Neuropeptide hormone bursicon mediates female reproduction in the whitefly, Bemisia tabaci (Hemiptera: Aleyrodidae).

Bursicon, a neuropeptide hormone comprising two subunits-bursicon (burs) and partner of burs (pburs), belongs to the cystine-knot protein family. Bursicon heterodimers and homodimers bind to the lucine-rich G-protein coupled receptor (LGR) encoded by rickets to regulate multiple physiological processes in arthropods. Notably, these processes encompass the regulation of female reproduction, a recent revelation in Tribolium castaneum. In this study we investigated the role of burs/pburs/rickets in mediating female vitellogenesis and reproduction in a hemipteran insect, the whitefly, Bemisia tabaci. Our investigation unveiled a synchronized expression of burs, pburs and rickets, with their transcripts persisting detectable in the days following eclosion. RNAi-mediated knockdown of burs, pburs or rickets significantly suppressed the transcript levels of vitellogenin (Vg) and Vg receptor in the female whiteflies. These effects also impaired ovarian maturation and female fecundity, as evidenced by a reduction in the number of eggs laid per female, a decrease in egg size and a decline in egg hatching rate. Furthermore, knockdown of burs, pburs or rickets led to diminished juvenile hormone (JH) titers and reduced transcript level of Kruppel homolog-1. However, this impact did not extend to genes in the insulin pathway or target of rapamycin pathway, deviating from the results observed in T. castaneum. Taken together, we conclude that burs/pburs/rickets regulates the vitellogenesis and reproduction in the whiteflies by coordinating with the JH signaling pathway.

Partner of neuropeptide bursicon homodimer pburs mediates a novel antimicrobial peptide Ten3LP via Dif/Dorsal2 in Tribolium castaneum.

Bursicon is a cystine knot family neuropeptide, composed of two subunits, bursicon (burs) and partner of burs (pburs). The subunits can form heterodimers to regulate cuticle tanning and wing maturation and homodimers to signal different biological functions in innate immunity, midgut stem cell proliferation and energy homeostasis, and reproductive physiology in the model insects Drosophila melanogaster or Tribolium castaneum. Here, we report on the role of the pburs homodimer in signaling innate immunity in T. castaneum larvae. Through transcriptome analysis we identified a set of immune-related genes that respond to pburs RNAi. Treating larvae with recombinant-pburs protein led to up-regulation of antimicrobial peptide (AMP) genes in vivo and in vitro. The upregulation of most AMP genes was dependent on the NF-κB transcription factor Relish. Most importantly, we identified a novel AMP, Tenecin 3-like peptide (Ten3LP), regulated by pburs via NF-κB transcription factor Dorsal-related immunity factor (Dif)/Dorsal2, but not Relish. We conducted Ten3LP RNAi, synthesized recombinant Ten3LP protein for microbial inhibition assays and functionally characterized Ten3LP as an AMP specific for fungi and Gram-positive bacteria. We demonstrate that expression of Ten3LP is activated by pburs via the Toll pathway. These findings identify new molecular targets for development of potential antibiotics for treating microbial infections and perhaps for RNAi based pest management technology.

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.

DNA G-quadruplex structure participates in regulation of lipid metabolism through acyl-CoA binding protein.

G-quadruplex structure (G4) is a type of DNA secondary structure that widely exists in the genomes of many organisms. G4s are believed to participate in multiple biological processes. Acyl-CoA binding protein (ACBP), a ubiquitously expressed and highly conserved protein in eukaryotic cells, plays important roles in lipid metabolism by transporting and protecting acyl-CoA esters. Here, we report the functional identification of a G4 in the promoter of the ACBP gene in silkworm and human cancer cells. We found that G4 exists as a conserved element in the promoters of ACBP genes in invertebrates and vertebrates. The BmACBP G4 bound with G4-binding protein LARK regulated BmACBP transcription, which was blocked by the G4 stabilizer pyridostatin (PDS) and G4 antisense oligonucleotides. PDS treatment with fifth instar silkworm larvae decreased the BmACBP expression and triacylglycerides (TAG) level, resulting in reductions in fat body mass, body size and weight and growth and metamorphic rates. PDS treatment and knocking out of the HsACBP G4 in human hepatic adenocarcinoma HepG2 cells inhibited the expression of HsACBP and decreased the TAG level and cell proliferation. Altogether, our findings suggest that G4 of the ACBP genes is involved in regulation of lipid metabolism processes in invertebrates and vertebrates.

