity on Pardosa pseudoannulata.

Cadmium (Cd) pollution is intractable heavy metal pollution in the farmland ecosystem, posing a life-threatening challenge to the paddy field organisms. Spiders are riveting animal biomarkers for evaluating Cd-induced toxicity, yet the effects of long-term Cd toxicity on spider reproductive function and its underlying mechanism remain unclear. In the present study, we found that Cd exposure impaired the antioxidant enzyme system in the wolf spider Pardosa pseudoannulata and decreased the concentration of four antioxidant enzymes (catalase, glutathione peroxidase, superoxide dismutase, and peroxidase) (p < 0.05). The content of vitellogenin and the number of hatched spiderlings were also dramatically reduced under Cd stress (p < 0.05), indicating that Cd stress could vitiate the fecundity of P. pseudoannulata. Moreover, a total of 10,511 differentially expressed genes (DEGs) and 391 proteins (DEPs) were yielded from the ovarian transcriptome and proteome, and a mass of genes and proteins involved in protein processing in endoplasmic reticulum (ER) were significantly down-regulated. DEGs and DEPs directly encoding the antioxidant enzyme system and/or vitellogenesis were also distinctively down-regulated. In addition, we illustrated that the PI3K-AKT signaling pathway might play a crucial role in regulating protein synthesis, cell cycle, growth, differentiation and survival in P. pseudoannulata. The effects of protein processing in ER and PI3K-AKT pathways could further trigger transcriptional factor Forkhead shackling the protein synthesis and cell growth process. Collectively, this integrated analysis identified the Cd-induced reproductive toxicity on P. pseudoannulata and provided multifaceted insights to investigate the molecular mechanisms of spiders to Cd pollution.

Comparative analysis unveils the cadmium-induced reproductive toxicity on the testes of Pardosa pseudoannulata.

Cadmium (Cd) pollution is one of the most serious heavy metal pollutions in the world, which has been demonstrated to cause different toxicities to living organisms. Cd has been widely suggested to cause reproductive toxicity to vertebrates, yet its reproductive toxicity to invertebrates is not comprehensive. In this study, the wolf spider Pardosa pseudoannulata was used as a bioindicator to evaluate the male reproductive toxicity of invertebrates under Cd stress. Cd stress had no effect on the color, size and length of testis. However, Cd significantly increased the contents of catalase, glutathione peroxidase and malondialdehyde, the antioxidants in the testis of P. pseudoannulata. Then we analyzed the transcriptome of testis exposed to Cd, and identified a total of 4739 differentially expressed genes (DEGs) compared to control, with 2368 up-regulated and 2371 down-regulated. The enrichment analysis showed that Cd stress could affect spermatogenesis, sperm motility, post-embryonic development, oxidative phosphorylation and metabolism and synthesis of male reproductive components. At the same time, the protein-protein interaction network was constructed with the generated DEGs. Combined with the enrichment analysis of key modules, it revealed that Cd stress could further affect the metabolic process in testis. In general, the analysis of testicular damage and transcriptome under Cd stress can provide a novel insight into the reproductive toxicity of Cd on rice filed arthropods and offer a reference for the protection of rice filed spiders under Cd pollution.

Molecular response uncovers neurotoxicity of Pardosa pseudoannulata exposed to cadmium pressure.

Cadmium (Cd) is a widely distributed heavy metal in south of China. Growing evidence indicates that systemic exposure to Cd, particularly the long-term exposure, may cause neurotoxic effects. Nevertheless, mechanisms underlying Cd neurotoxicity remain not completely understood. In this report, we investigated the neural alterations in the spider Pardosa pseudoannulata (Bösenberg and Strand, 1906) exposed to long-term Cd (LCd) and short-term Cd (SCd) pressure. Cd stress lowered foraging ability and prey consuming time in the spiders. In addition, enzymatic analysis results indicated that Cd exposure reduced the level of acetylcholinesterase at subcellular level. We then identified differentially expressed genes (DEGs) in the Cd exposed spiders using pairwise comparisons and found that a large number of DEGs were related to neurotransmitter receptors and ion transport and binding proteins. Notably, LCd exposure harbored more altered genes in ion transporter activity comparing with SCd exposure. From six K-means clusters, 53 putative transcriptional factors (TFs) belonging to 21 families were characterized, and ZBTB subfamily displayed the most distinctive alterations in the characterized genes, which is assumed to play a key role in the regulation of ion transmembrane process under Cd stress. A protein-to-protein interaction network constructed by the yielded DEGs also showed that ion and receptor binding activities were affected under long-term Cd exposure. Four key modules from the network indicated that Cd may further down-regulate energy metabolism pathway in spiders. Collectively, this comprehensive analysis provides multi-dimensional insights to understand the molecular response of spiders to Cd exposure.

