Alterations and resilience of intestinal microbiota to increased water temperature are accompanied by the recovery of immune function in Nile tilapia

Abstract

In the context of ongoing global warming, fish, as aquatic ectotherms, are highly vulnerable to increased water temperature caused by climate change and extreme heatwaves because of their inability to maintain their body temperature. After prolonged coevolution, the intestinal microbiota has become an integral part of fish and plays a pivotal role in immunity and metabolism. To date, however, little is known about the effects of increased water temperature on the intestinal microbiota of fish, particularly the intestinal mucosa-associated microbiota. Here, we investigated the variation patterns of the intestinal microbiota and immune status in Nile tilapia (Oreochromis niloticus; 125.02 ± 4.55 g) under increased water temperature. The results showed that the microbial diversity, structure, dominant microbes, and predicted function of fish intestinal microbiota were resilient to low-level warming (increasing by 2 °C) but not to high-level warming (increasing by 8 °C) and that fish immune parameters (serum lysozyme content and bactericidal activity) recovered simultaneously. Notably, along with compromised immune function, short-term warming (7 days) drove a significant increase in the microbial richness and diversity of fish intestinal mucosae, in which the overgrowth of opportunistic pathogens such as Romboutsia ilealis, Escherichia–Shigella, Fusobacterium, Streptococcus, Acinetobacter, and Enterobacter inhibited the colonization of potential probiotics such as Cetobacterium, ultimately resulting in a significant reduction in metabolic pathways and a significant increase in the potentially pathogenic phenotype. After long-term warming (37 days), the above alterations disappeared in low-level warming but remained in high-level warming. Critically, long-term warming disrupted the network complexity and stability of the intestinal mucosa- and digesta-associated microbiota to different extents. Collectively, this study revealed that the alterations and resilience of intestinal microbiota to increased water temperature coincided with the recovery of immune function in fish. Our findings extend the understanding of how the intestinal microbiota in aquatic ectotherms respond to increased water temperature, providing important implications for harnessing the potential benefits of host-associated microorganisms to enhance their resilience to climate change.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-87980-y.

Introduction

In recent decades, continuous climate change, together with extreme heatwaves, has posed serious threats to human health, global food security, and biological diversity^1–3. Climate scientists have warned that extreme heatwaves will become more frequent and hotter in the future as global warming continues³. Because the anticipation of climate change can improve management capacity, it is essential to increase our understanding of how the main food sources, including plants and animals, respond to climate change². Numerous animals and plants harbor commensal microbes inside or outside their bodies that play a vital role in maintaining host health⁴. Environmental temperature is one of the most important factors affecting the host-associated microbiome⁵. Given the interdependence between hosts and their associated microbes, the effects of climate change on plants and animals cannot be considered effects on a single entity but rather on host-microbe symbionts⁴. However, how climate change affects the host-associated microbiota and its interconnections with host physiological states remains unclear.

Currently, a growing amount of evidence reveals that environmental temperature shapes the intestinal microbiota in many animal taxa, including amphibians⁶, reptiles⁵, arthropods⁷, mammals⁸, and fish⁹. Increased temperature can directly affect the host-colonized microbial reservoir by changing environmental microbial communities and may also indirectly affect the host-associated microbiota through its effects on host physiology¹⁰. Changes in the host-associated microbiota caused by climate change may have serious negative impacts on host health. Worryingly, even if environmental conditions return to normal, the loss of host-associated microbial symbionts may not be recoverable. These negative impacts may eventually affect host fitness and phenotype, as it was previously reported that microbial diversity loss is strongly associated with reduced animal survival under elevated temperature conditions⁵. A major concern is that changes in microbial diversity and function impair the resilience of host organisms, thereby affecting their ability to cope with climate change¹¹.

Complex microbial communities can maintain their states and recover from perturbations, and this resilience gives microbial ecosystems functional resilience to resist environmental stresses and maintain the ecological services they support¹². Microbes with shorter generation times can adapt on a shorter timescale than the host and may provide fitness advantages that allow the host to cope with climate change¹³. Thus, host-associated microbial communities may enable organisms to react and cope with adversities such as extremely high temperatures on a short timescale, especially for species with long generations, such as plants and animals¹⁴. The finding that the host-associated microbiome can mitigate the environmental stress faced by the host raises new hope for sustainable agriculture and wildlife conservation in the context of climate change¹⁵. Recently, the use of host-associated microbes to improve agricultural sustainability and mitigate the impacts of climate change on global food security has received increasing attention¹¹. However, before active microbiome management can be performed, the potential role of the host-associated microbiome in host adaptation to climate change stress urgently needs to be revealed and clarified.

Compared with endotherms, ectotherms tend to have narrower thermal tolerance ranges and are therefore more severely affected by temperature fluctuations¹⁶. The basic physiological functions of fish, which are typical examples of aquatic ectotherms, are vulnerable to increased water temperature caused by climate change or heatwave events¹⁷. Prior studies have shown that warming affects multiple aspects of fish, including body size¹⁸, community structure¹⁹, and physiological function²⁰. Although many fish can withstand temperature fluctuations in daily or seasonal cycles, elevated water temperatures induced by heatwaves certainly challenge the thermal tolerance limits of some fish, especially those living near the upper threshold of their thermal limits²⁰. Fisheries are important parts of ensuring world food security, and their response to climate change is receiving increasing attention²¹. Given the significant effects of heat extremes on surface water warming²², one of the most important issues is how to make farmed fish more resilient to high temperatures and even heatwave shocks. Nevertheless, a crucial but currently unresolved question in climate change research is how the complex gut microecosystems of aquatic animals respond to increased water temperature. Therefore, it is necessary to better understand the complex interactions between fish physiology and the intestinal microbiota under increased water temperature and their role in alleviating warming pressures.

Tilapia are eurythermal fish with a strong tolerance to water temperature²³, which provides an ideal research object for elucidating the effects of increased water temperature on the intestinal microbiota of ectothermic vertebrates. Although some advances have been made in studying the effects of temperature on fish intestinal microbiota^24,25, to our knowledge, the response patterns of fish intestinal microbiota, particularly the intestinal mucosa-associated microbiota, to increased water temperature caused by climate change or heatwave events and its correlation with fish immune status remain unknown. To address these issues, we explored the variation patterns of tilapia intestinal mucosa- and digesta-associated microbiota under simulated warming in a laboratory temperature-controlled system. Given the vital role of fish intestinal microbiota in regulating immune function²⁵, the simultaneous alterations in fish immune function and the intestinal microbiota under increased water temperature were also examined. Based on the resilience of the host intestinal microbiota to environmental stresses^26–28, the scientific hypothesis is that the fish intestinal microbiota is resilient to increased water temperature; that is, the fish intestinal microbiota changes after short-term warming and recovers to the initial state after long-term warming, which coincides with the recovery of immune function. To verify this, a strict warming experiment was designed as follows: the initial temperature was 18 °C, and then the water temperature was increased to 20 °C and 26 °C. Ultimately, the variation patterns of intestinal microbiota and immune parameters in Nile tilapia were detected after short-term (7 days) and long-term (37 days) warming.

