# The Potential Implications of Sex-Specific Differences in the Intestinal Bacteria of the Overwintering Wolf Spider Pardosa astrigera (Araneae: Lycosidae)

## TL;DR

This study shows that male and female wolf spiders have different gut bacteria during winter, which may affect their ability to survive cold temperatures.

## Contribution

The study reveals sex-specific differences in the gut microbiome of overwintering Pardosa astrigera and identifies potential key bacterial species.

## Key findings

- Female Pardosa astrigera have higher Chao1 and Shannon diversity indices but lower Simpson index compared to males.
- Males have higher relative abundances of Proteobacteria and Actinobacteriota, while females have more Bacteroidota and Firmicutes.
- Six key metabolic pathways were more active in females, while five were less active compared to males.

## Abstract

There are differences in the cold resistance and anti-freeze compound levels in the two sexes of Pardosa astrigera, driving us to explore the differences in their intestinal microbiome. In this study, intestinal bacterial communities of P. astrigera in winter were compared using 16S rRNA high-throughput sequencing. The results showed significant differences in most alpha diversity indices and the relative abundances of dominant bacteria between males and females. Our research reveals the significant role of sex in shaping the diversity and composition of intestinal bacteria in overwintering P. astrigera. We suggest that Pseudomonas versuta (belonging to Proteobacteria) and Rhodococcus erythropolis (belonging to Actinobacteriota) may have the potential to play key roles in overwintering P. astrigera.

Gut microbiota can promote the resistance of host arthropods to low-temperature stress. Female Pardosa astrigera have a lower anti-freeze compound level and weaker resistance to cold temperatures than the males in winter, which implies that their intestinal bacteria may be different during overwintering. This study primarily compared the intestinal bacterial communities between the two sexes of P. astrigera in a temperate region using 16S rRNA gene sequencing. Our findings indicated that the Chao1 and Shannon indices of intestinal bacteria in females were significantly higher than those in males, while the Simpson index in females was significantly lower than that in males. The male intestinal bacterial community was characterized by Proteobacteria and Actinobacteriota at the phylum level and by Pseudomonas and Rhodococcus at the genus level, with total relative abundances of 89.58% and 85.22%, respectively, which were also significantly higher than those in females, whose total relative abundances were 47.49% and 43.68%, respectively. In contrast, the total relative abundances of Bacteroidota and Firmicutes were significantly lower in males (4.26% and 4.75%, respectively) than in females (26.25% and 22.31%, respectively). Noteworthy divergences in bacterial communities were also found through an LEfSe analysis between females and males. Additionally, the results of the PICRUSt2 analysis showed that six out of eleven level-2 pathways related to key metabolic functions were significantly (or marginally significantly) higher in females than males, and five other level-2 pathways were significantly (or marginally significantly) lower in females than males. Our results imply that significant gender differences exist in intestinal bacterial communities of overwintering P. astrigera. We suggest that Pseudomonas versuta (belonging to Proteobacteria) and Rhodococcus erythropolis (belonging to Actinobacteriota) may have the potential to play key roles in overwintering P. astrigera.