# Long-Term Heat Stress Triggers Immune Activation and Cell Death Remodeling in the Brain of Largemouth Bass (Micropterus salmoides)

## TL;DR

Long-term heat stress in largemouth bass causes brain changes, including immune activation and cell death, suggesting a new way the brain adapts to high temperatures.

## Contribution

Reveals brain-specific immune activation as a compensatory strategy in fish under chronic heat stress.

## Key findings

- Heat stress caused cytoarchitectural changes and up-regulated immune-related pathways in the brain.
- 1240 differentially expressed genes were identified, with immune and cell death pathways notably affected.
- Inhibition of chromosome segregation genes and activation of ribosome biogenesis and stress response genes were observed.

## Abstract

The impacts of global warming on fish physiology, particularly on the brain—a tissue highly sensitive to environmental temperature fluctuations—remain incompletely understood. In this study, we subjected largemouth bass to long-term heat stress and subsequently conducted histopathological examinations and RNA-seq analysis. Our results revealed distinct cytoarchitectural alterations in certain brain regions. Additionally, we identified 1240 differentially expressed genes. Bioinformatics analysis showed that multiple immune-related pathways were unexpectedly up-regulated, while cell death pathways were slightly activated. Concurrently, disturbances of gene sets related to microtubule dynamics and chromosome segregation were observed, leading to the inhibition of cell proliferation under heat stress. Protein–protein interaction analysis showed that 10 hub genes associated with chromosome segregation were generally inhibited, whereas ribosome biogenesis and stress response processes were enhanced in the treatment group. Collectively, our findings suggest that chronic heat stress may induce cytoarchitectural remodeling in the largemouth bass brain, accompanied by the activation of innate immunity to facilitate acclimation to environmental changes.

Heat stress typically suppresses systemic immunity in fish; however, its effects on the brain—an organ traditionally regarded as immune-privileged—remain unclear. In this study, we performed histopathological examination and RNA-seq analysis on the brains of juvenile largemouth bass (Micropterus salmoides) exposed to control (28 °C) and elevated (36.5 °C) water temperatures for 8 weeks. Histological analysis revealed distinct cytoarchitectural and pathological changes in specific brain regions. RNA-seq analysis identified a total of 1240 differentially expressed genes, with 22 heat shock protein genes notably showing significant up-regulation. The immune system-associated genes emerged as the most prominently affected category. Gene set enrichment analysis (GSEA) based on Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotations revealed that up-regulated genes were enriched in immunity-related pathways, including the NOD-like receptor (NLR) signaling pathway, Toll-like receptor (TLR) signaling pathway, and cytosolic DNA-sensing pathway. Additionally, the levels of apoptosis and necroptosis were moderately increased. GSEA based on Gene Ontology (GO) terms indicated that down-regulated genes were primarily associated with cell division. Protein–protein interaction (PPI) and clustering analysis identified 41 core genes in the top three clusters, encompassing those related to nuclear chromosome segregation, ribosome biogenesis, and stress response. The inhibition of genes involved in nuclear chromosome segregation may disrupt cellular homeostasis by significantly impairing microtubule dynamics. In contrast, genes associated with ribosome biogenesis and stress response were up-regulated, which could counteract the adverse effects caused by long-term heat stress. We propose that brain-specific immune activation, particularly via the NLR and TLR signaling pathways, acts as a compensatory strategy to counterbalance heat-induced cell death, thereby revealing a novel neuro-immune adaptation axis.