Heat stress induces pathological and molecular responses in the gills of largemouth bass (Micropterus salmoides) revealed by histology, transcriptomics, and DNA methylomics

Largemouth bass (Micropterus salmoides), an important species in both aquatic ecosystems and aquaculture, is increasingly affected by heat extremes. Heat stress poses significant challenges to fish health, particularly affecting key organs like the gills. This study investigated the effects of heat stress (37 °C) on the gills of juvenile largemouth bass, focusing on histopathology and molecular adaptations, including genome-wide gene expression and DNA methylation. After the heat stress experiment, histological analysis of hematoxylin and eosin-stained gill tissues revealed significant alterations, including increased blood cell density, epithelial cell proliferation, and curling of gill lamellae. Ultrastructural analysis via transmission electron microscopy showed irregular cell shapes of the secondary lamellae. TUNEL staining demonstrated a significant increase in apoptotic cells, from 14.53 % in control fish versus 69.58 % in heat-stressed fish, which correlated with upregulation of apoptosis-related genes, such as caspase3 and p53. Transcriptomic analysis using RNA sequencing identified 608 differentially expressed genes. KEGG enrichment analysis revealed significant enrichment of the “protein processing in endoplasmic reticulum” pathway. Additionally, whole-genome bisulfite sequencing and methylomic analysis identified 12,505 differentially methylated regions. GO enrichment analysis of genes containing promoter DMRs identified 44 significantly enriched terms, including “metabolic process,” “organic substance metabolic process,” “binding” and “protein interaction.” Notably, genes associated with apoptosis, such as capn3a, dok4, rassf7a, and tusc2a, exhibited significant changes in both gene expression and DNA methylation in their promoters. These findings provide insights into the molecular responses of fish gills to heat stress and mechanisms of resilience in cultured fish species under changing environmental conditions.