Materials and methods

Ethical approval of experimental animals

This project was approved by the Animal Ethical and Welfare Committee of the Institute of Hydrobiology, Chinese Academy of Sciences, China (approval number: IHBCAS-Y53Z151301). All experiments were performed in accordance with relevant guidelines and regulations in the ARRIVE guidelines²⁹. All the authors consented to participate in the experimental project.

Experimental design and protocol

Three hundred adult tilapia (NEW GIFT strain of Oreochromis niloticus) of nearly the same size were transported to the laboratory and placed in a large indoor fish tank with a volume of approximately 3000 L. All tilapia were selected from the same hatching batch and sampled from Mingde Fish Fry Incubation Co., Ltd., located in Yingshan County, Huanggang City, Hubei Province, China. The fish were acclimated for 15 days at a water temperature of approximately 18 °C. Following acclimation, ninety healthy fish were transferred to nine small tanks (volume of 200 L per tank), with ten fish in each tank. The experimental tanks were cylindrical containers made of white plastic with removable mesh lids on the top to prevent fish from escaping. The water inlet and outlet holes were located on the upper side and the drain valves were located at the bottom of each tank, which could conduct water changes in time and discharge feces and feed residue. Each tank was equipped with an automatic aerator to continuously maintain the dissolved oxygen content in the water, and a temperature-control device to precisely regulate the water temperature.

The experiment was a completely randomized design, and the nine tanks were divided into three groups (three tanks per group): the control group (18 °C), the low-level warming group (20 °C), and the high-level warming group (26 °C). Before warming, the water temperatures of all tanks were stabilized at 18 °C, and the fish were acclimated for another 7 days so that their intestinal microbiota remained stable under the control temperature. During the warming process, the water temperature of the two warming groups gradually increased by 2 °C every day until the water temperature reached 20 °C and 26 °C, respectively, and then remained stable. The experimental water was preheated and aerated tap water. The prepared water was heated with temperature-controlled heating devices until it was consistent with the water temperature of the corresponding tank and was continually aerated to maintain adequate dissolved oxygen. Following the temperature of each tank, one-third of the water was replaced daily to maintain the water quality within the appropriate range³⁰. During the acclimation and experimental periods, the fish were fed at a daily rate of 3% body weight, with the commercial diet provided by Huai’an TianShen Feed Co., Ltd, China (crude protein ≥ 30.0%; crude fat ≥ 4.5%; crude fiber ≥ 9.0%; crude ash ≥ 12.0%; total phosphorus ≥ 0.8%; sodium chloride, 0.4–4.0%; lysine ≥ 1.7%; moisture ≤ 12.5%). After 1.5 h of feeding, the drain valves were opened to discharge the feed residue and fish feces, and then an appropriate amount of preheated water was replenished into the fish tanks. During the experiment, the water level of the fish tanks was adjusted according to the number and weight of the fish to keep the stocking density stable. The water temperature of the fish tanks was measured and recorded every day, and mortality was observed and recorded every day. The experiment began on March 7, 2018, and ended on April 22, 2018, lasting a total of 45 days. During the experiment, common water quality indicators were tested weekly as follows: the concentration of dissolved oxygen was 6.52 ± 0.14 mg L⁻¹, the pH was 7.19 ± 0.08, and the ammonia nitrogen content was 0.13 ± 0.01 mg L⁻¹.

Sample collection and processing

After acclimation for another 7 days, six fish were randomly collected from three control tanks as the control group (Z). The initial body weight of tilapia was 125.02 ± 4.55 g. After warming for 7 days, two fish were collected from each warming tank as the short-term warming group (F). At the end of the experiment, another two fish were collected from each warming tank as the long-term warming group (T). The collected fish samples were labeled in time for subsequent sample processing. After that, all tilapia were weighed after being lightly anesthetized with a low dose (20 mg L⁻¹) of MS-222 (Sigma, Germany). Blood samples were then immediately collected from the caudal vein of the fish with 5 mL syringes. After coagulation at 4 °C for 4 h, the collected blood samples were centrifuged at 2000 × g for 5 min to collect the serum, which was stored at − 20 °C. After blood collection, the fish were euthanized with a high-dose (100 mg L⁻¹) MS-222 (Sigma, Germany) solution. Before dissection, the fish surface was disinfected with 75% ethanol. The abdominal cavity was opened with sterile scissors, and the posterior intestine (hindgut) was removed. The hindgut was dissected longitudinally to expose the lumen, and the intestinal contents were collected by sterile forceps in sterile centrifuge tubes. The intestinal lumen was cleaned with sterile 0.9% saline, and the visible intestinal contents were gently removed. The posterior intestine was subsequently immersed three times in sterile 0.9% saline to completely remove the adherent contents. Finally, the hindgut segments were cut into pieces and homogenized on a vortex oscillator. The collected intestinal contents (C) and mucosae (M) were immediately frozen in liquid nitrogen and then stored at − 80 °C.

Four samples were randomly selected from each group for intestinal microbiota detection, and the specific groups were as follows: in the control group, the intestinal contents were labeled ZC1–ZC4 and the intestinal mucosae were labeled ZM1–ZM4; in the short-term low-level warming group, the intestinal contents were labeled FLC1–FLC4 and the intestinal mucosae were labeled FLM1–FLM4; in the short-term high-level warming group, the intestinal contents were labeled FMC1–FMC4 and the intestinal mucosae were labeled FMM1–FMM4; in the long-term low-level warming group, the intestinal contents were labeled TLC1–TLC4 and the intestinal mucosae were labeled TLM1–TLM4; in the long-term high-level warming group, the intestinal contents were labeled TMC1–TMC4 and the intestinal mucosae were labeled TMM1–TMM4.

