Immunological mechanisms of Hsp70-Tlr4 and Hsp90-Akt signaling pathways in Percocypris pingi during chronic heat stress.

Percocypris pingi was classified as a National Class II Key Protected Wild Animal in 2021, exclusively for wild populations. Due to habitat degradation, the population size and distribution range of P. pingi have been drastically reduced. Climate warming threatens this stenothermal species by restricting suitable habitats. This study investigated spleen immune responses of juvenile P. pingi exposed to temperature gradients (0 d-9 °C, 0 d-15 °C, 0 d-21 °C, 0 d-27 °C, 7 d-27 °C and 14 d-27 °C). The results showed that 27 °C stress significantly induced macrophage proliferation in a time-dependent manner. During water temperature maintenance at 27 °C, the positive area percentages of M1 and M2 macrophages presented the reverse pattern. RNA-seq analysis revealed that multiple immune-related pathways and MAPK signaling pathway were clustered in the elevated process of water temperature based on the KEGG enrichments for 471 and 485 differentially expressed genes. Compared with 0 d-9 °C, Hsp70, Tlr4, Myd88, p38, Jnk and Nf-κb were significantly upregulated at 0 d-21 °C, and Hsp70, p38 and Erk were remarkably upregulated at 0 d-27 °C. Then, akt and s6k1/2 expression elevated at 0 d-21 °C, and hsp90, pi3k and eif4e were low at 0 d-15 °C when compared to 0 d-9 °C. Furthermore, Hsp70-Tlr4 pathway correlated with M1 polarization, while Hsp90-Akt pathway correlated with M2 polarization. Validation (0 d-18 °C, 0 d-28 °C, 0 d-33 °C and 14 d-33 °C) in zebrafish confirmed conserved in hsp70l, mapk8b and mapk3 expression patterns. Thus, Hsp70-Tlr4 and Hsp90-Akt pathways are essential for temperature adaptation in P. pingi spleen.