Profiling Substrate Specificity of the SUMO Protease Ulp1 by the YESS-PSSC System to Advance the Conserved Mechanism for Substrate Cleavage.

SUMO modification is a vital post-translational regulation process in eukaryotes, in which the SUMO protease is responsible for the maturation of the SUMO precursor and the deconjugation of the SUMO protein from modified proteins by accurately cleaving behind the C-terminal Gly-Gly motif. To promote the understanding of the high specificity of the SUMO protease against the SUMO protein as well as to clarify whether the conserved Gly-Gly motif is strictly required for the processing of the SUMO precursor, we systematically profiled the specificity of the S. cerevisiae SUMO protease (Ulp1) on Smt3 at the P2-P1↓P1' (Gly-Gly↓Ala) position using the YESS-PSSC system. Our results demonstrated that Ulp1 was able to cleave Gly-Gly↓ motif-mutated substrates, indicating that the diglycine motif is not strictly required for Ulp1 cleavage. A structural-modeling analysis indicated that it is the special tapered active pocket of Ulp1 conferred the selectivity of small residues at the P1-P2 position of Smt3, such as Gly, Ala, Ser and Cys, and only which can smoothly deliver the scissile bond into the active site for cleavage. Meanwhile, the P1' position Ala of Smt3 was found to play a vital role in maintaining Ulp1's precise cleavage after the Gly-Gly motif and replacing Ala with Gly in this position could expand Ulp1 inclusivity against the P1 and P2 position residues of Smt3. All in all, our studies advanced the traditional knowledge of the SUMO protein, which may provide potential directions for the drug discovery of abnormal SUMOylation-related diseases.

Co-infection with Wolbachia and Cardinium may promote the synthesis of fat and free amino acids in a small spider, Hylyphantes graminicola.

Associations between endosymbiotic bacteria and their hosts are widespread in nature and have been demonstrated extensively; however, only a few studies have examined how facultative symbionts affect host nutrition and metabolism. To gain insight into the associations between facultative symbionts and host nutrition and metabolic activity, we detected endosymbiotic infection in a small spider species, Hylyphantes graminicola, and established two infectious strains, i.e., W-C+ (Wolbachia negative, Cardinium positive) and W+C+ (Wolbachia positive, Cardinium positive). We then determined the content of fat and free amino acids in W-C+ and W+C+ spiders, respectively. We also detected the transcriptome of H. graminicola and the expression of genes involved in fat and amino acid metabolism at different host ages. Results showed that fat content in W+C+ spiders was higher than that in W-C+ spiders, and free amino acid content was higher in W+C+ males than W-C+ males, with no difference observed in females. Transcriptome analysis identified 144 (W-C+ vs W+C+) differentially expressed genes (DEGs). Moreover, the expression of five genes involved in fat and amino acid metabolism were significantly up-regulated in the third, fourth, and fifth instar stages in W+C+ spiders. This study indicated that Wolbachia and Cardinium co-infection had a pivotal effect on fat and amino acid synthesis in hosts. Moreover, our results provide strong evidence explaining the long-term coexistence of hosts and endosymbionts.

Bacillus thuringiensis protein Vip3Aa does not harm the predator Propylea japonica: A toxicological, histopathological, biochemical and molecular analysis.

The ladybeetle Propylea japonica is a widely distributed natural enemy in many agricultural systems. P. japonica is often used as a test organism for safety assessments of transgenic Bacillus thuringiensis crops. Plant varieties expressing the Vip3Aa insecticidal protein are not currently commercially available in China. In this study, protease inhibitor E-64 was used as a positive control to examine the responses of P. japonica larvae to a high concentration of Vip3Aa proteins. Larvae that were fed E-64 had increased mortality and prolonged developmental period, but these parameters were unaffected when larvae were fed Vip3Aa. The epithelial cells of midguts were intact and closely connected with the basal membrane when larvae were fed Vip3Aa, but the epithelial cells degenerated in the E-64 treatment. The activities of antioxidative enzymes and expression levels of detoxification-related genes in P. japonica larvae were not altered after exposure to Vip3Aa; however, these biochemical and molecular parameters were significantly changed in the E-64 treatment. The results demonstrate that Vip3Aa protein is not harmful to the predator P. japonica.

Antioxidant responses of the pest natural enemy Hylyphantes graminicola (Araneae: Linyphiidae) exposed to short-term heat stress.

