Multiomics profiling reveals the adaptive responses of largemouth bass to high temperature stress
Wenzhi Guan, Yongqing Yu, Jinpeng Zhang, Jieliang Jian, Baolong Niu, Bao Lou, Dayan Hu, Xiaojun Xu
TL;DR
This study explores how largemouth bass respond to high temperatures, revealing tissue damage and recovery mechanisms linked to specific biological pathways.
Contribution
The study identifies novel regulatory pathways and the role of Hspa9 in largemouth bass during heat stress recovery.
Findings
Heat stress causes oxidative damage to gill, brain, and liver tissues in largemouth bass.
Recovery involves PPAR, focal adhesion, ErbB, retinoid metabolism, and cytochrome P450 pathways.
Hspa9 is shown to play a pivotal protective role during heat stress.
Abstract
Largemouth bass (Micropterus salmoides) is among the most economically important freshwater fish species. High temperature is a major abiotic stressor, leading to increased mortality and significant economic losses. However, research on the regulatory mechanisms of heat stress response in largemouth bass is limited. This study aims to elucidate the mechanisms of adaptability in largemouth bass during heat stress and subsequent recovery. The morphobiochemical alterations and adaptive mechanisms induced by high water temperature in the gill, brain and liver tissues of largemouth bass are investigated through biochemical blood analysis, haematoxylin and eosin staining, transmission electron microscopy and transcriptomic and proteomic profiles. The results reveal that heat stress-induced oxidative stress causes severe damage to the gill, brain and liver tissues, as well as to the…
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Figure 9- —National Key Research and Development Program of China
- —Zhejiang Provincial Natural Science Foundation of China
- —Huzhou Agricultural New Quality Productivity R&D and Promotion Project
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TopicsPhysiological and biochemical adaptations · Heat shock proteins research · Effects of Environmental Stressors on Livestock
Background
In recent years, global warming has intensified, leading to a gradual increase in water temperatures. Elevated water temperatures have profound effects on aquatic ecosystems, disrupting biological interactions and inducing physiological changes in fish and other organisms [1, 2]. High water temperature has a particularly significant effect on the physiology of fish, resulting in diminished reproduction, reduced immunity, and slower growth rates, which directly affect the economic viability of aquaculture [3–5]. Consequently, understanding the genetic mechanisms underlying high temperature adaptation in fish has become a research hotspot. To date, in-depth studies have investigated the effects of high environmental temperatures on various fish species, including Oreochromis mossambicus, Danio rerio, Scophthalmus maximus, Oncorhynchus mykiss, and Carassius auratus [6–10]. Exploring the physiological and biochemical mechanisms underlying fish responses to acute high temperature is essential for guiding the development of healthy fish breeding practices.
Largemouth bass (Micropterus salmoides) is a key economic freshwater fish species in China. Owing to its tender meat, lack of intermuscular spines, and fast growth rate, it has rapidly become an important aquaculture species, with cultured production surpassing 0.94 million tons in 2024 (Ministry of Agriculture and Rural Affairs China, 2025). In recent years, numerous studies have focused on the exploration of breeding modes, including breeding practices, disease prevention, feed nutrition, fast growth, and sex differentiation, many of which have yielded significant findings [11–16]. The optimal growth temperature for largemouth bass is between 26 and 29 °C [17]. However, with the increasing frequency of extreme high temperature due to global climate change, largemouth bass are facing severe thermal stress (Fig. 1A–C). High temperature during the summer negatively impacts the physiological and biochemical parameters of fish, leading to reduced feeding, slowed growth, frequent disease outbreaks, and elevated mortality rates, all of which result in considerable economic losses [18–20]. Despite these challenges, few studies have investigated the molecular mechanisms underlying the response of the largemouth bass to high temperature stress, which severely restricts the process of heat-tolerant breeding. Understanding the regulatory mechanisms of the adaptation of the largemouth bass to high temperature is thus crucial, as it has important implications for the breeding of heat-tolerant species and for supporting the sustainable development of the aquaculture industry.Fig. 1. Water temperature variation of largemouth bass aquaculture ponds in the summer (A), water temperature data statistics (B). The test site is at Rongsheng Fishery Co., LTD., Changxing County, Huzhou, Zhejiang Province. The test time was from July 19 to September 12 in both 2023 and 2024, with the pond water depth maintained at 1 m. C Schematic diagram of high temperature stress in largemouth bass. D Schematic illustration of experimental design. Samples were collected at the red dots
Although liver transcriptomes have been investigated in largemouth bass under high temperature stress, integrated transcriptome-proteome analyses remain rare in fish, particularly across multiple tissues during heat stress and recovery [21]. Comprehensive studies combining morphobiochemical, transcriptomic and proteomic analyses have not yet been reported, especially concerning the high temperature adaptation mechanisms in largemouth bass. Thus, the aim of the present study was to elucidate the morphobiochemical modulations induced by high water temperature through transcriptomics and proteomics in the gill, brain and liver tissues of largemouth bass. This research seeks not only to deepen the understanding of fish responses to climate change but also to improve survival at high temperature and provide a scientific foundation for aquaculture.