Detection of the serum lysozyme content and bactericidal activity

Following our previous study³⁰, the serum lysozyme activity (SLA) was determined via the turbidimetric method of self-contrast with a commercial test kit (Nanjing Jiancheng Bioengineering Institute, China). In addition, a previous method³¹ was modified for the detection of serum bactericidal activity (SBA) in this study. Aeromonas hydrophila, Streptococcus agalactiae and Edwardsiella tarda are three common pathogens of tilapia. Thus, three strains of these bacterial species previously isolated from diseased tilapia were used to detect serum bactericidal activity in this study. First, three bacterial strains at the logarithmic growth stage were centrifuged at 5000 r/min for 10 min to collect bacterial deposits, respectively. The above deposits were suspended in sterile phosphate-buffered saline (PBS), and the concentration of the bacterial mixture was adjusted so that the absorbance value at 570 nm was 0.3–0.5. Then, 30 µL of each serum sample was added to 3 mL of bacterial mixture, which was incubated in a 37 °C water bath for 30 min. The mixture was quickly immersed in an ice bath, and the absorbance value (A) at 570 nm was measured. After that, 30 µL of PBS was used as a blank control, and the absorbance value (A0) at 570 nm was measured after the same operation. The serum bactericidal activity was calculated according to the following formula: U = √((A0-A)/A). A0 is the absorbance value of the blank control at 570 nm, and A is the absorbance value of the serum sample at 570 nm.

16 S rRNA gene amplicon sequencing

Microbial DNA was extracted from all intestinal samples with the QIAamp® DNA Stool Mini Kit (Qiagen, Germany), and the DNA concentration and quality were tested. In this study, the V3-V4 hypervariable regions of the bacterial 16S rRNA gene were amplified with the universal primers 338F (5’-ACTCCTACGGGAGGCAGCAG-3’) and 806R (5’-GGACTACHVGGGTWTCTAAT-3’). The PCR amplification conditions were as follows: denaturation at 95 ◦C for 3 min; 27 cycles at 95 ◦C for 30 s, annealing at 55 ◦C for 30 s, and elongation at 72 ◦C for 45 s; final extension at 72 ◦C for 10 min; and incubation at 10 ◦C until halted. PCR was performed in triplicate 20 µL mixture containing 4 µL of 5 × FastPfu Buffer, 2 µL of 2.5 mM dNTPs, 0.8 µL of each primer (5 µM), 0.4 µL of FastPfu Polymerase, 0.2 µL of BSA,10 ng of template DNA, and sterile water added up to 20 µL. The PCR reactions were conducted in triplicate, and then the PCR products were further purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA) and quantified using the QuantiFluorTM-ST (Promega, USA) by the manufacturer’s protocol. Finally, the purified amplicons were pooled in equimolar and paired-end sequenced simultaneously on the Illumina MiSeq PE300 platform (Illumina, San Diego, USA) at the Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China).

Bioinformatics and statistical analyses

Raw fastq files were quality-controlled by Trimmomatic and FLASH based on our previous description³². Operational taxonomic units (OTUs) were clustered with a 97% similarity cutoff, and then chimeric sequences were removed. The taxonomy of each representative sequence was analyzed via the RDP Classifier (http://rdp.cme.msu.edu/) against the Bacterial Silva 16 S rRNA database (SILVA SSU 138). To eliminate differences in sequencing depth, all samples were randomly resampled to the minimum number of valid sequences (32761 reads). The evaluation indices of alpha diversity included observed species richness (OTUs), the Chao richness index, the Shannon index, the Shannon evenness index, the Simpson index, and Good’s coverage. For example, a higher Shannon index value indicates greater community diversity, whereas a higher Simpson index value indicates lower community diversity. Beta diversity was analyzed via principal coordinate analysis (PCoA) and analysis of similarity (ANOSIM) based on the Bray‒Curtis metric. Based on the relative abundance of microbial taxa, the enterotype was studied by calculating the Jensen‒Shannon distance and performing partitioning around medoids. The optimal cluster K value was calculated via the Calinski‒Harabasz (CH) index and then visualized via principal coordinate analysis (PCoA, K ≥ 2). The microbiome data were analyzed on the free online platform of the Majorbio Cloud Platform (http://www.majorbio.com).

Before calculating the correlation coefficients, we selected the top 500 most abundant OTUs. We then used SparCC to calculate Spearman correlation coefficients for these OTUs, selecting results with an absolute value of the correlation coefficient > 0.5 and p< 0.05³³. The networks were visualized in Gephi (v0.10.1) via the Fruchterman-Reingold layout³⁴. The network topological parameters were calculated via NetworkX (v2.6.3) in Python. In this study, the functional profiles and pathways of the intestinal microbiota were predicted using the PICRUSt2³⁵from the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways³⁶. The accuracy of the functional predictions was assessed through the computation of the nearest sequenced taxon index (NSTI)³⁷. BugBase (https://bugbase.cs.umn.edu/index.html) was used to identify high-level phenotypes present in microbial samples³⁸. Welch’s t-test (SPSS, version 19.0) was used to identify differences between two independent groups, and the Kruskal–Wallis H test was used to identify differences among three independent groups. The p-value was corrected via the false discovery rate, and the significance level was set at 0.05.

Results

Effects of increased water temperature on the growth performance and survival rate of tilapia

During the experiment, the actual temperatures in the fish tanks fluctuated within 1 °C above or below the designed temperature values (Table S1), indicating that the temperature control was relatively stable throughout the experiment. After short-term warming, the body weight of tilapia in the high-level warming group was significantly greater than that in the low-level warming group (Welch’s t-test, p < 0.05). After long-term warming, no significant difference (Welch’s t-test, p > 0.05) in tilapia body weight was detected between the low-level and high-level warming groups (Table 1). In addition, after short-term warming, the cumulative survival rate of tilapia in the low-level warming group was greater than that in the high-level warming group. However, after long-term warming, the cumulative survival rate of tilapia in the high-level warming group was much greater than that in the low-level warming group (Table 1). These results indicated that warming amplitude affected the growth performance and survival rate of tilapia.

Table 1.

Growth performance and survival rate of tilapia in different groups. The same letter after the values represents no significant difference (p > 0.05), and the different letters represent significant differences (p < 0.05).