Temperature is a critical abiotic factor that causes physiological changes in arthropods. However, little is known about the effect of heat stress on the antioxidant responses of Araneae species. Hylyphantes graminicola is a dominant predator in many cropping systems in China. In the present study, the effect of short-term heat stress (36, 38, 40 or 42 °C) on the reactive oxygen species (ROS) levels, the activities of antioxidant enzymes (superoxide dismutase [SOD], catalase [CAT], peroxidases [POD] and glutathione-S-transferases GST]), total antioxidant capacity (TAC), malondialdehyde (MDA) concentrations and survival of H. graminicola spiderlings and adults were investigated. The results showed that H. graminicola adults had a significantly higher survival rate compared to spiderlings at 40 °C. The heat stress increased ROS contents in H. graminicola. The SOD, CAT, POD and GST activities increased in spiderlings and adults under heat stress. These data suggest a defensive function for these enzymes in alleviating oxidative damage. Specifically, SOD plays a key role in reducing the high level of superoxide radicals in spiderlings and adults. Moreover, the POD and CAT capabilities for scavenging H2O2 in spiderlings were similar, and CAT may play a more important role than POD in scavenging H2O2 in adults at 42 °C. The spiderling TAC increased significantly at 40 and 42 °C, and the adult TAC was stable at 36-40 °C but decreased at 42 °C. These data suggest that TAC was insufficient in H. graminicola adults under more severe stress conditions. These results further our understanding of the physiological response of Araneae species exposed to heat stress.

Experimental replacement of an obligate insect symbiont.

Symbiosis, the close association of unrelated organisms, has been pivotal in biological diversification. In the obligate symbioses found in many insect hosts, organisms that were once independent are permanently and intimately associated, resulting in expanded ecological capabilities. The primary model for this kind of symbiosis is the association between the bacterium Buchnera and the pea aphid (Acyrthosiphon pisum). A longstanding obstacle to efforts to illuminate genetic changes underlying obligate symbioses has been the inability to experimentally disrupt and reconstitute symbiont-host partnerships. Our experiments show that Buchnera can be experimentally transferred between aphid matrilines and, furthermore, that Buchnera replacement has a massive effect on host fitness. Using a recipient pea aphid matriline containing Buchnera that are heat sensitive because of an allele eliminating the heat shock response of a small chaperone, we reduced native Buchnera through heat exposure and introduced a genetically distinct Buchnera from another matriline, achieving complete replacement and stable inheritance. This transfer disrupted 100 million years (∼ 1 billion generations) of continuous maternal transmission of Buchnera in its host aphids. Furthermore, aphids with the Buchnera replacement enjoyed a dramatic increase in heat tolerance, directly demonstrating a strong effect of symbiont genotype on host ecology.

Transcriptome Profiling Analysis of Wolf Spider Pardosa pseudoannulata (Araneae: Lycosidae) after Cadmium Exposure.

Pardosa pseudoannulata is one of the most common wandering spiders in agricultural fields and a potentially good bioindicator for heavy metal contamination. However, little is known about the mechanisms by which spiders respond to heavy metals at the molecular level. In the present study, high-throughput transcriptome sequencing was employed to characterize the de novo transcriptome of the spiders and to identify differentially expressed genes (DEGs) after cadmium exposure. We obtained 60,489 assembled unigenes, 18,773 of which were annotated in the public databases. A total of 2939 and 2491 DEGs were detected between the libraries of two Cd-treated groups and the control. Functional enrichment analysis revealed that metabolism processes and digestive system function were predominately enriched in response to Cd stress. At the cellular and molecular levels, significantly enriched pathways in lysosomes and phagosomes as well as replication, recombination and repair demonstrated that oxidative damage resulted from Cd exposure. Based on the selected DEGs, certain critical genes involved in defence and detoxification were analysed. These results may elucidate the molecular mechanisms underlying spiders' responses to heavy metal stress.

The Effects of Cadmium Exposure on Fitness-Related Traits and Antioxidant Responses in the Wolf Spider, Pardosa pseudoannulata.