Results
Changes in vital tissue and biochemical blood indices
We conducted surveys on temperature variations in largemouth bass aquaculture ponds from July 19 to September 12 in both 2023 and 2024. The results revealed that the average water temperature in the summer of 2024 was 2.3 °C higher than that in 2023, indicating a 7.7% increase (Fig. 1A) [22]. Additionally, in 2024, the water temperature exceeded 35 °C on 18 days, which was 8 more days than in 2023 (Fig. 1B).
Observations of gill, brain and liver tissues under high temperature stress at 0 h, 24 h and 48 h were analysed. Histological analyses (HE staining) revealed that heat stress caused great damage to the gill, brain and liver tissues at 24 h compared to 0 h in the control groups (Fig. 2A). The gill tissue sections showed that the filaments were congested and swollen, and the diameters of the secondary lamellae thickened significantly (P < 0.05), but the area of the secondary lamellae and the diameters of the gill filaments decreased significantly at 24 h and 48 h (P < 0.05), respectively (Fig. 2C). Additionally, the secondary lamellae were severely disrupted, and the epithelial cells exhibited hypertrophy and rupture. Under heat stress, pericellular oedema, severe vacuolation and nuclear aggregation were observed in the brain tissue sections, and the liver tissue sections exhibited cell swelling, vacuolar deformation, nuclear pyknosis and karyolysis (Fig. 2A and Additional file 1: Table S1).Fig. 2. Histological sections (A) and TEM (B) for the effects of water temperature change on gill, brain, liver tissues of largemouth bass at different times, gill morphological change data statistics (C), and biochemical blood index analysis (D). HE staining of gill: SL, secondary lamella. BC, blood cell. MC, mitochondria-rich cell. PVC, pavement cell. Ep, epithelial cells. Brian: V, vacuolation. NA, nuclear aggregation. Liver: Cv, central vein. HC, hepatocyte. HV, hepatocellular vacuolation. Pn, nuclear pyresis. scale bar = 100 μm. TEM, MT, mitochondria (blue arrow). ER, endoplasmic reticulum (purple arrow). GA, golgi apparatus (yellow arrow). orange scale bar = 1 μm, black scale bar = 200 nm. * P < 0.05, ** P < 0.01, *** P < 0.001
Transmission electron microscopy (TEM) was used to assess the ultrastructures of the gill, brain and liver tissues (Fig. 2B). The structures of the mitochondria, endoplasmic reticulum and Golgi apparatus were intact, and the mitochondrial cristae were clear at 0 h. After 24 h of heat stress, these structures were severely damaged, the membrane structures were partially dissolved, and mitochondrial vacuolation increased significantly. In the heat recovery group at 48 h, the damage to some organelles was alleviated across different tissues.
Biochemical blood indices results revealed that the levels of superoxide dismutase (SOD) and triglycerides (TG) were significantly lower (P < 0.05), while total protein (TP) levels were significantly higher (P < 0.05) under heat stress at 24 h than at 0 h. In the recovery group at 48 h, the levels of glutamic oxalacetic transaminase (GOT), SOD, and TG were significantly lower (P < 0.05), whereas the level of catalase (CAT) was significantly greater (P < 0.05) than at 0 h (Fig. 2D). These findings indicate that high temperature stress severely damages key tissues in fish, with oxidative stress being a major factor contributing to apoptotic damage.
Transcriptomic analysis
A total of 213.48 GB of clean data were generated from RNA-seq libraries prepared from gill, brain, and liver tissues. The average Q30 value exceeded 94.93% (Additional file 1: Table S2), indicating high sequencing quality. Quality-trimmed clean reads were obtained and mapped to the reference genome (GCF_014851395.1), with alignment rates ranging from 95.17% to 96.87%. Principal component analysis (PCA) of the transcriptomes revealed clear separation of the nine sample groups (Fig. 3A, B). A total of 84,635 transcripts were identified, comprising 52,065 previously annotated transcripts and 32,570 new transcripts. Differential expression analysis revealed 3,649, 2,963, and 6,496 differentially expressed genes (DEGs) across the gill, brain, and liver tissues, respectively (P-adjust < 0.05; |log_2_FC|≥ 1). Among these genes, 1,257, 46, 1,381, 445, 120, 541, 2,424, 98 and 2,651 DEGs showed upregulated expression, and 1,121, 105, 1,537, 1,059, 785, 1151, 2,548, 162 and 2,227 DEGs showed downregulated expression in the different groups (Fig. 3D).Fig. 3. Correlation analysis between gill (Gi), brain (Br) and liver (Li) samples (A), PCA analysis between transcriptome and proteome samples (B–C), the number of DEGs and DEPs in different comparison groups (D), Venn diagram illustrating DEGs (black digit)/DEPs (red digit) across different comparative groups (E–G)
