Aquaculture 590 (2024) 741041

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Aquaculture
journal homepage: www.elsevier.com/locate/aquaculture

Long-term thermal stress induces hepatic injury and alters the

thermotolerance response in Hong Kong catfish (Clarias fuscus)
Cunyu Duan a, b, Changxu Tian a, b, *, Yingyi Guan a, Hongfei Xu c, Lei Yang a, b, Yu Chen a, b,
Yong Liu a, b, Yijun Shen a, b, Yulei Zhang a, Shouxiong Cao c, Yang Huang a, b, Guangli Li a, b, *
a
Fisheries College, Guangdong Ocean University, Zhanjiang 524088, China
b
Guangdong Research Center on Reproductive Control and Breeding Technology of Indigenous Valuable Fish Species, Guangdong Provincial Engineering Laboratory for
Mariculture Organism Breeding, Guangdong Provincial Key Lab of Pathogenic Biology and Epidemiology for Aquatic Economic Animals, Zhanjiang 524088, China
c
Guangxi Introduction and Breeding Center of Aquaculture, Nanning 530001, China

A R T I C L E I N F O A B S T R A C T

Keywords: Global climate change has a significant impact on fish survival and the aquaculture industry. Even highly
Catfish adaptable organisms like Siluriformes fish are susceptible to these effects. This study investigates the conse­
Heat stress quences of extreme high temperature on Hong Kong catfish (Clarias fuscus), a species known for its long-term
Oxidative stress
habitation in tropical and subtropical regions. In this study, C. fuscus were cultivated for 90 days at high tem­
Histopathology
Transcriptome
perature (HT, 34 ◦ C) and normal temperature (NT, 26 ◦ C), followed by subjecting the two groups to high
temperature stress (34 ◦ C) and temperature recovery (26 ◦ C). The hepatic histology, biochemical, and tran­
scriptomic characteristics of the species were examined before acute high temperature stress (NT-C, HT-C), after
acute high temperature stress (NT-T, HT-T), and after temperature recovery (NT-R, HT-R). The histological
analysis revealed that long-term heat stress damaged the liver tissue of C. fuscus. Furthermore, significant
damage was observed in the liver tissues of the NT group under acute high temperature stress, while no further
damage was observed in the HT group. The biochemical analysis showed that 90 days of heat stress altered the
body’s immune and oxidative balance, resulting in an increase in alkaline phosphatase (ALP), alanine amino­
transferase (ALT), catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPX) activities, and
a decrease in malondialdehyde (MDA) content. Upon acute heat stress, the liver function of the NT group was
disordered and unable to produce enough total-bilirubin (T-BIL), while the aspartate aminotransferase (AST)
activity of the HT group was more susceptible to being affected. Long-term heat stress led to significant changes
in gene expression, with the NT group showing three times more differentially expressed genes (DEGs) identified
in the liver compared to the HT group under acute heat stress conditions. Analysis of DEG enrichment during the
same treatment period in both groups revealed significant differences in amino acid metabolism and lipid
metabolism pathways. These results suggest that long-term heat stress caused liver tissue damage in C. fuscus,
altering immune and oxidative balance, enzyme activity sensitivity, as well as the body’s response to acute heat
stress pathways and intensity. This study lays the groundwork for future investigations into fish adaptability to
sudden temperature changes and aquaculture strategies to mitigate heat stress in fish facing extreme
temperatures.

1. Introduction 2022). Rising temperatures can elevate water temperatures, causing

In recent years, the cumulative emission of greenhouse gases has led
to global warming (Guo et al., 2018). Studies have shown that ambient
temperatures are projected to increase by 3.3–5.7 ◦ C by 2100, resulting
in more frequent and intense temperature fluctuations (Meehl and
Tebaldi, 2004; Kiarsi et al., 2023; Messina et al., 2023; Kuan et al.,
increased stress on aquatic organisms especially in freshwater ecosys­
tems (He et al., 2023a; Shi et al., 2020; Guo et al., 2018). This heat stress
can result in metabolic disorders and reduced immunity in aquatic or­
ganisms, potentially leading to widespread mortality among aquacul­
ture organismspecies (Liu et al., 2019; Gao et al., 2021). This is a
challenge for the sustainable and stable development of the aquaculture

* Corresponding authors at: Fisheries College, Guangdong Ocean University, Zhanjiang 524088, China.
E-mail addresses: tiancx@gdou.edu.cn (C. Tian), ligl@gdou.edu.cn (G. Li).