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.

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.

G-Quadruplex Regulation of VEGFA mRNA Translation by RBM4.

In eukaryotes, mRNAs translation is mainly mediated in a cap-dependent or cap-independent manner. The latter is primarily initiated at the internal ribosome entry site (IRES) in the 5'-UTR of mRNAs. It has been reported that the G-quadruplex structure (G4) in the IRES elements could regulate the IRES activity. We previously confirmed RBM4 (also known as LARK) as a G4-binding protein in human. In this study, to investigate whether RBM4 is involved in the regulation of the IRES activity by binding with the G4 structure within the IRES element, the IRES-A element in the 5'-UTR of vascular endothelial growth factor A (VEGFA) was constructed into a dicistronic reporter vector, psiCHECK2, and the effect of RBM4 on the IRES activity was tested in 293T cells. The results showed that the IRES insertion significantly increased the FLuc expression activity, indicating that this G4-containing IRES was active in 293T cells. When the G4 structure in the IRES was disrupted by base mutation, the IRES activity was significantly decreased. The IRES activity was notably increased when the cells were treated with G4 stabilizer PDS. EMSA results showed that RBM4 specifically bound the G4 structure in the IRES element. The knockdown of RBM4 substantially reduced the IRES activity, whereas over-expressing RBM4 increased the IRES activity. Taking all results together, we demonstrated that RBM4 promoted the mRNA translation of VEGFA gene by binding to the G4 structure in the IRES.

Large-scale screening of i-motif binding compounds in the silkworm, Bombyx mori.

DNA secondary structure i-motif involves in gene transcription and considered as a novel target for cancer gene therapy. I-motif-binding compounds can either stabilize or destroy the structure, resulting in change in target gene transcription. In this study, a large-scale screening of binding compounds was conducted using the i-motif structure of BmPOUM2, a transcription factor in silkworm, Bombyx mori. Surface plasmon resonance imaging (SPRi) high-throughput binding screening of 3642 compounds found 60 compounds with an binding affinity Kd of 10-7-10-6 M. SPRi and circular dichroism (CD) double screening demonstrated that the BmPOUM2 i-motif structure bound the compounds IF1, IF3, IF4, IF6 and IF7 with Kd of 10-7 M, and the compounds IF2 and tetrakis (4-N-methylpyridyl) porphine (TMPyP4) with a Kd of 10-8 M. Interestingly, IF2, IF3, IF4, IF6 and IF7 promoted the binding of the i-motif-binding protein BmILF with the i-motif structure, whereas TMPyP4 inhibited the binding. This study provided a list of compounds that have potential applications in functional analysis of i-motif structure and in pesticide and drug development through gene transcription regulation by i-motif structure.

Neuropeptide Bursicon Influences Reproductive Physiology in Tribolium Castaneum.

Bursicon is a neuropeptide belonging to the cystine knot family and is composed of burs and partner of burs (pburs) subunits. It can form heterodimers or homodimers to execute different biological functions. Bursicon heterodimers regulate cuticle sclerotization and wing maturation, whereas bursicon homodimers mediate innate immunity and midgut stem cell proliferation. A recent study has shown that bursicon potentially induces the expression of vitellogenin (Vg) in the black tiger shrimp Penaeus monodon; however, the underlying mechanism remains unknown. In this study, we investigated the role of bursicon in the reproductive physiology of the red flour beetle, Tribolium castaneum. The knockdown of burs, pburs, or its receptor T. castaneum rickets (Tcrk) in 2-day pupae significantly downregulated the expression levels of Vg1, Vg2, and Vg receptor (VgR) genes in females 3- and 5-day post-adult emergence, leading to abnormal oocytes with limited Vg content. The silencing of burs repressed the number of eggs laid and completely inhibited egg hatch, whereas the silencing of pburs dramatically decreased the number of eggs laid, hatch rate, and offspring larval size, and this RNA interference (RNAi) effects persisted to the next generation. Furthermore, the knockdown of burs or pburs downregulated the expression of the insulin/insulin-like signaling/target of rapamycin (TOR) signaling genes encoding insulin receptor (InR), protein kinase B (Akt), TOR, and ribosomal protein S6 kinase (S6K). Most importantly, the injection of recombinant pburs (r-pburs) protein was able to upregulate the expression of Vg, VgR, InR, Akt, TOR, S6K, JH synthesis (JHAMT), Methoprene-tolerant (Met), and Taiman (Tai) in normal females and rescue the expression of Vg and VgR in pburs RNAi females but failed to rescue Vg and VgR in Tcrk knockdown females. We infer that bursicon homodimers influence Vg expression via the receptor Tcrk, possibly by mediating the expression of the juvenile hormone (JH) and IIS/TOR pathway genes, thereby regulating reproduction in T. castaneum.