Notably, 15, 23, and 47 DEGs were shared among the three groups in the gill, brain and liver, respectively, strongly suggesting that distinct functional pathways were impacted at each stage (Fig. 3E‒G). Functional enrichment analysis using the GO and KEGG databases revealed significant enrichment in pathways such as the cell cycle process (GO:0022402), DNA replication initiation (GO:0006270), haemoglobin complex (GO:0005833), oxygen carrier activity (GO:0005344), oxygen binding (GO:0019825), DNA replication (GO:0006260), cell cycle, p53 signalling pathway, ECM-receptor interaction, protein digestion and absorption, HIF-1 signalling pathway, PI3K-Akt signalling pathway, PPAR signalling pathway, MAPK signalling pathway, protein processing in the endoplasmic reticulum, and xenobiotic metabolism by cytochrome P450 (Fig. 4). Interestingly, overlapping pathways were identified among the comparisons between different groups within the same tissue, highlighting the dynamic regulation of gene expression under high temperature stress.Fig. 4GO-enriched pie (A‒I) charts and KEGG-enriched bubble diagram (J‒L) of transcriptome analysis of DEGs in largemouth bass’s different groups. Gi, gill. Br, brain. Li, liver
4D-DIA protein analysis
To gain insights into the differentially expressed proteins (DEPs) in the gill, brain, and liver tissues of largemouth bass under heat stress, proteomic analyses were conducted on tissue samples. PCA of the proteomic profiles revealed clear separation among the nine sample groups (Fig. 3C). Following data preprocessing and de-redundancy, 59,712 peptides and 7,040 proteins were identified. Among these, the numbers of DEPs with upregulated expression in the different groups were 357, 377, 456, 445, 423, 335, 280, 489, and 444, whereas the numbers of DEPs with downregulated expression were 502, 468, 433, 461, 332, 337, 740, 684, and 435, respectively (Fig. 3D). Notably, 154, 102, and 167 DEPs were shared among the gill, brain, and liver samples, respectively (Fig. 3E–G). GO enrichment analysis revealed significant enrichment of terms such as regulation of hormone levels (GO:0010817), cell communication (GO:0007154), hormone metabolic process (GO:0042445), hormone biosynthetic process (GO:0042446), chemical synaptic transmission (GO:0007268), synaptic signalling (GO:0099536), and regulation of neurotransmitter levels (GO:0001505) across different tissues. KEGG enrichment results revealed that the neuroactive ligand-receptor interaction pathway was significantly co-enriched in all three gill tissue groups, whereas mucin type O-glycan biosynthesis was significantly co-enriched in the brain tissues, and both the neuroactive ligand-receptor interaction pathway and cell adhesion molecules were significantly co-enriched in the liver tissues (Additional file 2: Fig. S1).
Integrated analysis of DEGs and DEPs
To assess the consistency between gene expression at the transcript and protein levels, comparative transcriptomic and proteomic analyses were performed on tissues under heat stress. A total of 96 and 100 DEGs/DEPs were identified in the gill tissues at 24 h vs. 0 h and 48 h vs. 24 h, respectively. Among these, 68 DEGs (42 upregulated and 26 downregulated) and 38 DEGs (18 upregulated and 20 downregulated), respectively, exhibited expression patterns consistent with those of their corresponding DEPs. Similarly, 93 and 34 DEGs/DEPs were identified in the brain tissues, with 63 DEGs (11 upregulated and 52 downregulated) and 26 DEGs (13 upregulated and 13 downregulated), respectively, showing congruent expression with the DEPs. In the liver tissues, 317 and 185 DEGs/DEPs were identified, with 203 DEGs (70 upregulated and 133 downregulated) and 65 DEGs (41 upregulated and 24 downregulated), respectively, showing congruent expression with the DEPs (Fig. 5A‒F) [23]. Notably, compared with the other tissues, the liver presented greater numbers of up- and downregulated DEGs/DEPs in the 24 h vs. 0 h group, likely reflecting the complex and multifunctional role of the liver in response to heat stress.Fig. 5. Quantitative Venn diagrams of the transcriptome and proteome association and clustering heat map of co-expression of DEGs and DEPs in gill (A, B), brain (C, D) and liver (E, F) tissues across the different groups. Gi, gill. Br, brain. Li, liver