https://doi.org/10.1016/j.aquaculture.2024.741041
Received 8 March 2024; Received in revised form 30 April 2024; Accepted 4 May 2024
Available online 6 May 2024
0044-8486/© 2024 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
C. Duan et al.
industry. Temperature, as one of the most significant external factors,
affects the survival of aquatic organisms by influencing various biolog­
ical processes (Huang et al., 2020; Liu et al., 2022a). Fish, being the
oldest vertebrates, are mostly poikilothermic animals and highly sensi­
tive to water temperature changes (Han et al., 2023). While they can
usually adapt to temperature variations through physiological plasticity
(Yu et al., 2023; Xu et al., 2018; Lyu et al., 2018; Brionne et al., 2023),
temperatures exceeding their tolerance limit can induce stress, dis­
rupting their internal homeostasis and ultimately leading to fish mor­
tality (Liu et al., 2022c; Bellard et al., 2012). The ability of fish to
withstand drastic temperature changes and recover from stress is crucial
for their survival.
While the change of temperature affects the biological processes of
fish, fish can also adapt to the change of ambient temperature by
actively regulating the metabolic mechanisms, so as to maintain a
relatively constant activity level in a wide range of ambient tempera­
tures (Luo et al., 2022). Previous studies have demonstrated that
Atlantic salmon (Salmo salar) exhibit different acute cortisol stress re­
sponses at different culture temperatures and changes in isozymes and
Hsp70 due to variations in culture temperature, which in turn affect
metabolic recombination (Luo et al., 2022; Madaro et al., 2018; Dalvi
et al., 2017). Nevertheless, our understanding of fish’s response capa­
bilities and adaptive mechanisms to acute heat shock under different
rearing temperatures remains limited.
Liver plays a vital role in the metabolic response to stress in fish,
affecting immune defense, protein synthesis, metabolic detoxification,
and other regulatory processes (Sun et al., 2019). It is also a highly
susceptible organ, as even brief heat stress can result in oxidative stress
and mild liver damage. Therefore, alterations in liver indicators can
serve as physiological markers in response to sudden temperature
changes (Wang et al., 2019). Numerous studies have demonstrated that
acute high temperature affects the tissue status, physiological and
biochemical indicators, and expression levels of metabolic and immune-
related pathways in various fish species. For example, investigations on
pikeperch (Sander lucioperca) (Wang et al., 2019; Liu et al., 2022b),
Chinese giant salamander (Andrias davidianus) (Wang et al., 2018) and
Nile tilapia (Oreochromis niloticus) (Esam et al., 2022) revealed that
acute heat stress caused histological damage and dysfunction in liver,
leading to the activation of the antioxidant system to counteract
oxidative stress. Similarly, studies on largemouth bass (Micropterus sal­
moides) (Yu et al., 2023; Zhao et al., 2022), Horabagrus brachysoma
(Dalvi et al., 2017), and Black Rockfsh (Sebastes schlegelii) (Lyu et al.,
2018) indicated that high temperature influenced protein and lipid
metabolic pathways. Specifically, the study of Glyptosternum maculatum
(Sisoridae: Siluriformes) (He et al., 2023b) indicated that endoplasmic
reticulum (ER) stress and the subsequent activation of the unfolded
protein response (UPR) and ER-associated degradation (ERAD) path­
ways were identified as crucial mechanisms used by the liver to combat
heat stress. The current research mainly focuses on studying the histo­
logical changes, oxidative stress, and transcriptomic characteristics of
fish when subjected to acute heat stress under normal physiological
conditions. However, there is a lack of studies that explore how fish
respond to acute heat stress after being exposed to high-temperature
environments for an extended period.
The Siluriformes are widely studied at all biogeographic scales from
regional to global (Sullivan et al., 2006). Relevant studies include the
effects of environmental factors such as temperature on tissue meta­
bolism, immunity, nutrition and other aspects (da Silva Ribeiro et al.,
2021; de Souza et al., 2020; Kumar et al., 2022; Conte et al., 2023; Liu
et al., 2022c). Hong Kong catfish (Clarias fuscus) is the only native fish of
Clariidae in China (Tian et al., 2023), which is widespread in tropical
and subtropical environments. Its survival water temperature is
10–32 ◦ C, and the optimum growth temperature is 24–28 ◦ C. It is one of
the preferred economic fish for aquaculture in the south, with delicious
meat, strong adaptability and stress resistance (Xu et al., 2021; Lisachov
et al., 2023; Lin et al., 2022). Despite their well-adapted and well-