Octopamine signaling is involved in the female postmating state in Nilaparvata lugens Stål (Hemiptera: Delphacidae).

Mating triggers physiological and behavioral changes in female insects. In many species, females experience postmating behavioral and physiological changes that define a post-mated state. These changes are comprised of several conditions, including long-term refractoriness to re-mating and increased production and laying of eggs. Here, we report that mating led to several changes in brown planthopper (BPH) females, including increased octopamine (OA), cAMP concentrations, and activities of several enzymes. Mating also led to changes in the expression of several genes acting in female physiology, including those in the cAMP/PKA signal transduction pathway. OA injections into virgin females led to similar changes. RNAi silencing of the gene encoding tyramine ß-hydroxylase, involved in the final step in OA synthesis, led to decreased expression of these genes, and reduced the cAMP/PKA signaling. At the whole-organism level, the RNAi treatments led to reduced fecundity, body weights, and longevity. RNAi silencing of genes acting in OA signaling led to truncated ovarian development, egg maturation, and ovarian vitellogenin (Vg) uptake. The impact of these decreases is also registered at the population level, seen as decreased population growth. We infer that OA signaling modulates the postmating state in female BPH and possibly other hemipterans.

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.

Characterization of cadmium-responsive transcription factors in wolf spider Pardosa pseudoannulata.

Transcription factors (TFs) act on the regulation of gene expression, which is prevalent in all organisms, and their characterization may provide important clues for understanding the regulatory mechanism of gene expression. In this research, inhibited growth (delayed developmental time and decreased body weight) and increased activities of antioxidant enzymes (peroxidase, superoxide dismutase, and catalase) were recorded in Pardosa pseudoannulata in response to cadmium burden. Expression profiles of TFs were analyzed based on the transcriptome profiling of P. pseudoannulata, and 1711 TFs genes were differentially expressed with 995 up-regulated and 716 down-regulated. Most of the differentially expressed TFs belonged to zf-C2H2, ZBTB, Homeobox, and bHLH families. Interestingly, hub genes smads, TCF7L2, EGR1, and GATA5 were identified to be the candidate Cd-responsive TFs related to growth of spider. The expression level of Sod2 (superoxide dismutase) was regulated by the up-regulated TF foxo3, implying its important role in the antioxidant defense of spider. Moreover, sequence analysis demonstrated that smads and foxo3 were conserved among spiders and insects. This study revealed for the first time the role of TFs in molecular response of P. pseudoannulata to Cd stress, providing the basis for the protection of tarantula under Cd stress.

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.

Transcriptome sequencing reveals the effects of cadmium toxicity on the cold tolerance of the wolf spider Pirata subpiraticus.

As the predominant predator of pests in rice fields, spiders have been exposed to cadmium (Cd) pollution for a long time. The livability of spiders during the overwintering period is closely related to population growth in spring, but the effects of Cd on spider's survival of cold hardness and the underlining mechanism remain unclear. In the present study, we found that some growth parameters (body length, width, mass and livability) in the wolf spider Pirata subpiraticus were altered distinctively under Cd stress. To investigate the effects of Cd toxicity on the spider at molecular levels, RNA-sequencing was performed on the spiderlings undergoing ambient temperature alterations. Transcriptome data showed that a total of 807 differentially expressed genes (DEGs) were yielded in the comparison. The obtained DEGs were mainly linked with metabolism-related process, including oxidoreductase activity and lipid transport, and 25 DEGs were associated with the reported cryoprotectants, including glycerol, arginine, cysteine, heat shock protein, glucose and mannose. Growth factors (insulin growth factor, platelet-derived growth factor and transforming growth factor) and cytochrome P450 encoding genes were dramatically expressed in the spider. Furthermore, transcriptional factors (TFs) family were characterized according to the transcriptomic profile, and ZBTB TFs were represented the most distinctive alterations in the characterized genes. Collectively, our study illustrated that Cd poses disadvantageous effects on the growth of P. subpiraticus at cold ambient temperature, and the spiders are capable of responding to the adverse Cd stress by expressing the genes involved in the metabolism of energy substances, cryoprotectants and immune-related components.