Cadmium exposure alters expression of protective enzymes and protein processing genes in venom glands of the wolf spider Pardosa pseudoannulata.

Cadmium (Cd) pollution is currently the most serious type of heavy metal pollution throughout the world. Previous studies have shown that Cd elevates the mortality of paddy field spiders, but the lethal mechanism remains to be explored profoundly. In the present study, we measured the activities of protective enzymes (acetylcholinesterase, glutathione peroxidase, phenol oxidase) and a heavy metal chelating protein (metallothionein) in the pond wolf spider Pardosa pseudoannulata after Cd exposure. The results indicated that Cd initially increased the enzyme activities and protein concentration of the spider after 10- and 20-day exposure before inhibiting them at 30-day exposure. Further analysis showed that the enzyme activities in the cephalothorax were inhibited to some extent. Since the cephalothorax region contains important venom glands, we performed transcriptome sequencing (RNA-seq) analysis of the venom glands collected from the spiders after long-term Cd exposure. RNA-seq yielded a total of 2826 differentially expressed genes (DEGs), and most of the DEGs were annotated into the process of protein synthesis, processing and degradation. Furthermore, a mass of genes involved in protein recognition and endoplasmic reticulum (ER) -associated protein degradation were down-regulated. The reduction of protease activities supports the view that protein synthesis and degradation in organelles and cytoplasm were dramatically inhibited. Collectively, our outcomes illustrate that Cd poses adverse effects on the expression of protective enzymes and protein, which potentially down-regulates the immune function in the venom glands of the spiders via the alteration of protein processing and degradation in the ER.

The antibiotic jinggangmycin increases brown planthopper (BPH) fecundity by enhancing rice plant sugar concentrations and BPH insulin-like signaling.

The brown planthopper (BPH), Nilaparvata lugens, is a resurgent pest with an unexpected response to jinggangmycin (JGM), a broadly applied antibiotic used to control rice sheath blight disease. JGM stimulates BPH fecundity, but the underlining molecular mechanisms remain unclear. Here we report that JGM sprays led to increased glucose concentrations, photosynthesis and gene expression, specifically Rubsico, sucrose phosphate synthase, invertase 2 (INV2) and INV3 in rice plants. JGM sprays led to high-glucose rice plants. Feeding BPH on these plants led to increased insulin-like signaling and vitellogenin synthesis. Treating BPH with metformin, a gluconeogenesis inhibitor, reversed the influence of feeding on high-glucose rice, which was rescued by glucose injections. Silencing insulin-like peptide 2 using per os dsRNA led to reduction in juvenile hormone (JH) III titers and other fecundity parameters, which were reversed by topical applications of the JH analog, methoprene. We infer that JGM acts via two broad mechanisms, one through increasing rice plant sugar concentrations and a second by upregulating BPH insulin-like signaling.

Biogenic amine biosynthetic and transduction genes in the endoparasitoid wasp Pteromalus puparum (Hymenoptera: Pteromalidae).

Biogenic amines (BAs), such as octopamine, tyramine, dopamine, serotonin, and acetylcholine regulate various behaviors and physiological functions in insects. Here, we identified seven genes encoding BA biosynthetic enzymes and 16 genes encoding BA G protein-coupled receptors in the genome of the endoparasitoid wasp, Pteromalus puparum. We compared the genes with their orthologs in its host Pieris rapae and the related ectoparasitic wasp Nasonia vitripennis. All the genes show high (>90%) identity to orthologs in N. vitripennis. P. puparum and N. vitripennis have the smallest number of BA receptor genes among the insect species we investigated. We then analyzed the expression profiles of the genes, finding those acting in BA biosynthesis were highly expressed in adults and larvae and those encoding BA receptors are highly expressed in adults than immatures. Octα1R and 5-HT7 genes were highly expressed in salivary glands, and a high messenger RNA level of 5-HT1A was found in venom apparatuses. We infer that BA signaling is a fundamental component of the organismal organization, homeostasis and operation in parasitoids, some of the smallest insects.

Genome-wide characterization and transcriptomic analyses of neuropeptides and their receptors in an endoparasitoid wasp, Pteromalus puparum.