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Aquaculture 590 (2024) 741041
tolerated nature, Siluriformes have been affected to some extent by
the increasing water temperature resulting from global climate change
(Khieokhajonkhet et al., 2022; He et al., 2023b). Previous research on
C. fuscus has shown that chronic heat stress can lead to tissue damage
and trigger protective reactions to maintain intracellular homeostasis
(Liu et al., 2024). However, it is still unknown whether different living
environment temperatures will affect the response of Siluriformes to
acute high temperature. Therefore, it is of great significance to study the
response degree and molecular mechanism of C. fuscus to acute tem­
perature changes at different culture temperatures.
This study aims to explore the response of C. fuscus to acute heat
stress following exposure to varying temperature cultures focusing on
histology, enzyme activity, and transcriptome analysis. By examining
the response mechanisms to heat stress under different environmental
temperature conditions, this research aims to offer scientific insights
into the study of high-temperature adaptability of C. fuscus and aqua­
culture strategies to mitigate fish heat stress in the face of global
warming.
2. Materials and methods
2.1. Ethics statement
All experimental protocols in this study were performed according to
the criterion of the Animal Research and Ethics Committee of Guang­
dong Ocean University (NIH Pub. No.85–23, revised 1996). The subjects
of this study are not endangered or protected species.
2.2. Animals
Juveniles of C. fuscus (1 month old) were purchased from Guangxi
Hongtai Aquatic Product Farm, Guangxi Province, China. Following the
methodology outlined in a previous study by Liu et al. (2024), the initial
breeding temperature and duration of the experiment were carefully
controlled. The fish were divided into two groups: the normal temper­
ature (NT) group and the high temperature (HT) group, as shown in
Fig. 1. Each group had three replicates. The NT group was cultured at 26
± 2 ◦ C for 90 days, and the HT group was cultured at 34 ± 0.5 ◦ C for the
same duration. Both groups were fed twice daily at 9:00 and 17:00. To
conduct acute high temperature stress and temperature recovery ex­
periments, the NT and HT groups were temporarily raised at 26 ◦ C for a
week. These groups were then referred to as the NT-C and HT-C groups,
respectively. The fish in each group had an average body weight of
77.62 ± 6.55 g and a body length of 19.01 ± 0.50 cm. For the acute high
temperature treatment, 50 fish per barrel were quickly transferred to
corresponding barrels with a volume of 700 L at 34 ◦ C for 72 h. After
that, they were transferred to barrels at 26 ◦ C for 72 h for the temper­
ature recovery treatment. Six serum and liver tissue samples were
collected at 0 h (C), 24 h (T24), 48 h (T48) and 72 h (T72) of acute high
temperature treatment, and 24 h (R24), 48 h (R48) and 72 h (R72) of
temperature recovery treatment. One portion of the liver tissue was
stored in liquid nitrogen at − 80 ◦ C, while another portion was preserved
in Bouin’s liquid for subsequent experiments (Fig. 1).
2.3. Liver histology
Histological analysis was performed on liver tissues obtained from a
random selection of 6 individuals from each group. Following 24 h of
fixation with Bouin’s solution, liver samples were subjected to gradient
dehydration with ethanol, embedded in paraffin, and sliced. Subse­
quently, histopathological changes in the liver were visualized and
documented under a microscope after hematoxylin-eosin (HE) staining.
To ensure accuracy, five non-overlapping fields of view were randomly
selected under a 40× objective to count the total number of nuclei.
C. Duan et al.
Fig. 1. The schematic diagram of acute heat stress and temperature recovery experiment of C. fuscus under different culture temperature conditions.
2.4. Serum and liver indices
Under the guidance of the manufacturer ‘s instructions, the activities
of alkaline phosphatase (ALP), aspartate aminotransferase (AST),
alanine aminotransferase (ALT), catalase (CAT), superoxide dismutase
(SOD), glutathione peroxidase (GPX), malonaldehyde (MDA), and total
bilirubin (T-BIL) contents were measured using ELISA detection kits
from Enzyme-linked Biotechnology, Shanghai, China. To minimize
interference, each serum and liver sample was subjected to three bio­
logical replicates along with two technical replicates.
2.5. RNA extraction,library construction, sequencing and assembly
RNA extraction from liver tissues was performed on six groups (NT-
C, NT-T72, NT-R72, HT-C, HT-T72, HT-R72), with three replicates in
each group. TRIzol Reagent Kit was used to determine RNA degradation