The objective of the present study was to assess the ecotoxicological responses of Pardosa pseudoannulata to a common environmental pollutant, cadmium. Third-instar spiderlings and adult spiders were exposed to sublethal concentrations of CdCl2 solution in their drinking water. The Cd content in P. pseudoannulata adults increased significantly with the number of days of exposure to a 0.2 mM CdCl2 solution, when exposed to 2 mM CdCl2 solution, the Cd content in the spiders increased sharply in the first two (male) or three (female) weeks, and then no significant changes were recorded following with the next three (male) or two (female) weeks exposure. Exposure of spiders to Cd contaminated drinking water resulted in reduced body mass, delayed development, fewer eggs and increased mortality. Significantly higher activities of superoxide dismutase, catalase and glutathione-S-transferase were recorded in the spiders after 7 day exposure to 0.2 mM CdCl2 solution. However, longer-term exposures or increased Cd concentrations did not result in significantly higher antioxidant enzyme activity relative to control treatment.

Characterizing interactions of endoplasmic reticulum resident proteins in situ through the YST-PPI method.

The mutual interactions of endoplasmic reticulum (ER) resident proteins in the ER maintain its functions, prompting the protein folding, modification, and transportation. Here, a new method, named YST-PPI (YESS-based Split fast TEV protease system for Protein-Protein Interaction) was developed, targeting the characterization of protein interactions in ER. YST-PPI method integrated the YESS system, split-TEV technology, and endoplasmic reticulum retention signal peptide (ERS) to provide an effective strategy for studying ER in situ PPIs in a fast and quantitative manner. The interactions among 15 ER-resident proteins, most being identified molecular chaperones, of S. cerevisiae were explored using the YST-PPI system, and their interaction network map was constructed, in which more than 74 interacting resident protein pairs were identified. Our studies also showed that Lhs1p plays a critical role in regulating the interactions of most of the ER-resident proteins, except the Sil1p, indicating its potential role in controlling the ER molecular chaperones. Moreover, the mutual interaction revealed by our studies further confirmed that the ER-resident proteins perform their functions in a cooperative way and a multimer complex might be formed during the process.

Endophytic bacteria for Cd remediation in rice: Unraveling the Cd tolerance mechanisms of Cupriavidus metallidurans CML2.

The utility of endophytic bacteria in Cadmium (Cd) remediation has gained significant attention due to their ability to alleviate metal-induced stress and enhance plant growth. Here, we investigate C. metallidurans CML2, an endophytic bacterial strain prevalent in rice, showing resilience against 2400 mg/L of Cd(II). We conducted an in-depth integrated morphological and transcriptomic analysis illustrating the multifarious mechanisms CML2 employs to combat Cd, including the formation of biofilm and CdO nanoparticles, upregulation of genes involved in periplasmic immobilization, and the utilization of RND efflux pumps to extract excess Cd ions. Beyond Cd, CML2 exhibited robust tolerance to an array of heavy metals, including Mn2+, Se4+, Ni2+, Cu2+, and Hg2+, demonstrating effective Cd(II) removal capacity. Furthermore, CML2 has exhibited plant growth-promoting properties through the production of indole-3-acetic acid (IAA) at 0.93 mg/L, soluble phosphorus compounds at 1.11 mg/L, and siderophores at 22.67%. Supportively, pot experiments indicated an increase in root lengths and a decrease in Cd bioaccumulation in rice seedlings inoculated with CML2, consequently reducing Cd translocation rates from 43% to 31%. These findings not only contribute to the understanding of Cd resistance mechanisms in C. metallidurans, but also underscore CML2's promising application in Cd remediation within rice farming ecosystems.

Bioaccumulation of mercury and its effects on survival, development and web-weaving in the funnel-web spider Agelena labyrinthica (Araneae: Agelenidae).

This study investigated the bioaccumulation and effects of mercury (Hg) in funnel-web spiders, Agelena labyrinthica, following exposure to sublethal concentrations of Hg(NO3)2 in their drinking water. The results showed that the Hg content in adult A. labyrinthica increased rapidly with the number of days exposed to the Hg(NO3)2 solution, and the mortality of adult spiders within 30 days increased with increased concentrations of Hg(NO3)2 in the drinking water. The total developmental duration of A. labyrinthica exposed to Hg(NO3)2 was significantly longer than in the control spiders, but there were no significant differences in the total developmental duration of spiders among the three treatment groups (exposed to 10, 20 and 50 mg/L Hg(NO3)2 solution). We also compared the web-weaving of the control and treated spiders, and found no significant differences in shape, structure, color, or size of the webs between the control and treated spiders; however, there was a significant difference in web placement between the treatment and control groups. The spiders in the control group appeared to have an episodic-like memory, choosing to weave their five webs in the same corner in the five time periods allowed.