Group	Average weight (g)	Cumulative survival rate (%)
Control group (Z)	125.02 ± 4.55 a	NA
Short-term low-level warming group (FL)	177.73 ± 30.72 b	96.67
Short-term high-level warming group(FM)	215.42 ± 12.88 c	93.33
Long-term low-level warming group (TL)	361.98 ± 42.99 d	83.33
Long-term high-level warming group (TM)	369.27 ± 40.97 d	93.33

Effects of increased water temperature on the serum immune parameters of tilapia

In this study, serum bactericidal activity and lysozyme activity were measured to evaluate the nonspecific immune function of fish. After short-term warming, the serum bactericidal activity against three common pathogens (Aeromonas hydrophila, Streptococcus agalactiae, and Edwardsiella tarda) tended to decrease, and the serum bactericidal activity against Streptococcus agalactiae decreased significantly (Welch’s t-test, p < 0.05) (Fig. 1A, B, and C). After long-term warming, the serum bactericidal activity against these common pathogens tended to increase, and most values were significantly greater (Welch’s t-test, p < 0.05) than those in the short-term warming group or even in the control group (Fig. 1A, B, and C). In addition, the serum lysozyme activity tended to decrease after short-term warming but then tended to increase after long-term warming, but these changes were not significant (Welch’s t-test, p > 0.05) (Fig. 1D). Overall, the two immune parameters of tilapia decreased after short-term warming, increased after long-term warming, and finally recovered to normal levels. These findings indicated that the immune function of tilapia was compromised after short-term warming and then recovered with gradual adaptation to the long-term warming environment.

Fig. 1.

The variation trend of immune parameters in tilapia after warming: (A) The variation trend of serum bactericidal activity (SBA) against Aeromonas hydrophila; (B) The variation trend of serum bactericidal activity against Streptococcus agalactiae; (C) The variation trend of serum bactericidal activity against Edwardsiella tarda; (D) The variation trend of serum lysozyme activity (SLA). The same letter after the values represents no significant difference (p > 0.05), and the different letters represent significant differences (p < 0.05). Z: the control group, FL: the short-term low-level warming group, TL: the long-term low-level warming group, FM: the short-term high-level warming group, TM: the long-term high-level warming group.

Effects of increased water temperature on the diversity and structure of the intestinal microbiota

After quality filtering and control, a total of 1,684,155 valid reads (32761–54232 reads per sample) were obtained from all the samples. After random resampling, the retained reads were clustered into 2364 OTUs, and the Good’s coverage of all the samples ranged from 98.67 to 99.98%. The corresponding rarefaction curves tended to plateau, and the Shannon curves remained stable (Fig. S1A and B), indicating that most microbes present in the intestinal samples were detected. In the low-level warming group, statistical analyses revealed that the microbial richness, diversity, and evenness of the intestinal contents and mucosae increased significantly after short-term warming, decreased significantly after long-term warming (Welch’s t-test; p < 0.05), and then recovered to the initial level (Fig. 2A, B, C, and D). In the high-level warming group, the microbial richness, diversity, and evenness of the intestinal contents decreased significantly (Welch’s t-test; p < 0.05) after short-term warming and then remained at this low level after long-term warming, whereas these indices of the intestinal mucosae increased significantly (Welch’s t-test; p < 0.05) after short-term warming and remained at this high level after long-term warming (Fig. 2A, B, C, and D).

Fig. 2.

Comparison of richness and diversity indices of intestinal microbiota in different groups: (A) Comparison of observed species richness index of intestinal microbiota in different groups; (B) Comparison of Shannon index of intestinal microbiota in different groups; (C) Comparison of Simpson index of intestinal microbiota in different groups; (D) Comparison of Shannoneven index of intestinal microbiota in different groups. The significance of Welch’s t-test: * represents p < 0.05. ZC: intestinal contents in the control group, FLC: intestinal contents in the short-term low-level warming group, TLC: intestinal contents in the long-term low-level warming group, FMC: intestinal contents in the short-term high-level warming group, TMC: intestinal contents in the long-term high-level warming group; ZM: intestinal mucosae in the control group, FLM: intestinal mucosae in the short-term low-level warming group, TLM: intestinal mucosae in the long-term low-level warming group, FMM: intestinal mucosae in the short-term high-level warming group, TMM: intestinal mucosae in the long-term high-level warming group.

In the low-level warming group, ANOSIM revealed that the microbial structures of the intestinal contents and mucosae changed significantly (R = 0.9792, p = 0.034; R = 0.9792, p = 0.034) after short-term warming and then altered significantly again (R = 1, p = 0.034; R = 1, p = 0.034) after long-term warming until there were no significant differences (R = − 0.0521, p = 0.496; R = 0.1979, p = 0.150) from those in the control group (Table S2). In the high-level warming group, the microbial structures of the intestinal contents and mucosae changed significantly (R = 0.7083, p = 0.034; R = 1, p = 0.034) after short-term warming and then showed no significant changes (R = 0.4167, p = 0.072; R = 0.5938, p = 0.080) after long-term warming until they remained significantly different (R = 0.6563, p = 0.034; R = 1, p = 0.034) from those in the control group (Table S2). The PCoA plots visualized the ANOSIM results, showing that the integral structure of the intestinal microbiota changed significantly after short-term warming (Fig. 3A and B). After long-term warming, the structure of the intestinal microbiota in the low-level warming group recovered to the initial state, but no such structural recovery occurred in the high-level warming group (Fig. 3A and B). Overall, the two principal coordinates explained 95.32% and 88.49% of the variation in the microbial structures of the intestinal contents and mucosae, respectively (Fig. 3A and B).

Fig. 3.

Comparison of the overall structure of intestinal microbiota in different groups: (A) Principal coordinate analysis of intestinal digesta-associated microbiota; (B) Principal coordinate analysis of intestinal mucosa-associated microbiota; (C) Enterotype analysis of intestinal digesta-associated microbiota; (D) Enterotype analysis of intestinal mucosa-associated microbiota. ZC: intestinal contents in the control group, FLC: intestinal contents in the short-term low-level warming group, TLC: intestinal contents in the long-term low-level warming group, FMC: intestinal contents in the short-term high-level warming group, TMC: intestinal contents in the long-term high-level warming group; ZM: intestinal mucosae in the control group, FLM: intestinal mucosae in the short-term low-level warming group, TLM: intestinal mucosae in the long-term low-level warming group, FMM: intestinal mucosae in the short-term high-level warming group, TMM: intestinal mucosae in the long-term high-level warming group.

In addition, after short-term low-level warming, the intestinal digesta-associated microbiota clustered together into the same enterotype (Type 1), whereas the intestinal mucosa-associated microbiota clustered together with the intestinal mucosa-associated microbiota after high-level warming into the same enterotype (Type 4) (Fig. 3C and D). After long-term low-level warming, the intestinal digesta-associated and mucosa-associated microbiota clustered together with those in the control group into the same enterotypes (Type 3 and Type 5, respectively) (Fig. 3C and D). After high-level warming, the intestinal digesta-associated microbiota clustered together into the same enterotype (Type 2), whereas the intestinal mucosa-associated microbiota clustered together into the same enterotype (Type 4) (Fig. 3C and D). Collectively, these results indicated that the richness, diversity, structure, and enterotype of the intestinal microbiota were resilient to low-level warming but not to high-level warming.