and contamination by 1% agarose gel electrophoresis. The quality of
RNA was evaluated by the Agilent Bioanalyzer 2100 system, including
integrity and amount (Wang et al., 2023). The RNA library was estab­
lished using the NEBNext ® Ultra TM RNA Library Prep Kit (NEB, USA)
guided by the manufacturer ‘s protocol. After library construction,
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Aquaculture 590 (2024) 741041
quantified the library by Qubit2.0 Fluorometer and diluted it to 1.5 ng/
μl. The library’s insert size was then evaluated by the Agilent 2100
bioanalyzer. Subsequently, qRT-PCR accurately quantified the effective
concentration of the library (higher than 2 nM) and ensured its quality.
Sequencing was performed using the Illumina NovaSeqTM 6000 (Illu­
mina, USA), generating 150 bp paired-end reads. The low-quality reads
in the original reads of the fastq format were removed, and the filtered
reads were assembled using the default parameter Trintey software
(Garber et al., 2011) to obtain clean reads. Paired end clean reads were
mapped to the reference genome (Tian et al., 2023) using HISAT2 v
2.0.5. RNA-Seq data can be viewed in NCBI’s SRA database under the
accession number PRJNA1025152.
2.6. Differentially expressed genes (DEGs) and functional annotation
The gene expression of each sample was analyzed by the featur­
eCounts tool software in the subread software (Liao et al., 2014). The
expression level was calculated by fragment per kilobase of transcript
per million mapped reads (FPKM) based on the gene length. Using
DESeq2 software (1.20.0) (Anders and Huber, 2010) to identify differ­
entially expressed genes (DEGs) with padj <0.05 and |log2FoldChange|
C. Duan et al.
≥ 2.0 for each comparison group. Finally, clusterProfiler software
version 3.8.1 (Yu et al., 2012) was employed for Gene Ontology (GO)
and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment
analysis.
2.7. Short time-series expression miner (STEM) analysis
The change trend of DEGs was analyzed based on the time node. The
log2(FPKM +1) values of each gene in the NT group and the HT group at
different treatment periods were normalized to 0, v1-v0, and v2-v0 in
chronological order. STEM software (Ernst and Bar-Joseph, 2006) was
used to cluster the genes with similar log2(FPKM +1) value change
patterns in the two groups, respectively. The analysis parameters were
set as follows: maximum unit of change between time points was 2,
maximum output trend clustering figure was 20, minimum fold change
of log2(FPKM +1) was 2. Clusters with a P < 0.05 were considered
significant.
2.8. Validation of gene expression by qRT-PCR
To ensure the accuracy of the sequencing results, qRT-PCR was
conducted on 9 randomly selected DEGs for each treatment period in
both the NT and HT groups. Specific primers were designed using Primer
5.0 and synthesized by Sangon Biotechnology Co., Ltd. (Shanghai,
China), as outlined in Table S1. Total RNA from liver samples at each
treatment period (a total of 18 samples) from both groups was extracted
using the TRIzol reagent kit. Subsequently, reverse-transcribed cDNA
was synthesized following the instructions provided by the TransScript®
Fly First-Strand cDNA Synthesis SuperMix kit from Beijing TransGen
Biotech Co. Ltd. (Beijing, China). According to manufacturer ‘s in­
structions, qRT-PCR was performed using the PerfectStart ® Green qPCR
SuperMix (Beijing TransGen Biotech Co., Ltd., Beijing, China) kit in a
Roche LightCycler 96 real-time PCR system. The reaction procedure is as
follows: 95 ◦ C pre-denaturation for 5 min, followed by 40 cycles of 95 ◦ C
for 30 s and 52 ◦ C for 30s. β-actin was used as a standardized internal
reference for gene expression level, and the fold change of target gene
expression was calculated by 2-ΔΔCt method.
2.9. Statistical analysis
All statistical analyses were conducted by using SPSS 26.0, with re­
sults presented as mean ± SD. A two-tailed unpaired t-test was utilized
to analyze the significance of the differences in mean data between the
NT group and HT group under the same temperature treatment stage. A
P-value of <0.05 indicates a statistical difference, < 0.01 signifies a
significant statistical difference, and < 0.001 indicates a highly signifi­
cant statistical difference between the two groups.
3. Results
3.1. Effects of acute hyperthermia and temperature recovery on liver
histological structure
Histological analysis of liver tissue using HE staining revealed that
prior to acute high temperature stress, hepatocytes in the NT-C group
exhibited regular arrangement and distinct boundaries, whereas those in
the HT-C group showed disorganized arrangement, vacuolar degenera­
tion, inflammatory infiltration and significantly smaller nucleus
