Influence of Cry1Ab protein on growth and development of a predatory spider, Pardosa pseudoannulata, from protective perspectives.

The expression of Cry proteins in genetically modified rice varieties safeguards the crop from lepidopteran pests. These proteins have the potential to be transferred through the food chain to arthropods like planthoppers and predatory spiders, triggering defensive responses in these unintended organisms. Hence, we hypothesized that Cry protein might influence the growth and development of spiders by altering protective enzyme activities. The results showed that Cry1Ab protein could accumulate in tissues and subcellular organelles of Pardosa pseudoannulata from Nilaparvata lugens. Cry1Ab protein exposure prolonged the developmental duration in the 5th and 7th instar spiderlings but induced no alterations of other growth indicators, such as body length, median ocular area, and survival rate. In addition, Cry1Ab protein exerted no adverse impacts on several detoxifying enzymes (i.e., superoxide dismutase, catalase, glutathione peroxidase, and acetylcholine esterase) in muscle, midgut, ganglia, and hemolymph at subcellular components (i.e., microsome and cytoplasm). To further explore the effects of Cry1Ab protein on the spiderlings, we performed an integrated transcriptome analysis on spiderlings exposed to Cry1Ab protein. The results showed that Cry1Ab protein might prolong the development duration of P. pseudoannulata via the altered cuticle metabolism (e.g., chitin metabolic process and structural constituent of cuticle). In addition, the gene expression profile associated with detoxifying enzymes and three stress-responsive pathways (JAK/STAT, JNK/SAPK, and Hippo pathways) also displayed no significant alterations under Cry1Ab exposure. Collectively, this integrated analysis generates multidimensional insights to assess the effects of Cry1Ab protein on non-target spiders and demonstrates that Cry1Ab protein exerts no toxicity in P. pseudoannulata.

Rhizoctonia solani transcriptional activator interacts with rice WRKY53 and grassy tiller 1 to activate SWEET transporters for nutrition.

INTRODUCTION: Rhizoctonia solani, the causative agent of the sheath blight disease (ShB), invades rice to obtain nutrients, especially sugars; however, the molecular mechanism via which R. solani hijacks sugars from rice remains unclear. OBJECTIVES: In this study, rice-R. solani interaction model was used to explore whether pathogen effector proteins affect plant sugar absorption during infection. METHODS: Yeast one-hybrid assay was used to identify Activator of SWEET2a (AOS2) from R. solani. Localization and invertase secretion assays showed that nuclear localization and secreted function of AOS2. Hexose transport assays verified the hexose transporter activity of SWEET2a and SWEET3a. Yeast two-hybrid assays, Bimolecular fluorescence complementation (BiFC) and transactivation assay were conducted to verify the AOS2-WRKY53-Grassy tiller 1 (GT1) transcriptional complex and its activation of SWEET2a and SWEET3a. Genetic analysis is used to detect the response of GT1, WRKY53, SWEET2a, and SWEET3a to ShB infestation. Also, the soluble sugar contents were measured in the mutants and overexpression plants before and after the inoculation of R. solani. RESULTS: The present study found that R. solani protein AOS2 activates rice SWEET2a and localized in the nucleus of tobacco cells and secreted in yeast. AOS2 interacts with rice transcription factor WRKY53 and GT1 to form a complex that activates the hexose transporter gene SWEET2a and SWEET3a and negatively regulate rice resistance to ShB. CONCLUSION: These data collectively suggest that AOS2 secreted by R. solani interacts with rice WRKY53 and GT1 to form a transcriptional complex that activates SWEETs to efflux sugars to apoplast; R. solani acquires more sugars and subsequently accelerates host invasion.

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.

Integrative analysis uncovers response mechanism of Pirata subpiraticus to chronic cadmium stress.

Soil cadmium (Cd) pollution is global environmental pollution and adversely affects paddy field organisms. Wolf spider grants a new insight to evaluate the toxicity triggered by Cd, yet the impact of chronic Cd exposure on the spider and its molecular mechanism remains unclear. The present study found that the wolf spider Pirata subpiraticus fed with Cd-accumulated flies for 5 weeks presented lower catalase, peroxidase, and acetylcholinesterase activities and higher malonaldehyde content than the control spiders (p < 0.05). An in-depth transcriptomic analysis yielded a total of 5995 differentially expressed genes (DEGs, with 3857 up-regulated and 2138 down-regulated genes) from the comparison, and 19 DEGs encoding three enzymatic indicators were down-regulated. Further enrichment analysis indicated that Cd stress could inhibit the expression of cuticle and chitin-encoding genes via the down-regulation of several key enzymes, such as chitin synthase, glutamine-fructose-6-phosphate transaminase, and chitinase. In addition, our findings suggested that hedgehog and FoxO signaling pathways might play an essential role in regulating survival, cell cycle, and autophagy process in spiders, which were primarily down-regulated under Cd stress. An intensely interactive network displayed that Cd exposure could repress key biological processes in P. subpiraticus, particularly peptide metabolic process and peptide biosynthetic process. To sum up, this integrative investigation confirmed an effective bioindicator for assessing Cd-induced toxicity; provided a mass of genes, proteins, and enzymes for further validation; and granted novel perspectives to uncover the molecular responses of spiders to Cd pollution.