Effect of increased water temperature on the taxonomic composition of the intestinal microbiota

At the phylum level, the relative abundances of intestinal dominant microbes fluctuated significantly with increased water temperature (Fig. 4A and B). In the low-level warming group, the relative abundance of Fusobacteriota in the intestinal contents decreased significantly after short-term warming and then increased significantly after long-term warming, whereas the relative abundances of other dominant phyla in the intestinal contents, including Firmicutes, Proteobacteria, Bacteroidota, Cyanobacteria, and Spirochaetota, increased significantly after short-term warming and then decreased significantly after long-term warming (Fig. 4A; Fig. S2A). A comparison of the intestinal mucosae after low-level warming revealed that the relative abundance of Fusobacteriota decreased significantly after short-term warming and then increased significantly after long-term warming, whereas the relative abundances of Firmicutes, Bacteroidota, Cyanobacteria, and Campilobacterota increased significantly after short-term warming and then decreased significantly after long-term warming. In addition, the relative abundance of Proteobacteria remained stable after short-term warming and then decreased significantly after long-term warming, whereas the relative abundance of Desulfobacterota decreased significantly throughout the warming period (Fig. 4B; Fig. S2B). In the high-level warming group, the relative abundance of Fusobacteria in the intestinal contents increased significantly after short-term warming and then decreased slightly after long-term warming but increased significantly overall. The relative abundance of Proteobacteria in the intestinal contents decreased significantly after short-term warming and then increased significantly after long-term warming, whereas the relative abundances of other dominant phyla, including Firmicutes, Bacteroidota, Cyanobacteria, and Spirochaetota, decreased significantly throughout the warming period (Fig. 4A; Fig. S2C). Concerning the intestinal mucosae after high-level warming, the relative abundances of most dominant phyla, such as Firmicutes, Bacteroidota, Cyanobacteria, Campilobacterota, and Actinobacteriota, increased significantly throughout the warming period, whereas the relative abundances of Fusobacteriota and Desulfobacterota decreased significantly throughout the warming period (Fig. 4B; Fig. S2D).

Fig. 4.

Comparison of taxonomic composition of intestinal microbiota: (A) Comparison of dominant phyla of intestinal digesta-associated microbiota after warming; (B) Comparison of dominant phyla of intestinal mucosa-associated microbiota after warming; (C) Comparison of dominant genera of intestinal digesta-associated microbiota after low-level warming; (D) Comparison of dominant genera of intestinal mucosa-associated microbiota after low-level warming; (E) Comparison of dominant genera of intestinal digesta-associated microbiota after high-level warming; (F) Comparison of dominant genera of intestinal mucosa-associated microbiota after high-level warming. The significance of Welch’s t-test: * represents p < 0.05. ZC: intestinal contents in the control group, FLC: intestinal contents in the short-term low-level warming group, TLC: intestinal contents in the long-term low-level warming group, FMC: intestinal contents in the short-term high-level warming group, TMC: intestinal contents in the long-term high-level warming group; ZM: intestinal mucosae in the control group, FLM: intestinal mucosae in the short-term low-level warming group, TLM: intestinal mucosae in the long-term low-level warming group, FMM: intestinal mucosae in the short-term high-level warming group, TMM: intestinal mucosae in the long-term high-level warming group.

At the genus (or species) level, in the low-level warming group, the relative abundance of Cetobacterium in the intestinal contents decreased significantly after short-term warming and then increased significantly after long-term warming, whereas the relative abundances of other dominant genera in the intestinal contents, including Romboutsia ilealis, Escherichia–Shigella, norank Chloroplast, Terrisporobacter, Clostridium sensu stricto 1, Bacteroides, and Treponema, increased significantly after short-term warming and then decreased significantly after long-term warming (Fig. 4C). A comparison of the intestinal mucosae after low-level warming revealed that the relative abundances of Cetobacterium and Plesiomonas shigelloides decreased significantly after short-term warming and then increased significantly after long-term warming, whereas the relative abundances of other dominant phyla, including Romboutsia ilealis, Escherichia–Shigella, Fusobacterium, and Clostridium sensu stricto 1, increased significantly after short-term warming and then decreased significantly after long-term warming (Fig. 4D). In the high-level warming group, the relative abundance of Cetobacterium in the intestinal contents increased significantly after short-term warming and then decreased slightly after long-term warming but increased significantly overall, whereas the relative abundance of Plesiomonas shigelloides in the intestinal contents decreased significantly after short-term warming but then increased significantly after long-term warming and increased significantly overall. In addition, the relative abundances of Romboutsia ilealis, Escherichia–Shigella, norank Chloroplast, and Terrisporobacter in the intestinal contents decreased significantly throughout the warming period, whereas the relative abundances of norank Barnesiellaceae and unclassified Tannerellaceae increased significantly throughout the warming period (Fig. 4E). In terms of the intestinal mucosae after high-level warming, the relative abundances of Cetobacterium, unclassified Mycoplasmataceae, and Plesiomonas shigelloides decreased significantly throughout the warming period, whereas the relative abundances of other dominant genera, including Romboutsia ilealis, Escherichia–Shigella, norank Chloroplast, Terrisporobacter, and Clostridium sensu stricto 1, increased significantly throughout the warming period (Fig. 4F). Specifically, after high-level warming, the relative abundances of dominant bacterial taxa containing common pathogens, such as Romboutsia ilealis, Escherichia–Shigella, Fusobacterium, Streptococcus, Acinetobacter, and Enterobacter, increased significantly in the intestinal mucosae, whereas the relative abundances of predominant beneficial taxa, such as Cetobacterium, decreased significantly in the intestinal mucosae (Table S3). Taken together, the relative abundances of most intestinal microbes were resilient to low-level warming but not to high-level warming.

Effects of increased water temperature on the co-occurrence networks of the intestinal microbiota

In the co-occurrence networks, after low-level warming, the number of nodes in the intestinal digesta-associated microbiota first increased slightly but then decreased significantly, whereas the number of edges, transitivity, average degree, and mean clustering coefficient decreased significantly throughout the warming period (Fig. 5A; Table S4). In addition, the mean clustering coefficient, average degree, and number of edges in the intestinal mucosa-associated microbiota increased significantly at first but then decreased significantly, whereas the number of nodes decreased slightly, but transitivity increased slightly (Fig. 5B; Table S4). Taken together, after low-level warming, the values of all network topological parameters decreased significantly in the intestinal digesta-associated microbiota, whereas the values of most network topological parameters in the intestinal mucosa-associated microbiota showed limited resilience but still decreased overall. After high-level warming, the values of all network topological parameters decreased drastically in the intestinal digesta- and mucosa-associated microbiota (Fig. 5A and B; Table S4). By comparing different sample types, we found that the network complexity of the intestinal mucosa-associated microbiota was much greater than that of the intestinal digesta-associated microbiota after water warming, indicating that the intestinal mucosa-associated microbiota was more stable to warming stress. However, notably, the network recovery of the intestinal mucosa-associated microbiota in response to low-level warming was incomplete, and long-term warming ultimately disrupted the network complexity and stability of the intestinal mucosa- and digesta-associated microbiota to different extents.

