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DNA methylation plays an important role in biological regulation in vertebrates, while its presence is variable among insects. However, only a few studies have reported such methylation in spiders. Here, we detected DNA methylation levels and patterns in the small-sized spider Hylyphantes graminicola (Araneae: Linyphiidae) through methylation-sensitive amplified fragment length polymorphism (MSAP). Given the prevailing rate of Wolbachia infection in H. graminicola and the potential impact on host DNA methylation, four H. graminicola groups, i.e., Wolbachia-uninfected (W−) males, Wolbachia-infected (W+) males, W− females, and W+ females, were used for detection of DNA methylation. In addition, quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression of the DNA methyltransferase 1 (Dnmt1) gene in the four groups. Results demonstrated the existence of genomic DNA methylation in H. graminicola. The hemi- or fully methylated ratios at the internal cytosine (HMeCG or MeCG) was significantly higher than the hemi-methylated ratio at the external cytosine (HMeCCG) in both W− and W+ spiders. Compared to W− male and female spiders, hemi- or fully methylated ratios at the internal cytosine in W+ males and females were significantly lower, and Dnmt1 expression was significantly lower in W+ males and females than in W− males and females. These results suggest that a potential association may exist between Wolbachia infection and DNA methylation in H. graminicola.

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.

Microorganisms in insect guts have been recognized as having a great impact on their hosts' nutrition, health, and behavior. Spiders are important natural enemies of pests, and the composition of the gut microbiota of spiders remains unclear. Will the bacterial taxa in spiders be same as the bacterial taxa in insects, and what are the potential functions of the gut bacteria in spiders? To gain insight into the composition of the gut bacteria in spiders and their potential function, we collected three spider species, Pardosa laura, Pardosa astrigera, and Nurscia albofasciata, in the field, and high‐throughput sequencing of the 16S rRNA V3 and V4 regions was used to investigate the diversity of gut microbiota across the three spider species. A total of 23 phyla and 150 families were identified in these three spider species. The dominant bacterial phylum across all samples was Proteobacteria. Burkholderia, Ralstonia, Ochrobactrum, Providencia, Acinetobacter, Proteus, and Rhodoplanes were the dominant genera in the guts of the three spider species. The relative abundances of Wolbachia and Rickettsiella detected in N. albofasciata were significantly higher than those in the other two spider species. The relative abundance of Thermus, Amycolatopsis, Lactococcus, Acinetobacter Microbacterium, and Koribacter detected in spider gut was different among the three spider species. Biomolecular interaction networks indicated that the microbiota in the guts had complex interactions. The results of this study also suggested that at the genus level, some of the gut bacteria taxa in the three spider species were the same as the bacteria in insect guts.

The increasing concentration of carbon dioxide in atmosphere is not only a major cause of global warming, but it also adversely affects the ecological diversity of invertebrates. This study was conducted to evaluate the effect of elevated CO2 concentration (ambient, 400 ppm and high, 800 ppm) and Wolbachia (Wolbachia‐infected, W+ and Wolbachia‐uninfected, W−) on Hylyphantes graminicola. The total survival rate, developmental duration, carapace width and length, body weight, sex ratio, net reproductive rate, nutrition content, and enzyme activity in H. graminicola were examined under four treatments: W− 400 ppm, W− 800 ppm, W+ 400 ppm, and W+ 800 ppm. Results showed that Wolbachia‐infected spiders had significantly decreased the total developmental duration. Different instars showed variations up to some extent, but no obvious effect was found under elevated CO2 concentration. Total survival rate, sex ratio, and net reproductive rate were not affected by elevated CO2 concentration or Wolbachia infection. The carapace width of Wolbachia‐uninfected spiders decreased significantly under elevated CO2 concentration, while the width, length and weight were not significantly affected in Wolbachia‐infected spiders reared at ambient CO2 concentration. The levels of protein, specific activities of peroxidase, and amylase were significantly increased under elevated CO2 concentration or Wolbachia‐infected spiders, while the total amino content was only increased in Wolbachia‐infected spiders. Thus, our current finding suggested that elevated CO2 concentration and Wolbachia enhance nutrient contents and enzyme activity of H. graminicola and decrease development duration hence explore the interactive effects of factors which were responsible for reproduction regulation, but it also gives a theoretical direction for spider's protection in such a dynamic environment. Increased activities of enzymes and nutrients caused by Wolbachia infection aids for better survival of H. graminicola under stress.