Toxic effects of the combined cadmium and Cry1Ab protein exposure on the protective and transcriptomic responses of Pirata subpiraticus.

Cadmium (Cd) pollution poses a serious threat to agricultural production and paddy field fauna. Crystalline proteins (e.g., Cry1Ab and Cry1Ac) are secreted by Bacillus thuringiensis, which can manage pests via a complicated toxic mechanism and have been widely used for pest control due to the commercialization of transgenic crops (e.g., cotton and rice) that expresses Bt insecticidal proteins. Nonetheless, studies on the effects of combined stress of Cd and Cry1Ab protein on field indicator species are limited. In the present study, we showed that spiders, Pirata subpiraticus, fed with Cd-containing flies+Cry1Ab had dramatically higher Cd accumulation than that in the spiders fed with Cd-containing flies (p < 0.05). In addition, the enrichment of Cd led to the activation of the protective mechanism by elevating the concentrations of glutathione peroxidase, glutathione S-transferase, and metallothionein in the spiders (p < 0.05). An in-depth transcriptome analysis revealed that the activities of ion metal binding proteins, transporters, and channels might play essential roles in the Cd accumulation process. More importantly, the higher Cd concentration in the combined Cd+Cry1Ab exposure prolonged developmental duration of P. subpiraticus, due to the down-regulated cuticle proteins (CPs) encoding genes involved in the molting process, which was regulated by a series of putative transcriptional factors such as ZBTB and zf-C2H2. Collectively, this integrated analysis illustrates that the combined Cd+Cry1Ab exposure increases the adverse effects of Cd stress on the growth, antioxidase, and CPs encoding genes of P. subpiraticus, thus providing a research basis and prospect for the rationality of transgenic Cry1Ab crops in the cultivation of heavy metal contaminated soil.

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.

A Traditional Chinese Medicine for the Treatment of Endometrial Hyperplasia via Regulating the HPO Axis in Rats.

Dysfunctional uterine bleeding, accompanied by endometrial hyperplasia (EH), is a common gynecological disease that seriously affects female physical and mental health. Some drugs have been prompted to cure the disease, but most medications have certain side effects and limitations. In the present study, we demonstrated an unexploited Chinese traditional medicine, a combination of Saururus chinensis, Celosia cristata, and Spatholobus suberectus (SCS), which could be used for the treatment of EH and associated complications in rats. We identified the active components from the three Chinese herbs via thin-layer chromatography and high-performance liquid chromatography methods. In addition, serum biochemical indexes and histologic section results found that acute high-dose SCS exerted no adverse impacts on the rats. We then showed that SCS shortened coagulation time (p=0.018) and degree of swelling (p=0.021) on rats at 30 min compared to blank control. Further studies proved that recovered endometrial thickness was associated with the modulation of four hormones (follicle-stimulating hormone, luteinizing hormone, estrogen, and progesterone). Specifically, follicle-stimulating hormone and progesterone contents increased gradually with time, and estrogen was decreased, whereas luteinizing hormone content was returned to normal after a short-term elevation (p < 0.05). Besides, SCS increased uterine endometrium's mRNA expression levels of matrix metalloproteinase-1 (p < 0.001) and tissue inhibitor of matrix metalloproteinase-1 (p < 0.001), promoting the repair of proliferating endometrium in the rats. Collectively, our study indicates that SCS harbors a profoundly curative effect on the treatment of EH and relative complications and uncovers the mechanism at molecular and gene expression levels.

Comparative analysis of cadmium-induced toxicity and survival responses in the wolf spider Pirata subpiraticus under low-temperature treatment.