Fig. 5.

The co-occurrence networks of intestinal microbiota after warming: (A) The co-occurrence networks of intestinal digesta-associated microbiota; (B) The co-occurrence networks of intestinal mucosa-associated microbiota. The nodes are colored according to modules. Node size is proportional to the betweenness centrality of each OTU, and edge thickness is proportional to the weight of each correlation. The red and green edges represent positive and negative correlations, respectively. ZC: intestinal contents in the control group, FLC: intestinal contents in the short-term low-level warming group, TLC: intestinal contents in the long-term low-level warming group, FMC: intestinal contents in the short-term high-level warming group, TMC: intestinal contents in the long-term high-level warming group; ZM: intestinal mucosae in the control group, FLM: intestinal mucosae in the short-term low-level warming group, TLM: intestinal mucosae in the long-term low-level warming group, FMM: intestinal mucosae in the short-term high-level warming group, TMM: intestinal mucosae in the long-term high-level warming group.

Effect of increased water temperature on the predictive function of the intestinal microbiota

PICRUSt2 prediction revealed that the mean weighted NSTI had a relatively low value of 0.1873 ± 0.0897. In the low-level warming group, ANOSIM revealed that the microbial functional structures of the intestinal contents and mucosae changed significantly (R = 0.9167, p = 0.016; R = 0.8958, p = 0.028) after short-term warming and then altered significantly again (R = 0.9583, p = 0.022; R = 0.9688, p = 0.033) after long-term warming until there were no significant differences (R = − 0.1042, p = 0.648; R = 0.1563, p = 0.237) from those in the control group (Table S2). In the high-level warming group, the microbial functional structures of the intestinal contents and mucosae changed significantly (R = 0.6563, p = 0.032; R = 0.9896, p = 0.037) after short-term warming and then did not significantly change (R = 0.3542, p = 0.068; R = 0.2813, p = 0.095) after long-term warming until they remained significantly different (R = 0.8125, p = 0.021; R = 0.9688, p = 0.030) from those in the control group (Table S2). The PCoA plots were used to visualize the ANOSIM results, which revealed that the functional structure of the intestinal microbiota changed significantly after short-term warming regardless of low-level or high-level warming (Fig. 6A). After long-term warming, the functional structure of the intestinal microbiota in the low-level warming group recovered to the initial state, but no such recovery of the functional structure occurred in the high-level warming group (Fig. 6A).

Fig. 6.

Comparison of microbial functions of intestinal microbiota predicted by PICRUSt2: (A) Comparison of functional structure of intestinal microbiota; (B) Comparison of functional composition of intestinal microbiota. ZC: intestinal contents in the control group, FLC: intestinal contents in the short-term low-level warming group, TLC: intestinal contents in the long-term low-level warming group, FMC: intestinal contents in the short-term high-level warming group, TMC: intestinal contents in the long-term high-level warming group; ZM: intestinal mucosae in the control group, FLM: intestinal mucosae in the short-term low-level warming group, TLM: intestinal mucosae in the long-term low-level warming group, FMM: intestinal mucosae in the short-term high-level warming group, TMM: intestinal mucosae in the long-term high-level warming group.

At KEGG level 2, a large proportion of microbial functions belonged to metabolism, mainly including global and overview maps, carbohydrate metabolism, amino acid metabolism, metabolism of cofactors and vitamins, energy metabolism, nucleotide metabolism, lipid metabolism, biosynthesis of other secondary metabolites, glycan biosynthesis and metabolism, metabolism of terpenoids and polyketides, and xenobiotics biodegradation and metabolism (Fig. 6B). In the low-level warming group, the functional categories related to these metabolic pathways in the intestinal contents and mucosae decreased significantly after short-term warming and then increased significantly after long-term warming, indicating strong resilience (Fig. 6B). In the high-level warming group, the microbial functions involved in these metabolic pathways increased significantly in the intestinal contents, whereas these microbial functions decreased significantly in the intestinal mucosae (Fig. 6B). In addition, after low-level warming, the relative abundance of the potentially pathogenic phenotype in the intestinal microbiota first increased significantly but then decreased significantly, indicating strong resilience (Fig. S3A). After high-level warming, the relative abundance of the potentially pathogenic phenotype decreased significantly in the intestinal contents but increased significantly in the intestinal mucosae (Fig. S3A). Notably, Romboutsia ilealis, Escherichia–Shigella, unclassified Chloroplast, Enterobacter, Clostridium sensu stricto 1, and Plesiomonas shigelloides contributed the most to the potentially pathogenic phenotype (Fig. S3B).

Discussion

To date, although it has been demonstrated that fish intestinal microbiota is affected by environmental temperature^24,25, whether it is resilient to increased water temperature remains unclear. This study evaluated the alterations and resilience of fish intestinal mucosa- and digesta-associated microbiota to increased water temperature and its correlation with immune function. We found that the intestinal microbiota of fish showed strong resilience to low-level warming but no resilience to high-level warming and that fish immune function recovered simultaneously. Considering the critical role of the intestinal microbiota in maintaining fish health and fitness, the resilience of fish intestinal microbiota, especially the intestinal mucosa-associated microbiota, to increased water temperature makes it a promising regulatory target for alleviating climate change stress, which provides an insightful perspective for devising management strategies for sustainable aquaculture and aquatic animal conservation in the context of global warming.