Long-term cadmium exposure affects cell adhesion and expression of cadherin in the male genital organ of Pardosa pseudoannulata (Bösenberg & Strand, 1906).

Pardosa pseudoannulata (Araneae: Lycosidae), as an important predator of crop pests, has served as a strong driver for ecological regulation of pests. Cadmium (Cd) is a toxic heavy metal widely distributed in the soil in China, which not only seriously pollutes the ecological environment, but also poses a great threat to the survival of organisms. Palpal bulbs are the genital organs of male spiders, playing an important role in reproductive physiology. However, the effects of long-term Cd stress on the genital organ of the primary pest predator were poorly understood. Therefore, we investigated the Cd effect on the male palpal organ of P. pseudoannulata at morphological and gene expression levels. The results showed that no obvious difference in the morphology between the Cd-treated and control groups was observed, but cell adhesion was affected at molecular level. Transcriptome sequencing analysis revealed that under long-term Cd stress, the biological processes including cell-cell adhesion via plasma-membrane adhesion molecules, cell-cell adhesion, and homophilic cell adhesion via plasma membrane adhesion molecules were the top three differentially expressed terms (p-adj < 0.001), and 51 unigenes were annotated into cadherin-related proteins, such as protocadherin, cadherin-87A, and cadherin-96Ca, among which, 18 unigenes were significantly upregulated under the Cd stress. Our outcomes indicate that the differentially expressed genes involved in cell adhesion may explain the negative effects of Cd stress on the spider genital organ, and the comprehensive transcriptome dataset will also provide a profound molecular information of the genital organ of P. pseudoannulata.

Bursicon homodimers induce the innate immunity via Relish in Procambarus clarkii.

Bursicon (burs) is a neuropeptide hormone consisting of two cystine-knot proteins (burs α and burs ß), and burs α-ß is responsible for cuticle tanning in insects. Further studies show that burs homodimers induce prophylactic immunity. Here, we investigated the hypothesis that burs homodimers act in regulating immunity in the red swamp crayfish Procambarus clarkii. We found that burs α and burs ß are expressed in neural system of crayfish. Treating crayfish with recombinant burs-homodimer proteins led to up-regulation of several anti-microbial peptide (AMP) genes, and RNAi-mediated knockdown of burs led to decreased expression of AMP genes. The burs proteins also facilitated bacterial clearance and decreased crayfish mortality upon bacterial infection. Furthermore, burs proteins activated the transcriptional factor Relish, and knockdown of Relish abolished the influence of recombinant burs homodimers on AMP induction. We infer the burs homodimers induce expression of AMP genes via Relish in crayfish and this study extends this immune signaling pathway from insects to crustaceans.

Mul-tiomics analysis of cadmium stress on the ovarian function of the wolf spider Pardosa pseudoannulata.

Cadmium (Cd) pollution is widespread in paddy filed soil in China. In this study, the toxicity of Cd with regard to the female reproductive system of paddy spider Pardosa pseudoannulata was investigated by means of multi-omics analyses (transcriptome, proteome, and miRNAs). Decreased activities of detoxifying enzymes including peroxidase (POD), Glutathione S-transferases (GST), and superoxide dismutase were detected in the ovary of P. pseudoannulata. Of these, GST and POD were consistently down-regulated at the transcriptional and translational levels. Vitellogenin content and fecundity of the spider were also reduced by Cd burden. Five vitellogenin encodes genes were down-regulated while only vitellogenin-6 protein was up-regulated. But protein lipovitellin-1, the main composition of vitellin, was down-regulated. In addition, the correlation between the mitogen-activated protein kinase (MAPK) signaling pathway and Cd stress was identified. A down-regulated gene that encoding connector of kinase to AP-1 in the MAPK signaling pathway was regulated by the up-regulated miRNA (miRNA id: miRNA dan-miR- 318>der-miR-318>dgr-miR-318>dme-miR-318-3p > dmo-miR-318>dpe-miR-318>dps-miR-318>dse-miR-318>dsi-miR-318>dvi-miR-318>dwi-miR-318>dya-miR-318). In conclusion, Cd stress possesses distinct female reproductive toxicity on P. pseudoannulata through impairing antioxidant system and synthesis of vitellin.