Diversity of Bacteria Associated with Guts and Gonads in Three Spider Species and Potential Transmission Pathways of Microbes within the Same Spider Host.

Microbial symbiosis plays a crucial role in the ecological and evolutionary processes of animals. It is well known that spiders, with their unique and diverse predatory adaptations, assume an indispensable role in maintaining ecological balance and the food chain. However, our current understanding of spider microbiomes remains relatively limited. The gut microbiota and gonad microbiota of spiders can both potentially influence their physiology, ecology, and behavior, including aspects such as digestion, immunity, reproductive health, and reproductive behavior. In the current study, based on high-throughput sequencing of the 16S rRNA V3 and V4 regions, we detected the gut and gonad microbiota communities of three spider species captured from the same habitat, namely, Eriovixia cavaleriei, Larinioides cornutus, and Pardosa pseudoannulata. In these three species, we observed that, at the phylum level classification, the gut and gonad of E. cavaleriei are primarily composed of Proteobacteria, while those of L. cornutus and P. pseudoannulata are primarily composed of Firmicutes. At the genus level of classification, we identified 372 and 360 genera from the gut and gonad bacterial communities. It is noteworthy that the gut and gonad bacterial flora of E. cavaleriei and L. cornutus were dominated by Wolbachia and Spiroplasma. Results show that there were no differences in microbial communities between females and males of the same spider species. Furthermore, there is similarity between the gut and ovary microbial communities of female spiders, implying a potential avenue for microbial transmission between the gut and gonad within female spiders. By comprehensively studying these two microbial communities, we can establish the theoretical foundation for exploring the relationship between gut and gonad microbiota and their host, as well as the mechanisms through which microbes exert their effects.

Interaction of High Temperature Stress and Wolbachia Infection on the Biological Characteristic of Drosophila melanogaster.

It was reported that temperature affects the distribution of Wolbachia in the host, but only a few papers reported the effect of the interaction between high temperature and Wolbachia on the biological characteristic of the host. Here, we set four treatment Drosophila melanogaster groups: Wolbachia-infected flies in 25 °C (W+M), Wolbachia-infected flies in 31 °C (W+H), Wolbachia-uninfected flies in 25 °C (W-M), Wolbachia-uninfected flies in 31 °C (W-H), and detected the interaction effect of temperature and Wolbachia infection on the biological characteristic of D. melanogaster in F1, F2 and F3 generations. We found that both temperature and Wolbachia infection had significant effects on the development and survival rate of D. melanogaster. High temperature and Wolbachia infection had interaction effect on hatching rate, developmental durations, emergence rate, body weight and body length of F1, F2 and F3 flies, and the interaction effect also existed on oviposition amount of F3 flies, and on pupation rate of F2 and F3 flies. High temperature stress reduced the Wolbachia vertical transmission efficiency between generations. These results indicated that high temperature stress and Wolbachia infection had negative effects on the morphological development of D. melanogaster.

Energy Allocation of the Wolf Spider Pardosa pseudoannulata under Dietary Restriction.

The phenomenon of food shortage is widespread in spider populations, which has a great impact on their growth, development, and survival. Pardosa pseudoannulata is a dominant spider species in rice fields and has an important controlling effect on rice pests. In this study, three feeding levels were tested at the juvenile stage (H, high feeding; M, medium dietary restriction; L, severe dietary restriction) and two at the adult stage (H and L). A total of six feeding levels were tested to explore the effects of dietary restriction on the development, longevity, nutrient content, and predation by P. pseudoannulata [HH (control group), HL, MH, ML, LH, LL]. The results showed that continuous dietary restriction (ML and LL groups) had negative impacts on the growth of P. pseudoannulata and positive impacts on longevity. Spiderlings suffered from dietary restrictions during the juvenile period, and when the restrictions were removed upon reaching adulthood (MH and LH groups), their lifespan started decreasing whilst their weight began returning to normal. This suggested that there might be a trade-off between the growth and longevity of the spider under dietary restrictions. The study also found that when food was severely restricted in the juvenile stage (LH and LL groups), the nutrient contents of the adult spider could return to the same level as the control group, but the predatory ability decreased. When food was moderately restricted in the juvenile stage (MH and ML groups), the predatory ability of the adult spiders improved, while nutrients of the adult spiders declined. Our results will provide an empirical basis for the protection and effective use of dominant spider species in agricultural fields.

Insights into the mechanism of shortened developmental duration and accelerated weight gain associated with Wolbachia infection in Hylyphantes graminicola.