A nine-quadrant map was constructed to analyse the associations between gene and protein expression. The results demonstrated that all gene and protein expression was positively correlated and showed consistent trends in the gill, brain and liver tissues (Fig. 6A–C). DEGs and DEPs with matching expression trends were selected for KEGG enrichment analysis [24]. In the gills, most DEGs and DEPs were enriched in pathways such as protein processing in the endoplasmic reticulum, apoptosis, RNA degradation, fatty acid metabolism, and the PPAR signalling pathway in the 24 h vs. 0 h comparison group. Similarly, focal adhesion, ErbB signalling, and fatty acid metabolism were the predominant pathways enriched in the 48 h vs. 24 h group (Fig. 6D, E). The top enriched pathways in the brain included the Toll-like receptor signalling pathway, DNA replication, the cytosolic DNA-sensing pathway, and cytokine–cytokine receptor interactions in the 24 h vs. 0 h group, as well as DNA replication, the cell cycle, the PPAR signalling pathway, and butanoate metabolism in the 48 h vs. 24 h group (Fig. 6F, G). In the liver, the most enriched pathways were steroid biosynthesis, retinol metabolism, and metabolic pathways in the 24 h vs. 0 h group, whereas ECM-receptor interaction, retinol metabolism, and drug metabolism-cytochrome P450 were the predominant pathways enriched in the 48 h vs. 24 h group (Fig. 6H, I). Notably, while the enriched pathways varied across tissues and temperature treatments, similarities and differences emerged, underscoring the complexity, flexibility, and diversity of gene regulatory pathways within the organism. These adaptations likely contribute to maintaining physiological homeostasis and ensuring normal function under varying temperature conditions. To more clearly illustrate the regulatory processes of largemouth bass in response to heat stress and heat recovery, we have constructed relevant pathway diagrams based on coexpression DEGs/DEPs (Fig. 7).Fig. 6. Nine-quadrant map of transcriptomic and proteomic associations for gill, brain and liver tissues (A–C) and KEGG enrichment diagram for the coexpression of DEGs and DEPs across the different groups (D–I). R, the pearson correlation coeficient between DEGs and DEPs. p*, p-value.* Gi, gill. Br, brain. Li, liverFig. 7Adaptation mechanisms of largemouth bass in respond to water temperature variations. Gill, protein processing in endoplasmic reticulum, apoptosis and necroptosis signalling pathways were displayed at heat stress treatment, ECM-receptor interaction, focal adhesion and ErbB signalling pathways were displayed at heat stress recovery. Brain, toll-like receptor and DNA replication signalling pathways were displayed at heat stress treatment, PPAR signalling pathway and cell cycle were displayed at heat stress recovery. Liver, steroid biosynthesis and retinol metabolism were displayed at heat stress treatment, endocytosis and ECM-receptor interaction were displayed at heat stress recovery. Red colors indicate up-regulation of DEGs/DEPs
Protein–protein interaction (PPI) network and functional enrichment
To explore the relationships between DEGs/DEPs during high temperature stress in largemouth bass, a protein–protein interaction (PPI) network was constructed using the STRING database and visualised with Cytoscape software. The resulting PPI network incorporated all 254 identified coexpressed DEGs and DEPs (duplicate and unnamed DEGs and DEPs across tissues were removed from Supplementary S2) across gill, brain, and liver tissues. The coexpression PPI network contained 72 DEGs and DEPs (Fig. 8A). HSPA9 was clearly positioned at the core of the network, where it interacts with key proteins such as HSP90, HSPA5, HSPBP1, ACO2, HSPD1, HSPA4A, HSPA4B, ACAA1, and COQ3. This central role suggests that HSPA9 may be a critical candidate protein that coordinates interactions with other genes to facilitate adaptation during high temperature stress. Functional enrichment analysis revealed that these interconnected DEGs and DEPs were significantly associated with pathways such as protein processing in the endoplasmic reticulum, fatty acid degradation, steroid biosynthesis, and the PPAR signalling pathway (Fig. 8B). These findings highlight the involvement of metabolic signalling pathways and their intricate interactions in orchestrating an organism’s response to high temperature stress.Fig. 8PPI network (A) and Sankey bubble diagrams (B) of coexpression of DEGs and DEPs associated in the three tissues
RT‒qPCR verification
Nine high temperature-related genes (Atp6, Cox1, Cytb, Hsp40, Hsp70, Hsp90b1, Hbae5, Pla2, and Pla2r) involved in thermogenesis, oxidative phosphorylation, protein processing in the endoplasmic reticulum and ether lipid metabolism were selected from the DEGs identified in the gill, brain, and liver for validation of the RNA-seq and protein profile data using RT-qPCR (Additional file 2: Fig. S2). The expression patterns of these genes across different samples demonstrated a degree of consistency between the RT-qPCR results and the RNA-seq results. Notably, among the tested genes, Hsp40, Hsp70, and Hsp90b1 exhibited the highest expression levels following 24 h of heat stress, followed by a gradual decline in the 48 h recovery group.
