# The Influence of Acute Cold Stress on Intestinal Health of the Juvenile Chinese Soft-Shelled Turtle (Pelodiscus sinensis)

## TL;DR

This study explores how sudden cold stress affects the intestines of Chinese soft-shelled turtles, revealing changes in gut health and molecular responses.

## Contribution

The study provides a comprehensive analysis of physiological, microbial, and molecular responses to acute cold stress in Chinese soft-shelled turtles.

## Key findings

- Acute cold stress at 7°C caused severe intestinal damage, including inflammation and tissue necrosis.
- Cold stress altered gut microbiota, with Helicobacter and Citrobacter dominating at 7°C.
- Transcriptomic and metabolomic analyses identified key pathways like Toll-like receptor signaling and linoleic acid metabolism affected by cold stress.

## Abstract

As a poikilothermic species, the Chinese soft-shelled turtle is highly sensitive to changes in environmental temperature. Sudden drops in temperature often lead to mass mortality, causing substantial economic losses to the aquaculture industry. This study aims to uncover the mechanisms of damage induced by low-temperature stress in the Chinese soft-shelled turtle. Through integrated analysis of intestinal microbiota, molecular expression, metabolite profiles, and related signaling pathways, we further elucidate the physiological and molecular responses of this species to cold stress, providing a scientific basis for understanding its low-temperature adaptability.

Sharp declines in temperature pose a significant risk for mass mortality events in the Chinese soft-shelled turtle (Pelodiscus sinensis). To assess the effects of acute cold stress on intestinal health, turtles were exposed to temperatures of 28 °C (control), 14 °C, and 7 °C for 1, 2, 4, 8, and 16 days. The results showed that acute cold stress at 14 °C and 7 °C induced time-dependent alterations in intestinal morphology and histopathology. The damage was more severe at 7 °C, characterized by inflammatory cell infiltration, lymphoid hyperplasia, and extensive detachment and necrosis across the villi, muscle layer, and submucosa. 16S rDNA sequencing revealed significant shifts in intestinal microbiota composition in the 7 °C group, dominated by Helicobacter and Citrobacter. Transcriptomic analysis identified differentially expressed genes (DEGs) that respond to acute cold stress and are involved in the Toll-like receptor signaling pathway (Tlr2, Tlr4, Tlr5, Tlr7, and Tlr8), the NOD-like receptor signaling pathway (Traf6, Traf2, Casr, Rnasel, Pstpip1, Plcb2, Atg5, and Mfn2), apoptosis (Tuba1c, Ctsz, Ctsb, Kras, Hras, Pik3ca, Bcl2l11, Gadd45a, Pmaip1, Ddit3, and Fos), and the p53 signaling pathway (Serpine1, Sesn2, Ccng2, Igf1, Mdm2, Gadd45a, Pmaip1, and Cdkn1a). Metabolomic profiling highlighted differentially expressed metabolites (DEMs) that cope with acute cold stress, such as organic acids (oxoglutaric acid, L-aspartic acid, fumaric acid, DL-malic acid, and citric acid) and amino acids (including L-lysine, L-homoserine, and allysine). The integrated analysis of DEGs and DEMs underscored three key pathways modulated by acute cold stress: linoleic acid metabolism, neuroactive ligand–receptor interaction, and the FoxO signaling pathway. This study provides a comprehensive evaluation of intestinal health in Chinese soft-shelled turtles under acute cold stress and elucidates the underlying mechanisms.