Response of fish immune function to increased water temperature

Fish are aquatic ectotherms, and their body temperature changes with water temperature; thus, water temperature directly affects the feeding and growth of fish^9,39. Our results revealed that after short-term warming, the body weight of tilapia in the high-level warming group was significantly greater than that in the low-level warming group (Table 1). In the appropriate temperature range, with increasing temperature, fish feed intake and digestive enzyme activity are increased, thereby increasing the metabolic rate and accelerating the growth rate of fish²⁰. However, fish growth rate subsequently declines as individuals struggle to maintain cardiac function and respiration in the face of increased metabolic demands⁴⁰. It explained our findings that after long-term warming, no significant difference in body weight was detected between the low-level and high-level warming groups. Previous studies have shown that increased water temperature affects fish survival and increases their mortality⁴¹. After short-term warming, the cumulative survival rate of tilapia in the low-level warming group was greater than that in the high-level warming group, indicating that the greater the degree of warming was, the lower the short-term survival rate. However, after long-term warming, the cumulative survival rate of tilapia in the high-level warming group was much greater than that in the low-level warming group, which was probably due to the effect of temperature itself rather than water warming⁴². The nonspecific immune system of fish, a lower vertebrate, plays a key role in defending against pathogens⁴³. Compared with the optimal temperature, both low and high temperatures greatly reduce the resistance of fish to pathogens and damage their immune function^44,45. Serum contains important immune molecules involved in the nonspecific immune response of fish, and serum immune parameters such as lysozyme activity and bactericidal activity are widely utilized to measure the level of nonspecific immune function in fish^46,47. We found that serum lysozyme activity and bactericidal activity decreased after short-term warming, increased after long-term warming, and finally recovered to normal levels. This confirmed that drastic changes in the aquaculture environment, such as a sharp rise in water temperature, usually lead to outbreaks of fish diseases, as acute stress leads to a decline in fish immunity and resistance to pathogens⁴⁸.

Response of intestinal microbial diversity and structure to increased water temperature

As a key influencing factor, environmental temperature affects the diversity, structure, and composition of fish intestinal microbiota^39,45. Our results revealed that the microbial richness and diversity of most intestinal samples increased significantly after short-term warming (Fig. 2A, B, C, and D), which was consistent with previous findings that the richness and diversity of fish intestinal microbiota at relatively high temperatures were greater than those at relatively low temperatures⁹. However, a previous study of rainbow trout showed that gut microbial richness and diversity were generally lower in warm than cold water⁴⁹. This inconsistency was probably related to differences in fish species as well as temperature. Given the crucial role of fish immunity in shaping the intestinal mucosal microbiota⁵⁰, the significant increase in microbial richness and diversity of the intestinal mucosae after short-term warming was undoubtedly closely related to compromised immune function detected after short-term warming. The latest findings indicate that an increase in microbial diversity causes an increased incidence of microbial consumption of different nutrients, which can effectively prevent the growth of pathogens and improve the colonization resistance of the intestinal microbiota⁵¹. Therefore, the significant increase in microbial richness and diversity of the intestinal mucosae after short-term warming could help improve the colonization resistance of the intestinal microbiota, which might play a role in alleviating the risk of pathogen infections when fish immunity is impaired⁵¹. Besides, after long-term high-level warming, the microbial richness and diversity of the intestinal mucosae remained high, which contributed to continuously reducing the risk of high-abundance potential pathogens invading the intestine. The above results supported the idea that the intestinal microbiota might provide health benefits for the host to cope with warming stress¹³. In addition, our results revealed significant alterations in the structure of the intestinal microbiota after short-term warming (Fig. 3A and B). The host immune system normally restricts the overgrowth of mucosal microorganisms and prevents pathogens from entering host tissues and organs, thereby maintaining the normal symbiotic relationship between the intestinal microbiota and the host⁵⁰. Thus, compromised immunity after short-term warming puts weak pressure on the intestinal microbiota, ultimately leading to structural changes in the intestinal microbiota. Previous studies revealed that the structure of fish intestinal microbiota changed after short-term exposure to pollutants and returned to its initial state after long-term exposure^28,52. After long-term low-level warming, we also found that the structure of fish intestinal microbiota recovered to the initial state.

Response of intestinal microbial co-occurrence networks to increased water temperature

Currently, it is generally accepted that the interaction between the microbial members of the intestinal microbiota maintains the stability of the intestinal microecosystem⁵³. We found that warming greatly disrupted the complexity and stability of the intestinal digesta-associated microbiota (Fig. 5A and B). Temperature changes strongly affect the growth and survival of environmental microbes, for example, relatively high temperatures generally favor relatively slow-growing species in bacterial communities⁵⁴. The intestinal digesta-associated microbiota was influenced mainly by microorganisms in the water environment, so warming could easily disrupt the interactions among its microbial members. Interestingly, short-term low-level warming improved the network complexity and stability of the intestinal mucosa-associated microbiota to some extent, suggesting that short-term low-level warming might contribute to stabilizing the intestinal mucosal barrier when fish immunity was impaired⁵⁵. This positive response of the intestinal mucosa-associated microbiota might promote the recovery of fish immune function, thereby alleviating heat stress in the intestine. In addition, the co-occurrence network of the intestinal mucosa-associated microbiota showed a certain degree of resilience to low-level warming, but its complexity and stability decreased and eventually changed. Combined with the severe disruption of the intestinal digesta-associated microbiota after low-level warming, we concluded that even low-level warming had potentially negative impacts on the stability of the intestinal microbiota. In addition, high-level warming dramatically disrupted the network complexity and stability of the intestinal mucosa-associated microbiota and altered its microbial interactions, raising concerns about the potential costs of long-term exposure to relatively high temperatures. Previous studies revealed that host symbiotic microbes were vulnerable to high-level warming and that heat-challenged hosts whose symbiotic systems were disrupted tended to exhibit increased mortality and other fitness defects^7,56. Predictably, more extreme warming events in the future could lead to severe disturbances in the host-associated microbiome⁶.

Response of the intestinal microbial composition to increased water temperature

Undoubtedly, the alterations and resilience of intestinal microbial diversity and structure to increased water temperature described above were due to changes in the composition and relative abundance of intestinal microbes. Our results revealed that the relative abundances of the most dominant phyla and genera were resilient to low-level warming but not to high-level warming (Fig. 4A and B). Previous studies of fish have shown that temperature changes radically alter the composition and relative abundance of intestinal microbes^9,24,25. Thus, the resilience of most dominant intestinal microbes to low-level warming we observed might have important implications for future aquaculture management in response to severe climate change⁵⁷. Although the relative abundances of most dominant microbes recovered to initial levels after long-term low-level warming, subtle changes in microbial taxa might still affect host health and fitness⁶. For example, after low-level warming, the relative abundances of Proteobacteria, Desulfobacterota, and Brevinema in the intestinal mucosae decreased significantly, whereas the relative abundance of Deinococcota increased significantly. The Proteobacteria phylum contains a variety of opportunistic pathogens, such as Plesiomonas shigelloides, and their existence may stimulate the development of the immune system and maintain normal intestinal mucosal immunity⁵⁰. Therefore, changes in the relative abundance of a few microbes after low-level warming might still affect the microbial barrier and immune function of the intestinal mucosae.