Bacterial endosymbionts, such as Wolbachia, are widespread in arthropods, with vertical transmission (from mother to offspring) deemed to be the dominant route. Although horizontal transmission of Wolbachia is considered possible as a conveyance route for infections to be introduced to new individuals, evidence for such transmission remains limited. Thus, to elucidate the horizontal transmission of Wolbachia among hosts, we conducted parasitism, predation, cannibalism, and mating experiments in small spider species Hylyphantes graminicola. While no direct evidence was found for horizontal transmission via parasitism or predation, cannibalism and mating behavior were shown to be potential Wolbachia transmission pathways, with copulatory transmission identified from Wolbachia-positive females to Wolbachia-negative males (and vice versa). Based on these findings, our study suggests that horizontal transmission of Wolbachia is more likely to occur within species than between species.

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., Wang et al. developed a fluorescence-assisted single-cell methodology (split protease-E. coli ClpXP (SPEC)) to characterise protein-protein interactions for both eukaryotic and prokaryotic species in E. coli cells. Their method can quantify these interactions with high sensitivity, easy manipulation, and broad temperature adaptability

Most spiders are natural enemies of pests, and it is beneficial for the biological control of pests to learn the relationships between symbionts and their spider hosts. Research on the bacterial communities of insects has been conducted recently, but only a few studies have addressed the bacterial communities of spiders. To obtain a complete overview of the microbial communities of spiders, we examined eight species of spider (Pirata subpiraticus, Agelena difficilis, Artema atlanta, Nurscia albofasciata, Agelena labyrinthica, Ummeliata insecticeps, Dictis striatipes, and Hylyphantes graminicola) with high‐throughput sequencing based on the V3 and V4 regions of the 16S rRNA gene. The bacterial communities of the spider samples were dominated by five types of endosymbionts, Wolbachia, Cardinium, Rickettsia, Spiroplasma, and Rickettsiella. The dominant OTUs (operational taxonomic units) from each of the five endosymbionts were analyzed, and the results showed that different spider species were usually dominated by special OTUs. In addition to endosymbionts, Pseudomonas, Sphingomonas, Acinetobacter, Novosphingobium, Aquabacterium, Methylobacterium, Brevundimonas, Rhizobium, Bradyrhizobium, Citrobacter, Arthrobacter, Pseudonocardia, Microbacterium, Lactobacillus, and Lactococcus were detected in spider samples in our study. Moreover, the abundance of Sphingomonas, Methylobacterium, Brevundimonas, and Rhizobium in the spider D. striatipes was significantly higher (p

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.

Arthropods are associated with various microorganisms which confer benefits to their hosts. Recently, research has been conducted on bacterial communities of insects to provide an insight into the potential interactions of the symbiotic bacteria and their hosts. Spiders are interesting to study as they are perceived to be natural enemies of pests. The effect of endosymbionts on spiders has been reported, but little is known about the overall bacterial communities present in spiders. Here, we report on the characterization of bacterial communities present in the whole body of the spider Marpiss magister using Illumina sequencing of 16S rRNA amplicons. Our study shows that the most abundant phyla of bacteria included Proteobacteria, Tenericutes, Bacteroidetes and Actinobacteria. At the genus level, the most abundant genera included Rickettsia, Wolbachia, Spiroplasma, and Cardinium. Besides these dominant endosymbionts, our study also showed the existence of bacteria in the genera Arthrobacter, Novosphingobium, Acinetobacter, Pseudomonas, Aquabacterium and Sphingomonas at an abundance ranging from 0.65 to 0.84%, and the existence of bacterial in genera Lactobacillus, Sphingobium, Methylobacterium, Bradyrhizobium, Propionibacterium, Brevundimonas, Achromobacter, Microbacterium, Corynebacterium, and Flavobacterium at a slightly lower abundance ranging from 0.1 to 0.5%. Therefore, our finding indicates that endosymbionts are not the only microbiota present in the spider M. magister, and other bacterial taxa also exist in its bacterial community.

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.

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 mechanism by which spiders respond to heavy metals at the molecular level. In this 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. Ultimately, 3450 cDNA simple sequence repeats were identified and validated as potential molecular markers in the unigenes. A total of 2939, 2491 and 3759 DEGs were detected among the three 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 mechanism underlying spiders' responses to heavy metal stress.

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.