Physiological Responses and Histopathological Changes in Narrow-Clawed Crayfish (Pontastacus leptodactylus) Under Acute Thermal Stress

Abstract

To investigate thermal tolerance, physiological responses, and molecular mechanisms of the narrow-clawed crayfish (Pontastacus leptodactylus) under acute thermal stress, the P. leptodactylus were acutely exposed to 4 different temperature groups-15 °C (control), 20 °C (T20), 25 °C (T25), and 30 °C (T30)-across 6 time points (3 h, 6 h, 12 h, 24 h, 48 h, and 72 h). Survival rates were recorded at each interval. Subsequent analyses comprised: (1) Hemolymph biochemical parameter determination; (2) hepatopancreatic antioxidant capacity assessment; (3) hepatopancreatic histopathology; and (4) comparative transcriptomics analysis of the hepatopancreas. The results showed that the survival rate in the T30 group significantly declined after 48 h of stress. The histological analysis of the hepatopancreas revealed tissue damage in both the T25 and T30 groups. The T25 group exhibited a notable increase in B-cell density and severe vacuolization, while the T30 group displayed disorganized hepatopancreatic cell arrangement, marked necrosis, and structural phenotypes in hepatopancreatic tubules, including lumen expansion and the loss of the star-shaped lumen structure. Biochemical analyses indicated pronounced declines in energy metabolism markers under elevated temperatures. Furthermore, the T30 group exhibited elevated levels of reactive oxygen species (ROS), malondialdehyde (MDA), superoxide dismutase (SOD), and catalase (CAT), alongside diminished total antioxidant capacity (T-AOC). Similarly, the T25 group displayed increased MDA and CAT levels but decreased T-AOC. Comparative transcriptomic analysis demonstrated that differentially expressed genes (DEGs) in the control vs. T25 group were predominantly enriched in metabolic pathways, whereas DEGs identified in control vs. T30 and T25 vs. T30 comparisons showed significant enrichment in energy metabolism and apoptotic processes. Based on these findings, we concluded that acute thermal stress induces mortality in P. leptodactylus through hepatopancreatic structural damage, energy metabolism dysregulation, and excessive ROS accumulation. Notably, P. leptodactylus should be excluded from aquaculture environments exceeding 25 °C. These results enhance understanding of the adaptive mechanisms of P. leptodactylus under acute thermal stress and provide actionable insights to advance its industrial cultivation.

Keywords: RNA-seq; energy metabolism; narrow-clawed crayfish; thermal stress.

Figure 1

Survival rate of P. leptodactylus under acute thermal stress during a 72 h.

Figure 2

Transverse sections of the hepatopancreas tubules of the P. leptodactylus under acute high-temperature stress for 48 h. (A) 15 °C 100×, (B) 15 °C 400×, (C) 25 °C 100×, (D) 25 °C 400×, (E) 30 °C 100×, (F) 30 °C 400×; B: Blister-like cells, F: Fibrillar cells; R: Resorptive cells; E: Embryonic cells; L: Lumen; BM: Basement membrane. The red arrows indicate highly vacuolated cells, with some of the vacuoles being fused. The blue arrow indicates extravasation of hemocytes. The blue asterisk indicates the dilatation of the hepatopancreatic tubule lumen. The brown arrow indicates partial cell degeneration and fusion. The purple arrow points to damage in the brush border.

Figure 2

Transverse sections of the hepatopancreas tubules of the P. leptodactylus under acute high-temperature stress for 48 h. (A) 15 °C 100×, (B) 15 °C 400×, (C) 25 °C 100×, (D) 25 °C 400×, (E) 30 °C 100×, (F) 30 °C 400×; B: Blister-like cells, F: Fibrillar cells; R: Resorptive cells; E: Embryonic cells; L: Lumen; BM: Basement membrane. The red arrows indicate highly vacuolated cells, with some of the vacuoles being fused. The blue arrow indicates extravasation of hemocytes. The blue asterisk indicates the dilatation of the hepatopancreatic tubule lumen. The brown arrow indicates partial cell degeneration and fusion. The purple arrow points to damage in the brush border.

Figure 3

Changes in hemolymph content of TG (A), T-CHO (B), GLU (C), HDL-C (D), and LDL-C (E) in P. leptodactylus under thermal stress. Each bar represents the mean ± SD (n = 6). Different letters above the bars indicate that there are significant differences between groups (p < 0.05).

Figure 4

Changes in hepatopancreatic ROS content (A), T-AOC (B), SOD (C) and CAT (D) activities, and MDA content (E) in P. leptodactylus exposed to thermal stress. Each bar represents the mean ± SD (n = 6). Different letters above the bars indicate that there are significant differences between groups (p < 0.05).

Figure 5

Differential gene expression analysis in the hepatopancreas of the P. leptodactylus under different temperature stress. Statistical analysis of DEGs of the Control vs. T25 (A), Control vs. T30 (B), and T25 vs. T30 (C). Volcano plot of DEGs of the Control vs. T25 (D), Control vs. T30 (E), and T25 vs. T30 (F).

Figure 6

GO enrichment analysis of DEGs in the hepatopancreas of the P. leptodactylus under different temperature stress. Control vs. T25 (A), Control vs. T30 (B), and T25 vs. T30 (C).

Figure 7

KEGG enrichment analysis of DEGs in the hepatopancreas of the P. leptodactylus under different temperature stress. Control vs. T25 (A), Control vs. T30 (B), and T25 vs. T30 (C).