Wolbachia infection is known to affect host reproduction and development. To date, however, the underlying mechanism related to the effects of Wolbachia on host development has not yet been reported. Here, we compared the developmental duration and body weight in different instars of Wolbachia-positive (W+ ) and Wolbachia-negative (W- ) spiders (Hylyphantes graminicola) and detected the relative expression levels of 6 insulin-related genes and 3 ecdysone-related genes using reverse transcription qPCR. Results showed that the developmental duration was significantly shortened in W+ spiders compared with W- spiders. Furthermore, W+ spiders were significantly heavier than W- spiders at the 3rd and 4th instars, although no significant differences in body weight were observed after maturity. We also found that the expression levels of insulin-like growth factor-2 mRNA-binding protein-1, insulin-degrading enzyme, and ecdysone-inducible protein-1 genes were significantly down-regulated in W+ spiders compared with W- spiders, whereas the expression levels of insulin-like growth factor-binding protein-1, insulin-like peptide receptor, insulin receptor substrate 2-B, insulin-like, ecdysone-induced protein-2, and ecdysone receptor genes were significantly up-regulated in W+ spiders. Our results suggest that Wolbachia may influence host development by affecting insulin and ecdysone signaling pathways.

Transmission of nanoplastics from Culex quinquefasciatus to Pardosa pseudoannulata and its impact on predators.

Many studies have explored the effects of plastic particles on aquatic organisms. To date, however, few studies have reported on the effects of plastic particles on terrestrial invertebrates. Here, Culex quinquefasciatus (southern house mosquito, prey) and Pardosa pseudoannulata (wolf spider, predator) were used to explore the transmission of nanoplastics (NPs) from aquatic to terrestrial invertebrates and to verify the effects of NPs in prey on predators. Mosquito larvae were exposed to 0, 200, and 1000 NPs mL-1 polystyrene, respectively, and then fed to spiders when they matured. Results showed that ingestion of NP-exposed mosquitoes affected the growth, development, and behavior of P. pseudoannulata, and the intestinal tissue structure, intestinal flora composition, and related enzymatic activities were also impacted. These results indicate that after spiders ingested NP-exposed mosquitoes, their growth, development, and predation ability were affected. This may prolong time to maturation and decrease the ability of spiders to survive and reproduce in the environment. Thus, plastic particles likely have a wide range of effects on organisms as well as the whole ecosystem.

Wolbachia strains typing in different geographic population spider, Hylyphantes graminicola (Linyphiidae).

The Wolbachia endosymbiont of spiders has not been extensively examined. In order to investigate the distribution, evolutionary history, and reproductive phenotype of Wolbachia in spiders in China, we tested 11 geographic populations of Hylyphantes graminicola. Wolbachia infection has been detected in each population. 10 Wolbachia strains have been characterized by multilocus sequence typing (MLST). Phylogenetic analyses indicated that eight Wolbachia strains in H. graminicola belonged to supergroup B, and two belonged to supergroup A. No correlation existed between Wolbachia diversity and host's geographic distance. The significant correlation was observed between pairwise distance of H. graminicola COI and genetic divergence of associated Wolbachia strains. We also found that Wolbachia infection frequencies in hosts varied over geographic space.

Elevated CO2 concentration affects survival, but not development, reproduction, or predation of the predator Hylyphantes graminicola (Araneae: Linyphiidae).

Elevated CO2 concentrations can change the multi-level nutritional relationship of the ecosystem through the cascading effect of the food chain. To date, few studies have investigated the effects of elevated CO2 concentration on the Araneae species through the tritrophic system. Hylyphantes graminicola (Araneae: Linyphiidae) is distributed widely in Asia and is a dominant predator in cotton fields. This study investigated chemical components in the food chain of cotton (Gossypium hirsutum)-cotton aphid (Aphis gossypii)-predator (H. graminicola) and compared the development, reproduction, and predation of H. graminicola under ambient (400 ppm) and elevated concentration of CO2 (800 ppm). The results showed that the elevated CO2 concentration increased the chemicals of cotton and cotton aphid, but it did not affect the nutrients, development, reproduction, and predation of the spider. However, the survival rate of the spider was significantly decreased in elevated CO2. The results will further our understanding of the role of natural enemies in an environment with elevated CO2 concentration.

A split protease-E. coli ClpXP system quantifies protein-protein interactions in Escherichia coli cells.