Cadmium (Cd) pollution is a serious heavy metal pollution in paddy fields, but its effect and underlying mechanism on soil arthropod overwintering and cold resistance are still unclear. In the present study, adult females of the wolf spider Pirata subpiraticus exposed to Cd stress underwent a simulated temperature process (25℃ → 16℃ → 8℃ → 4℃). The mortality rate and content of nutrients in the Cd-treated spiders were dramatically elevated after low-temperature treatment compared to those in the Cd-free control spiders under the same temperature condition. To uncover the putative modulatory mechanism of Cd on cold tolerance in P. subpiraticus, we employed an in-depth RNA sequencing analysis and yielded a total of 888 differentially expressed genes (DEGs). Besides, we characterized genes that participate in multiple cryoprotectant syntheses, including arginine, cysteine, glucose, glycerol, heat shock protein, and mannose. The enrichment analyses found that most of the DEGs involved in biological processes and pathways were related to carbohydrate, lipid, and protein metabolism. Notably, ten Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, such as starch and sucrose metabolism, arachidonic acid metabolism, amino acid metabolism, mineral absorption, and vitamin digestion and absorption, were distinctively enriched with downregulated genes. Meanwhile, we also identified that seven DEGs might inhibit the KEGG pathway of ovarian steroidogenesis and potentially cripple ovarian function and fecundity in the spider. The decreased egg sac weight, number of hatched spiderlings, and vitellin concentration further supported the view that Cd exposure vitiates the overwintering spider's fecundity. Collectively, the comparative analysis provides a novel perspective regarding the survival response and fecundity on the cold tolerance of spiders under Cd stress and offers a profound insight for evaluating Cd-induced toxicity on overwintering arthropods.

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.

Insights on bio-degumming of kenaf bast based on metagenomic and proteomics.

BACKGROUND: Microbes play important roles in kanef-degumming. This study aims at identifying the key candidate microbes and proteins responsible for the degumming of kenaf bast (Hibiscus cannabinus). Kenaf bast was cut into pieces and immersed into microbia fermentation liquid collected from different sites. Fermentation liquid samples were collected at 0, 40, 110 and 150 h and then subjected to the 16S/18S rRNA sequencing analysis and isobaric tag for relative and absolute quantitation (iTRAQ) analysis. The microbial (bacterial and fungal) diversity and the differentially expressed proteins/peptides (DEPs) were identified. RESULTS: With the prolonged degumming time, the weight loss rate increased, the bacterial diversity was decreased. [Weeksellaceae], Enterobacteriaceae and Moraxellaceae were rapidly increased at 0~40 h, and then decreased and were gradually replaced by Bacteroidaceae from 40 h to 150 h. Similarly, Chryseobacterium and Dysgonomonas were gradually increased at 0~110 h and then decreased; Acinetobacter and Lactococcus were increased at 0~40 h, followed by decrease. Bacteroides was the dominant genus at 150 h. Sequencing 18S rRNA-seq showed the gradually decreased Wallemia hederae and increased Codosiga hollandica during degumming. iTRAQ data analysis showed Rds1, and pyruvate kinase I was decreased and increased in the kanef-degumming, respectively. Other DEPs of ferredoxin I, superoxide dismutase and aconitatehydratase were identified to be related to the Glyoxylate and dicarboxylate metabolism (ko00630). CONCLUSIONS: Bacteria including Chryseobacterium, Dysgonomonas, Acinetobacter, Lactococcus and Bacteroidesand fungi like Wallemia hederae and Codosiga hollandica are key candidate microbes for kanef degumming.

Cry1Ab-expressing rice did not influence expression of fecundity-related genes in the wolf spider Pardosa pseudoannulata.

The impact of Bacillus thuringiensis (Bt) toxin proteins on non-target predatory arthropods is not well understood at the cellular and molecular levels. Here, we investigated the potential effects of Cry1Ab expressing rice on fecundity of the wolf spider, Pardosa pseudoannulata, and some of the underlying molecular mechanisms. The results indicated that brown planthoppers (BPHs) reared on Cry1Ab-expressing rice accumulated the Cry toxin and that reproductive parameters (pre-oviposition period, post-oviposition stage, number of eggs, and egg hatching rate) of the spiders that consumed BPHs reared on Bt rice were not different from those that consumed BPHs reared on the non-Bt control rice. The accumulated Cry1Ab did not influence several vitellin (Vt) parameters, including stored energy and amino acid composition, during one generation. We considered the possibility that the Cry toxins exert their influence on beneficial predators via more subtle effects detectable at the molecular level in terms of gene expression. This led us to transcriptome analysis to detect differentially expressed genes in the ovaries of spiders exposed to dietary Cry1Ab and their counterpart control spiders. Eight genes, associated with vitellogenesis, vitellogenin receptor activity, and vitellin membrane formation were not differentially expressed between ovaries from the treated and control spiders, confirmed by qPCR analysis. We infer that dietary Cry1Ab expressing rice does not influence fecundity, nor expression levels of Vt-associated genes in P. pseudoannulata.