Inductive expression patterns of Hspa9 under heat stress
To investigate the involvement of the Hspa9 gene in the heat stress response, expression levels of the Hspa9 mRNA and protein were analyzed across gill, brain and liver tissues of largemouth bass. Under 24 h heat stress, Hspa9 mRNA expression was significantly upregulated in the gill (P < 0.05), as well as in the brain (P < 0.05) and liver tissues (P < 0.001), with the most pronounced upregulation observed in the liver. Following 48 h of recovery, Hspa9 mRNA levels were significantly downregulated in these tissues, particularly in the liver (P < 0.01) (Fig. 9A). Consistently, the expression of HSPA9 protein displayed a similar trend (Fig. 9B, C), with the cytoplasm being the primary site of its expression (Fig. 9B).Fig. 9. Relative expression levels of Hspa9 under heat stress and the phylogenetic tree. A qPCR analysis of Hspa9 mRNA under heat stress (24 h) and recovery (48 h) in different tissues. * P < 0.05, ** P < 0.01, ***P < 0.001. B Immunohistochemistry analysis. red scale bar = 3 μm. C Western blot analysis, the polypeptide sequence of HSPA9 is CGRAPSKSVNPDEAV. D Phylogenetic tree of HSPA9 proteins (pentagons, largemouth bass). Accession numbers of amino acid sequences retrieved from GenBank are shown. The neighbor-joining method with MEGA 11 software and interactive tree of life online tool were used for drawing
The phylogenetic tree analysis revealed that the HSPA9 sequence of largemouth bass formed a clade with Pempheris klunzingeri HSPA9, which subsequently clustered with orthologs from Siniperca chuatsi, Chelmon rostratus, and Chaetodon trifascialis (Fig. 9D). This topology indicates strong evolutionary conservation of the HSPA9 coding sequence across teleost fishes.
Discussion
As poikilotherms, most fish are inherently influenced by water temperature, which regulates core physiological processes. Recent extreme heat events caused by climatic factors (especially prolonged summer heatwaves) pose serious threats to the survival and growth of fish. This urgency has accelerated research into thermal adaptation mechanisms, for which multiomics approaches have yielded profound mechanistic insights into piscine thermotolerance, leading to pivotal advances [25–28]. Heat stress can be a lethal stressor and can induce changes in fish behaviour and metabolism, including alterations in swimming speed, hormone secretion, and the regulation of energy and material metabolism. Our previous study revealed that largemouth bass lose balance and die when the temperature increases to 37.8 °C. In this study, heat-sensitive largemouth bass were subjected to acute high temperature treatment followed by defined recovery periods. With a focus on key tissues including the gill, brain, and liver, we investigated the effects of heat stress and elucidated the underlying mechanisms of thermal tolerance using integrated transcriptomic and proteomic analyses.
Heat stress has been shown to cause significant damage to important tissues and promote apoptosis in various fish species, which is consistent with the findings of our study (Fig. 2A, B). For example, under heat stress, gill filaments often exhibit congestion and swelling, with the gill lamellae showing severe bending and deformation. Additionally, mitochondrial swelling occurs, accompanied by a loss of clear cristae structure, disruption of the nuclear envelope’s double-layered structure, and slight expansion of the endoplasmic reticulum [25, 29]. These alterations in damage impair gill functions such as oxygen intake, osmoregulation, nitrogenous waste excretion and ion and acid–base balance [30]. High temperature can also induce a stress response in nerve cells within the fish brain, potentially resulting in cellular impairment or apoptosis [31]. Such impairments may compromise neural signal transmission, including pathways critical for thermoregulation and broader homeostasis maintenance. Similarly, heat stress can lead to cellular oedema, nuclear disappearance, dissolution, and even localised necrosis of liver tissue [25]. These damage changes may disrupt the liver metabolic disorders, the detoxification of exogenous substances, and the synthesis ability of bile acids, all of which are crucial pathways for maintaining metabolic homeostasis and overall physiological adaptability [10]. Further studies have shown that the tissue damage caused by heat stress in fish is directly related to the accumulation of ROS and oxidative stress [32, 33].
Heat stress disrupts mitochondrial structure in fish (e.g., cristae fragmentation and membrane potential decline), leading to reduced electron transport efficiency of mitochondrial electron transport chain complex I/III, and thereby generating ROS [34]. Excessive ROS affect lipid peroxidation, protein oxidation and DNA damage [35]. Enzymes (e.g., SOD, CAT, and GSH-Px) effectively alleviate oxidative stress by controlling the production of ROS [36]. SOD catalyses the dismutation of superoxide radicals into hydrogen peroxide, which is subsequently detoxified by CAT and GPX into water and molecular oxygen [37]. However, sustained heat stress causes the rate of ROS scavenging to be lower than that of their generation, ultimately leading to an insufficient content of antioxidant enzymes. Our data revealed that the levels of SOD and TG decreased in the treatment group (Fig. 2D). SOD level decreases might be due to overconsumption by excessive ROS and inhibited synthesis from transcriptional/translational impairment during the later stage of heat stress [38]. TG levels decrease as lipolysis (for energy) increases and synthesis is suppressed.