Characterizing protein-protein interactions (PPIs) is an effective method to help explore protein function. Here, through integrating a newly identified split human Rhinovirus 3 C (HRV 3 C) protease, super-folder GFP (sfGFP), and ClpXP-SsrA protein degradation machinery, we developed a fluorescence-assisted single-cell methodology (split protease-E. coli ClpXP (SPEC)) to explore protein-protein interactions for both eukaryotic and prokaryotic species in E. coli cells. We firstly identified a highly efficient split HRV 3 C protease with high re-assembly ability and then incorporated it into the SPEC method. The SPEC method could convert the cellular protein-protein interaction to quantitative fluorescence signals through a split HRV 3 C protease-mediated proteolytic reaction with high efficiency and broad temperature adaptability. Using SPEC method, we explored the interactions among effectors of representative type I-E and I-F CRISPR/Cas complexes, which combining with subsequent studies of Cas3 mutations conferred further understanding of the functions and structures of CRISPR/Cas complexes.

Biodiversity Survey of Flower-Visiting Spiders Based on Literature Review and Field Study.

Many arthropods exhibit flower-visiting behavior, including a variety of spider species. However, as spiders are assumed to be strictly predatory, flower-visiting spiders are an often neglected group. We conducted a systematic biodiversity study of flower-visiting spiders based on published papers and field surveys. Most previous studies have focused on the herbivorous behavior of flower-visiting spiders (nectivory or pollinivory) and their effects on host flowers (tritrophic interactions with flower-visiting insects). In our field survey, we utilized standard transect walks (active sampling) and colored pan traps (passive sampling) to investigate species occurrence, diurnal and seasonal variation, and flower color preference of flower-visiting spiders. From the transect walks, crab spider species were found to be the dominant flower-visiting spiders and, based on all spider species, juvenile visitors were significantly more common than adults. Furthermore, in terms of spider number and species richness, tulips were the preferred flower to visit. For the pan traps, wolf spiders were found to be the dominant spider species. No significant differences were observed in the number of spiders caught in different colored pans, suggesting that color may not be an important flower trait in regard to spider preference. To the best of our knowledge, this study is the first to propose the term 'flower-visiting spiders' and conduct a systematic investigation of their diversity. However, this is preliminary research and further studies are required, especially as biodiversity is often closely linked to survey sites and ecotopes.

1. Generally speaking, flower-visiting spiders focuses on the herbivorous behavior (nectivory or pollinivory) and their effects on host flowers (tritrophic interactions with flower-visiting insect). 2. The authenticity of flower-visiting spiders collected by active sampling was higher than passive trapping; juvenile spider visitors were significantly more common than adults. 3. Our study is the first to propose the term 'flower-visiting spiders', and further research is needed.

Transcriptome responses to elevated CO2 level and Wolbachia-infection stress in Hylyphantes graminicola (Araneae: Linyphiidae).

Hylyphantes graminicola is a resident spider species found in maize and cotton fields and is an important biological control agent of various pests. Previous studies have demonstrated that stress from elevated CO2 and Wolbachia infection can strongly affect spider species. Thus, based on CO2 levels (400 ppm, current atmospheric CO2 concentration and 800 ppm, high CO2 concentration) and Wolbachia status (Wolbachia-infected, W+ and Wolbachia-uninfected, W- ), we divided H. graminicola individuals into four treatment groups: W- 400 ppm, W- 800 ppm, W+ 400 ppm, and W+ 800 ppm. To investigate the effects of elevated CO2 levels (W- 400 vs W- 800), Wolbachia infection (W- 400 vs W+ 400), and the interactions between these two factors (W- 400 vs W+ 800), high-throughput transcriptome sequencing was employed to characterize the de novo transcriptome of the spiders and identify stress-related differentially expressed genes (DEGs). De novo assembly of complementary DNA sequences generated 86 688 unigenes, 23 938 of which were annotated in public databases. A total of 84, 21, and 157 DEGs were found among W- 400 vs W- 800, W- 400 vs W+ 400, and W- 400 vs W+ 800, respectively. Functional enrichment analysis revealed that metabolic processes, signaling, and catalytic activity were significantly affected by elevated CO2 levels and Wolbachia infection. Our findings suggest that the impact of elevated CO2 levels and Wolbachia infection on the H. graminicola transcriptome was, to a large extent, on genes involved in metabolic processes. This study is the first description of transcriptome changes in response to elevated CO2 levels and Wolbachia infection in spiders.