In insects, neuropeptides constitute a group of signaling molecules that act in regulation of multiple physiological and behavioral processes by binding to their corresponding receptors. On the basis of the bioinformatic approaches, we screened the genomic and transcriptomic data of the parasitoid wasp, Pteromalus puparum, and annotated 36 neuropeptide precursor genes and 33 neuropeptide receptor genes. Compared to the number of precursor genes in Bombyx mori (Lepidoptera), Chilo suppressalis (Lepidoptera), Drosophila melanogaster (Diptera), Nilaparvata lugens (Hemiptera), Apis mellifera (Hymenoptera), and Tribolium castaneum (Coleoptera), P. puparum (Hymenoptera) has the lowest number of neuropeptide precursor genes. This lower number may relate to its parasitic life cycle. Transcriptomic data of embryos, larvae, pupae, adults, venom glands, salivary glands, ovaries, and the remaining carcass revealed stage-, sex-, and tissue-specific expression patterns of the neuropeptides, and their receptors. These data provided basic information about the identity and expression profiles of neuropeptides and their receptors that are required to functionally address their biological significance in an endoparasitoid wasp.

Genomic and transcriptomic analyses of glutathione S-transferases in an endoparasitoid wasp, Pteromalus puparum.

Pteromalus puparum is a gregarious pupal endoparasitoid with a wide host range. It deposits eggs into pierid and papilionid butterfly pupae. Glutathione S-transferases (GSTs) are a family of multifunctional detoxification enzymes that act in xenobiotic metabolism in insects. Insect genome projects have facilitated identification and characterization of GST family members. We identified 20 putative GSTs in the P. puparum genome, including 19 cytosolic and one microsomal. Phylogenetic analysis showed that P. puparum GSTs are clustered into Hymenoptera-specific branches. Transcriptomic data of embryos, larvae, female pupae, male pupae, female adults, male adults, venom glands, carcass, salivary glands, and ovaries revealed stage-, sex-, and tissue-specific expression patterns of GSTs in P. puparum. This is the most comprehensive study of genome-wide identification, characterization, and expression profiling of GST family in hymenopterans. Our results provide valuable information for understanding the metabolic adaptation of this wasp.

The Pupal Ectoparasitoid Pachycrepoideus vindemmiae Regulates Cellular and Humoral Immunity of Host Drosophila melanogaster.

The immunological interaction between Drosophila melanogaster and its larval parasitoids has been thoroughly investigated, however, little is known about the interaction between the host and its pupal parasitoids. Pachycrepoideus vindemmiae, a pupal ectoparasitoid of D. melanogaster, injects venom into its host while laying eggs on the puparium, which regulates host immunity and interrupts host development. To resist the invasion of parasitic wasps, various immune defense strategies have been developed in their hosts as a consequence of co-evolution. In this study, we mainly focused on the host immunomodulation by P. vindemmiae and thoroughly investigated cellular and humoral immune response, including cell adherence, cell viability, hemolymph melanization and the Toll, Imd, and JAK/STAT immune pathways. Our results indicated that venom had a significant inhibitory effect on lamellocyte adherence and induced plasmatocyte cell death. Venom injection and in vitro incubation strongly inhibited hemolymph melanization. More in-depth investigation revealed that the Toll and Imd immune pathways were immediately activated upon parasitization, followed by the JAK/STAT pathway, which was activated within the first 24 h post-parasitism. These regulatory effects were further validated by qPCR. Our present study manifested that P. vindemmiae regulated the cellular and humoral immune system of host D. melanogaster in many aspects. These findings lay the groundwork for studying the immunological interaction between D. melanogaster and its pupal parasitoid.

Identification of novel antimicrobial peptides from rice planthopper, Nilaparvata lugens.

In this study, two novel antibacterial peptide genes, termed lugensin A and B were identified and characterized from a rice sap-sucking hemipteran insect pest, the brown planthopper, Nilaparvata lugens. Lugensin gene expression was significantly induced by Gram-negative and Gram-positive bacterial stains under the regulation of a signal receptor, the long peptidoglycan recognition protein (PGRP-LC) in the IMD pathway. Knockdown of PGRP-LC by RNAi eliminated bacterium induced Lugensin gene expression. Lugensins had the apparent antibacterial activities against Escherichia coli K12, Bacillus subtilis and the rice bacterial brown stripe pathogen Acidovorax avenae subsp. avenae (Aaa) strain RS-1. Lugensins inhibited bacterial proliferation by disrupting the integrity of the bacterial membranes. Scanning electron microscopy revealed abnormal membrane morphology of the recombinant Lugensin-treated bacteria. Lugensins induced complete cell disruption of E. coli K12 and B. subtilis strains while formed the holes on the cell surface of Aaa RS-1 strain. Immunofluorescence showed that Lugensins localized in the cell membrane of E. coli K12 while accumulated in the cytosol of B. subtilis. Differently, Lugensins remained in both the cell membrane and the cytosol of Aaa RS-1 strain, suggesting different action modes of Lugensins to different microbes. This is the first report of the novel antibacterial peptides found in the rice sap-sucking hemipteran insect species.