To adapt to heat stress, fish tissues leverage evolutionarily conserved signalling pathways that mitigate ROS-induced oxidative insult during heat stress. In the gill, the RNA-seq and protein-seq results revealed that most DEGs and DEPs were enriched in pathways related to protein processing in the endoplasmic reticulum, apoptosis, RNA degradation, fatty acid metabolism, and PPAR signalling during heat stress (Fig. 6D). Notably, protein processing in the endoplasmic reticulum plays a critical role in heat stress by regulating protein folding, synthesis and degradation [39]. If heat stress persists, the unfolded protein response may induce apoptosis and RNA degradation as protective mechanisms against cellular damage [40, 41]. Fatty acid metabolism affects membrane fluidity, energy production, oxidative stress management, and stress signalling [42]. PPAR-α enhances fatty acid utilisation for energy production by upregulating the expression of β-oxidation related genes, whereas PPAR-δ induces the expression of genes encoding antioxidant enzymes (such as SOD and CAT) to neutralise ROS and protect cells from oxidative damage [43]. Differently, the decrease in some antioxidant enzyme levels might be caused by the enzyme consumption due to excessive ROS, inhibition of the PPAR-δ regulatory pathway, as well as cellular metabolic disorders and impaired synthesis (Fig. 2D). In the brain, the key pathways identified included the toll-like receptor signalling pathway, DNA replication and the cytosolic DNA-sensing pathway (Fig. 6F). These pathways are essential for recognising stress signals and initiating immune responses, repairing damaged DNA to protect cells from mutation and apoptosis, and activating the cGAS-STING pathway to regulate immune responses to heat stress [44–46]. In the liver, most DEGs and DEPs were enriched in steroid biosynthesis, retinol metabolism and metabolic pathways (Fig. 6H). Previous studies have highlighted the importance of the ECM-receptor interaction pathway in the response of the largemouth bass liver to heat stress [21]. Collectively, these mechanisms contribute to survival under heat stress and help maintain homeostasis.
Although heat stress-induced oxidative damage inflicted substantial injury on the gill, brain, and hepatic tissues of largemouth bass, these pathological manifestations were significantly mitigated during the recovery phase upon cessation of thermal stress, which was primarily attributed to a pronounced upregulation of CAT levels at this stage. Recent studies have shown that CAT not only directly eliminates ROS but also may participate in signalling pathways related to heat stress by regulating the local concentration of H₂O₂ within cells [47, 48]. In the recovery phase, pathways such as focal adhesion, ErbB signalling, and fatty acid metabolism were prominently involved in the gill (Fig. 6E). The ErbB signalling pathway regulates the expression of heat shock proteins (HSPs) and mediates the heat stress response by activating downstream signalling cascades, such as MAPK/ERK and PI3K/Akt, enabling cells to adapt to temperature fluctuations [49]. DNA replication, cell cycle, and PPAR signalling pathways were prominently enriched in the brain tissues (Fig. 6G). The ECM-receptor interaction, retinol metabolism, and drug metabolism-cytochrome P450 pathways were enriched in the liver (Fig. 6I). Retinoid metabolism mitigates oxidative stress by activating the RAR/RXR-mediated transcription of antioxidant enzymes (e.g., SOD, CAT, and GPX) to scavenge ROS, while concurrently stabilising mitochondrial membranes to suppress heat-induced MPTP opening, thereby reducing electron leakage and endogenous ROS generation [50, 51]. CYP450 enzymes confer antioxidant protection by directly scavenging free radicals and lipid peroxides, while indirectly regenerating vitamin E and upregulating endogenous defence (e.g., GSH, HO-1, and SOD) via Nrf2 activation, collectively mitigating heat-induced oxidative insult to cellular structures [52]. Collectively, these recovery mechanisms coordinate to enable efficient damage repair, functional restoration, and homeostasis maintenance in cells and tissues after heat stress.
Heat shock proteins (HSPs), including sHSPs, HSP30, HSP40, HSP60, HSP70, and HSP90, are induced by abiotic stresses such as high temperature, high salt, and hypoxia and function in repairing/degrading damaged proteins, maintaining proper folding, and sustaining life under stress [53, 54]. In largemouth bass, PPI network analysis revealed that HSPA9 directly interacts with other DEPs, suggesting that it may play an important role and have a regulatory function in biological systems under heat stress. Hspa9 gene/Mortalin (a member of the HSP70 family) is a mitochondrial chaperone protein that is primarily localised to mitochondria and plays important roles in cell proliferation, stress responses, and maintenance of mitochondrial homeostasis [55, 56]. As the primary cellular source of ROS, mitochondria regulate their ROS production via alternative NAD(P)H dehydrogenases (type II NAD(P)H DHs) [57]. Interestingly, hspa9 regulates mitochondrial morphology by phosphorylating DRP1, and downregulation of hspa9 expression induces mitochondrial fragmentation and the generation of mitochondrial reactive oxygen species (mtROS) [55, 58]. In the recovery group, the damage to organelles (such as mitochondria) in the gill, brain and liver tissues was alleviated, which might be related to the protective effect by the strong upregulation of Hspa9 expression during heat stress, as observed in our study (Fig. 9A–C).
While this study comprehensively analysed heat stress adaptation mechanisms in largemouth bass, the number of DEGs far exceeded that of DEPs, a discrepancy linked to the duration of acute stress and temperature in our study. This is primarily because rapid transcriptional responses (via mRNA expression changes) precede slower protein synthesis/accumulation, with our brief stress period limiting the translation of many DEGs to detectable protein expression. Moreover, extreme heat disrupts mitochondria (reducing ATP levels for synthesis), the endoplasmic reticulum (inducing stress and translational repression), and the Golgi apparatus (impairing protein trafficking). These combined organelle dysfunctions hinder protein production and reduce DEP counts [59]. Finally, a key gene or protein (Hspa9/HSPA9) associated with heat stress was identified. However, further experiments, such as gene knockout or overexpression experiments, are needed to validate its function.