A Novel Iflavirus Was Discovered in Green Rice Leafhopper Nephotettix cincticeps and Its Proliferation Was Inhibited by Infection of Rice Dwarf Virus.

The green rice leafhopper, Nephotettix cincticeps (Hemiptera: Cicadellidae), is a key insect vector transmitting rice dwarf virus (RDV) that causes rice dwarf disease. We discovered a novel iflavirus from the transcriptomes of N. cincticeps and named it as Nephotettix cincticeps positive-stranded RNA virus-1 (NcPSRV-1). The viral genome consists of 10,524 nucleotides excluding the poly(A) tail and contains one predicted open reading frame encoding a polyprotein of 3,192 amino acids, flanked by 5' and 3' untranslated regions. NcPSRV-1 has a typical iflavirus genome arrangement and is clustered with the members of the family Iflaviridae in the phylogenetic analysis. NcPSRV-1 was detected in all tested tissues and life stages of N. cincticeps and could be transmitted horizontally and vertically. Moreover, NcPSRV-1 had high prevalence in the laboratory populations and was widely spread in field populations of N. cincticeps. NcPSRV-1 could also infect the two-striped leafhopper, Nephotettix apicalis, at a 3.33% infection rate, but was absent in the zigzag leafhopper, Recilia dorsalis, and rice Oryza sativa variety TN1. The infection of RDV altered the viral load and infection rate of NcPSRV-1 in N. cincticeps, for which it seems that RDV has an antagonistic effect on NcPSRV-1 infection in the host.

Expression and functional analysis of cytochrome P450 genes in the wolf spider Pardosa pseudoannulata under cadmium stress.

Cytochrome P450 enzymes (CYPs), encoded by Halloween genes, mediate the biosynthesis of molting hormone, ecdysteroids, in arthropods. In this report, the effect of heavy metal cadmium (Cd) stress on the expression of cytochrome P450 genes in the wolf spider Pardosa pseudoannulata was analyzed. The results showed the expression levels of genes encoding for Cd transporters including ABC transporters, zinc transporters, calcium channel proteins and calcium binding proteins were inhibited or induced by Cd stress. In addition, the increase in metallothionein (MT) content and glutathione peroxidase (GPX) activity and decrease in total acetylcholine esterase (AChE) activity were also detected. Apparently, these detoxification methods did not completely protect the spider from the cytotoxicity of Cd stress. Increased mortality of P. pseudoannulata was observed when they were under Cd tress. In total 569 CYP genes belonging to 62 CYP subfamilies were obtained from P. pseudoannulata RNA-seq databases. BlaxtX analysis showed that 150, 161, 11, and 40 CYP genes were similar to the genes dib, phm, sad and shd, respectively, which are thought to catalyze the biosynthesis of ecdysteroids. Gene expression analysis suggested that 25 dib encoding genes, 27 phm encoding genes, 2 sad encoding genes, and 6 shd encoding genes were differentially expressed in TS2 vs. S2 comparison (Cd-treated 2nd instar spider vs. 2nd instar spider), respectively. There were 70 dib, 70 phm and 19 shd encoding genes either upregulated or downregulated, while 3 sad encoding genes were upregulated in TS5 vs. S5 (Cd-treated 5nd instar spider vs. 5nd instar spider). Genes related to heme binding and essential for activating the CYPs were also differentially expressed. Expression levels of cuticle related genes were significant differentially expressed, implying the changes in activities of chitin synthases and chitinase. Therefore we assume that unsuccessful molting process may occur on P. pseudoannulata due to influenced ecdysteroids levels, thus increasing mortality of spider.