Higher water temperatures, especially extreme heatwaves in summer, favor the growth of potential pathogens, which can negatively affect host health⁵⁸. In this study, opportunistic pathogens, such as Romboutsia ilealis, Escherichia–Shigella, Fusobacterium, Streptococcus, Acinetobacter, and Enterobacter, were significantly enriched in the intestinal mucosae after short-term warming (Fig. 5D and F). Romboutsia ilealisis considered a potentially opportunistic pathogen that is associated with the worsening of type 2 diabetes and the incidence of neurodevelopmental disorders^59,60. Similarly, we found that Romboutsia ilealis was the microbial species that contributed the most to the potentially pathogenic phenotype of the intestinal microbiota. Thus, Romboutsia ilealis is highly likely an opportunistic pathogen in the intestinal mucosae of fish. The bacterial members within the genera Escherichia–Shigella and Fusobacteriumare common opportunistic pathogens in the gastrointestinal tract of fish^61,62. Streptococcus species, including Streptococcus agalactiae and Streptococcus iniae, are major bacterial pathogens of tilapia and can cause serious infectious diseases and high mortality⁶³. The genera Acinetobacter and Enterobacterbelong to the ESKAPE group, which contains various drug-resistant bacterial pathogens common to humans and fish^64,65. Ultimately, the overgrowth of these opportunistic pathogens in intestinal mucosae increased the risk of infectious diseases in tilapia⁵⁰, which was highly consistent with the phenotypic results that the relative abundances of potentially pathogenic phenotype in the intestinal mucosa-associated microbiota increased significantly after short-term warming. Moreover, the overgrowth of opportunistic pathogens might inhibit the colonization of some microbes that should have been normal commensals⁵⁰. Our results revealed that the relative abundance of Cetobacterium in the intestinal mucosae decreased significantly after short-term warming (Fig. 5D and F). It is widely believed that the bacterial species within Cetobacteriumcan promote intestinal digestion and metabolism by producing vitamin B12 and is a microbial indicator of intestinal health⁶⁶. A recent study revealed that vitamin B12 produced by Cetobacterium someraecould improve host resistance to pathogen infections through strengthening intestinal microbial interactions⁶⁷. Thus, the reduced abundance of Cetobacteriumin the intestinal mucosae might weaken intestinal metabolic functions, increasing the vulnerability of fish to intestinal dysfunction. Combined with fish immune fluctuations, the increase in opportunistic pathogens and the decrease in potentially beneficial microbes induced by increased water temperature illustrated the importance of assessing host-pathogen responses when assessing the impact of climate change on the outbreak and spread of infectious diseases⁶⁸.

Response of intestinal microbial function to increased water temperature

The function of the host-associated microbiota is the result of the microbiota–host coevolution and plays a key role in maintaining normal host physiological functions⁶⁹. In our study, the alterations and resilience of the intestinal microbiota to increased water temperature were apparent not only in the taxonomic structure of the intestinal microbiota but also in the predicted functional profile (Fig. 6A). Carbohydrate and amino acid metabolism predominated in the metabolic pathways at KEGG level 2, consistent with PICRUSt functional predictions in the intestinal microbiota of Atlantic salmon⁷⁰. Our results revealed that the dominant microbial genes involved in metabolism first decreased and then recovered to their initial abundances in both the intestinal mucosae and contents after low-level warming but decreased in the intestinal mucosae and increased in the intestinal contents after high-level warming (Fig. 6B). Previous studies have shown that warming affects the intestinal microbiota in ectotherms and that the resulting alterations in functional potential may affect host metabolism^5,6. Thus, high-level warming could disrupt the normal function of the intestinal microbiota, resulting in impaired intestinal metabolic function and heat-related phenotypic defects in the host⁶. Notably, the metabolic pathway involved in xenobiotics biodegradation and metabolism decreased significantly in the intestinal mucosae after short-term warming, indicating that warming might weaken the intestinal responses of fish to environmental pollution or pathogenic infections³⁰. In addition, the potentially pathogenic phenotype increased significantly in the intestinal mucosae after short-term warming. The intestine commonly harbors various opportunistic pathogens, which are commensal members of the intestinal microbiota and are not pathogenic to the host under normal circumstances⁵⁰. However, when the host’s immune system is compromised or its metabolism is dysfunctional, these microbes may become potential pathogens to infect the host²⁴. Thus, the significant increase in the potentially pathogenic phenotype suggested that warming increased the risk of intestinal mucosal pathogens infecting fish. Notably, the low sample size might miss several microbial and functional changes, thereby making the results of this study limited. Besides, the microbial communities in water and feed were not examined, so that errors in fish intestinal microbiota due to contamination or sequencing bias could not be eliminated. Despite these limitations, this study provided a glimpse into the effects of increased water temperature on the structure and function of fish intestinal microbiota.

Conclusions

In summary, this study elucidated that the alterations and resilience of fish intestinal microbiota to increased water temperature coincided with the recovery of immune function. The results showed that the microbial diversity, structure, dominant microbes, and predicted function of the intestinal microbiota were resilient to low-level warming but not to high-level warming. Notably, short-term warming significantly increased microbial richness and diversity in the intestinal mucosae, in which the overgrowth of opportunistic pathogens inhibited the colonization of potential probiotics, ultimately resulting in a significant decrease in metabolic pathways and a significant increase in the potentially pathogenic phenotype. These findings revealed that short-term warming induced compromised immune function and an imbalance in the intestinal mucosa-associated microbiota, increasing the risk of intestinal mucosal pathogens infecting fish. Further co-occurrence network analyses revealed that long-term warming reduced the network complexity and stability of the intestinal microbiota to different extents, suggesting that warming also negatively affected host health through the breakdown of symbiotic relationships. Overall, these observations provide valuable insights into the potential role of the intestinal microbiota in alleviating the warming stress faced by the host. Future studies should more specifically test how the host-associated microbiome affects the host’s ability to respond to climate change as well as extreme heatwave events.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Author contributions

The work was designed by A.L. and Z.W. The work was drafted by Z.W. The experiment was performed by Z.W. and Q.Z. The acquisition and analysis were performed by Z.W. and Q.Z., and X.W. substantively revised the manuscript. The interpretation of data was performed by Z.W. and A.L. All the authors read and approved the final manuscript.

Funding

This study was financially supported by the National Natural Science Foundation of China (No. 32073023) and the Wuhan Science and Technology Project (No. 2019020701011480).

Data availability

The raw reads were deposited in the NCBI Sequence Read Archive under accession number PRJNA1083750 (http://www.ncbi.nlm.nih.gov/sra).

Declarations

Competing interests

The authors declare no competing interests.