Conclusions
Using an integrated multiomics approach, this study clarifies the oxidative stress damage caused by heat stress to key tissues of the largemouth bass and systematically reveals the genetic regulatory networks in the gill, brain, and liver tissues in response to heat stress and heat recovery. Notably, while overlapping features exist among pathways across tissues and time points, distinct characteristics persist, which reflects the complexity, flexibility, and diversity of gene regulatory networks. This complexity facilitates systemic physiological homeostasis and ensures functional integrity under fluctuating temperature conditions. Additionally, this study identifies a key mitochondrial heat shock protein encoding gene (Hspa9), highlighting its critical regulatory role in biological systems under heat stress. Taken together, these findings provide valuable insights into the mechanisms underlying high temperature adaptation and the healthy culture of largemouth bass, while also offering a reference for investigating the regulatory mechanisms of heat tolerance in other fish species.
Methods
Fish and experimental design
To investigate the temperature variations in largemouth bass aquaculture ponds during the summer, we used temperature loggers (Elitech, RC-4) for real-time monitoring at Rongsheng Fishery Co., Ltd., Changxing County, Huzhou, Zhejiang Province, on July 19, 2023 and 2024 [22]. The water depth at 1 m below the pond surface was measured. To accurately assess the high temperature tolerance of largemouth bass in summer pond culture, an experiment was conducted in July 2023. After four months of feeding, a total of 100 experimental fish (weight 25.09 to 196.72 g) were collected from Huzhou Rongsheng Fishery Technology Co., Ltd., Zhejiang, China. After two weeks of acclimation in a lab culture vat, the heat stress experiment commenced on July 19, 2023. Sixty fish were placed in experimental tanks at an initial water temperature of 25 °C (0 h). The temperature was then gradually increased to 1 °C/h, until it reached 37 °C and was maintained at that temperature for 12 h (24 h) (in the pre-test, the maximum tolerance temperature was 37.8 °C), after which the temperature decreased to 1 °C/h, and was maintained at 25 °C for 12 h (48 h) (Fig. 1D) [60]. Twenty-seven live fish were euthanized with MS-222 (Yu fu Bao, Zhengzhou, China) and sampled at 0 h, 24 h, and 48 h (9 randomly selected per time point). Blood was collected from 3 fish per time point for antioxidant parameter analysis, followed by rapid isolation of gill, brain and liver tissues for transcriptome analysis, proteome sequencing and qPCR. In another 3 fish, gill, brain and liver tissues were dissected for HE staining and TEM observation, and the remaining 3 fish provided identical tissues for IHC and WB analyses.
Biochemical blood index analysis
The levels of alkaline phosphatase (AKP), glutamic-pyruvic transaminase (GPT), glutamic oxalacetic transaminase (GOT), superoxide dismutase (SOD), catalase (CAT), triglyceride (TG), glucose (GLU), and total protein (TP) in the serum were measured using commercial assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), following the manufacturer’s instructions.
Histological staining
In brief, gill, brain, and liver samples were fixed in 4% paraformaldehyde at room temperature for 24 h, subsequently dehydrated using 70–100% graded alcohol solutions and embedded in paraffin. The tissue sections were then cut to a thickness of 5–6 μm, stained with haematoxylin and eosin, and sealed with neutral resin. The results were observed under a microscope (Leica DM4B, Germany), and the images were processed using ImageJ software.
Transmission electron microscopy (TEM)
Tissue samples smaller than 1 mm^3^ were taken from the same locations in the gill, brain, and liver and immersed in 2.5% glutaraldehyde at 4 °C overnight fixation. The samples were then fixed in 1% osmium tetroxide at 4 °C for 2 h, dehydrated using graded ethanol concentrations, embedded in epoxy resin, sliced with an ultrathin microtome, and stained with 1% uranium acetate and lead citrate. The results were observed and photographed using a transmission electron microscope (HITACHIH-7650, Japan).
Transcriptome and 4D-DIA proteome analysis
The methods for transcriptome sequencing followed those described by Guan et al. [16, 61]. In brief, transcriptome sequencing involved total RNA extraction, library construction, RNA sequencing (depth of 16 ×/sample), de novo assembly, functional annotation, differential expression analysis, and functional enrichment analysis. The method for real-time quantitative PCR (RT-qPCR) verification also followed that of Guan et al. [16, 61], with the specific primers listed in Additional file 1: Table S3. β-actin was used as the internal control.
Protein lysis buffer (8 M urea, 1% SDS) was used to extract total protein from gill, brain and liver tissues, and a BCA protein assay kit (Thermo Scientific) was used to quantify the proteins by SDS-PAGE. Trypsin protease (Promega, Madison, WI), HLB and a Pierce Peptide Quantitation Kit (Thermo Fisher) were used to digest, desalt and quantify the peptides, respectively. Equal amounts of peptide were dissolved in mass spectrometry loading buffer, and DIA detection and analysis were performed after the addition of 10 × iRT peptides in proportion and mixing. EASY-nLC 1200 chromatography (Thermo, USA) and timsTOF Pro2 mass spectrometry (Bruker, Germany) were subsequentlyused to analyse the LC–MS/MS data. The established spectrum library was imported into Spectronaut™ to extract the daughter ion peak from the original DIA data, iRT correction retention time, and 6 peptides for each protein and 3 product ions for each peptide for quantitative analysis. The P value and fold change (FC) between groups were calculated using the T test function in RStudio. Differentially expressed proteins (DEPs) were identified with a screening threshold set at P < 0.05 and 1.2 < FC or < 0.83. DEPs were further used for GO and KEGG enrichment analysis.
Association analysis of the transcriptome and proteome
To analyse the transcriptome and proteome data between samples, the Pearson correlation coefficient was calculated to assess the correlation between DEGs and DEPs. Fisher’s exact test was employed to identify significantly enriched functional categories. Venn diagrams were used to visualise and calculate the numbers of DEGs and DEPs across various comparisons.
Immunohistochemistry (IHC) assays
IHC was performed on paraffin-embedded tissue blocks of gill, brain, and liver from different groups of largemouth bass. Tissue sections were dewaxed using an environment-friendly dewaxing and clearing solution (Cat. No. G1128, Servicebio). For inhibition of endogenous peroxidase activity, sections were immersed in 3% hydrogen peroxide. 3% BSA blocking solution was applied for incubation at room temperature for 30 min. Appropriately diluted HSPA9 antibody (synthesized by GenScript Biotech, Nanjing, China, 1:200) was added dropwise, followed by incubation at 4 °C overnight. Subsequently, biotin-labeled goat anti-rabbit IgG was added and incubated at 37 °C for 40 min. After washing with PBS, the reaction was developed using freshly prepared DAB chromogenic solution. Following counterstaining with hematoxylin, sections were thoroughly rinsed with distilled water, then subjected to dehydration, clearing, mounting, and observational analysis.
Western blot (WB) assay
Total proteins were extracted from gill, brain, and liver tissues of largemouth bass, followed by quantification. The total proteins were separated by SDS-PAGE and then transferred onto PVDF membranes. After blocking with 5% skimmed milk powder for 30 min, the membranes were sequentially incubated with rabbit anti-HSPA9 primary antibody (synthesized by GenScript Biotech, Nanjing, China, HSPA9 and GRP75-1:1000, GAPDH-1:10,000) at 4 °C overnight. Subsequently, the membranes were washed 3 times with TBST, and the corresponding secondary antibody, HRP-labeled goat anti-rabbit IgG (1:3000), was added for incubation at room temperature for 1 h, followed by another round of membrane washing. After visualization, grayscale analysis was performed using AIWBwell™ analysis software, with GRP75 and GAPDH serving as the internal reference.
Statistical analysis
The results are expressed as the mean ± standard deviation (SD). Data were analysed using SPSS 25 (IBM) software, and one-way analysis of variance (ANOVA) and Student’s t tests were performed (significance was accepted at P < 0.05). The figures were produced using Adobe Illustrator 2022 software.
Supplementary Information
Additional file 1: Supplementary Tables S1 to S3.Additional file 2: Supplementary Figures S1 to S2.
The reference list from the paper itself. Each links out to its DOI / PubMed record.
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- 2Guan WZ, Yu YQ, Zhang JP, Jian JL, Niu BL, Lou B, et al. Multiomics profiling reveals the adaptive responses of largemouth bass to high temperature stress. Zenodo; 2025. https://zenodo.org/records/18183125.10.1186/s 13059-026-03964-9PMC 1291118941572355 · doi ↗ · pubmed ↗
- 3Guan WZ, Yu YQ, Zhang JP, Jian JL, Niu BL, Lou B, et al. Multiomics profiling reveals the adaptive response of largemouth bass to high temperature stress. Zenodo; 2025. https://zenodo.org/records/18182713.10.1186/s 13059-026-03964-9PMC 1291118941572355 · doi ↗ · pubmed ↗
- 4Guan WZ, Yu YQ, Zhang JP, Jian JL, Niu BL, Lou B, et al. Multi-omics profiling reveals the adaptability response of high-temperature of largemouth bass (Micropterus salmoides). NCBI; 2024. https://www.ncbi.nlm.nih.gov/sra/PRJNA 1126404.
- 5Guan WZ, Yu YQ, Zhang JP, Jian JL, Niu BL, Lou B, et al. Multi-omics profiling reveals the adaptability response of high-temperature of largemouth bass (Micropterus salmoides). I Prox; 2024. https://www.iprox.cn/page/project.html?id=IPX 0009501000.