compared to the NT group. After 72 h of high temperature treatment,
hepatocytes in the NT group displayed a significant reduction in nuclei
size and number, unclear cell boundaries, and the onset of disordered
arrangement and vacuolar degeneration. Although the nucleus size
partially recovered after temperature recovery, it remained smaller than
pre-high temperature treatment levels. Conversely, in the HT group,
cells exhibited disorganized arrangement, vacuolar degeneration, in­
flammatory infiltration, and no significant changes in nucleus size
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Aquaculture 590 (2024) 741041
during both the acute high temperature treatment and temperature re­
covery phases (Fig. 2).
3.2. Effect of acute high temperature on biochemical indices in serum and
liver
The changes in biochemical indices of liver function in the serum of
the NT group and HT group during different treatment periods are
shown in Fig. 3A-D.It was observed that, under acute high temperature
stress, the activity of ALP decreased in both the NT and HT groups and
subsequently recovered after temperature recovery, particularly at the
beginning of the recovery period. In the early stage of acute high tem­
perature stress, ALT activity slightly decreased before increasing, and
then decreased again after temperature recovery. It is noteworthy that
the activities of ALP and ALT in the HT group consistently remained
higher than those in the NT group throughout the same periods. The
activity of AST did not exhibit significant changes between different
treatment stages, except in the HT group where it was affected by acute
heat stress faster than in the NT group. The content of TBIL increased
after acute high temperature stress in both groups, which subsequently
recovered after temperature recovery.
The changes in liver biochemical indices during different treatment
periods are shown in Fig. 3E-H. It was observed that the MDA content
increased after acute high temperature stress and decreased after tem­
perature recovery in both the NT and HT groups. Additionally, the MDA
content in each period of the HT group was lower than that of the NT
group. The activities of CAT, SOD and GPX slightly decreased in the
early stage of acute high temperature stress, then gradually increased
until the initial stage of temperature recovery, and finally decreased
after 24 h of temperature recovery. Furthermore, the activity of the three
enzymes in the HT group was higher than those in the NT group at
almost all stages.
3.3. Transcriptome sequencing analysis
3.3.1. Sequencing of reads
In this study, a total of 9 samples from the NT group were sequenced,
resulting in an average of 41.54 million clean reads per sample. The
percentage of Q30 bases exceeded 89.62%, and the GC content ranged
from 44.26% to 47.30%. These clean reads were then aligned with the
genome sequence of C. fuscus, with a comparison rate of over 86.71%
(Table S2). Similarly, for the HT group, 9 samples were sequenced and
yielded an average of 41.62 million clean reads per sample, with a Q30
bases percentage surpassing 88.74% and GC content ranging from
45.19% to 47.12%. The comparison rate to the C. fuscus genome
sequence was >89.14%. These results indicate that the sequencing data
generated in this experiment is of high quality and meets the standard
for subsequent analysis (Table S3).
3.3.2. Analysis of DEGs
The number of DEGs between the NT and HT groups at different
treatment times is shown in Fig. 4A-B. In the NT group, there were 1104
DEGs, while in the HT group, there were 372 DEGs. Specifically, NT-T72
vs. NT-C had 269 up-regulated genes and 267 down-regulated genes,
NT-R72 vs. NT-C had 101 up-regulated genes and 148 down-regulated
genes, HT-T72 vs. HT-C had 121 up-regulated genes and 67 down-
regulated genes, and HT-R72 vs. HT-C had 40 up-regulated genes and
29 down-regulated genes (Fig. 4A). Furthermore, HT-C vs. NT-C had 48
up-regulated genes and 75 down-regulated genes, HT-T72 vs. NT-T72
had 162 up-regulated genes and 217 down-regulated genes, and HT-
R72 vs. NT-R72 had 3 up-regulated genes and 11 down-regulated
genes. It is important to note that the number of DEGs was highest in
the two groups after acute high temperature treatment (Fig. 4B).
The commonality of DEGs in the NT group and the HT group was
determined using Venn diagrams, comparing the acute high tempera­
ture treatment and temperature recovery stage with the pre-acute high
C. Duan et al. Aquaculture 590 (2024) 741041

Fig. 2. The liver structure of C. fuscus in different treatment stages of NT group and HT group. A, B and C represent NT-C group (A), NT-T72 group (B) and NT-R72
group (C); D, E and F represent HT-C group (D), HT-T72 group (E) and HT-R72 group (F); G represent the number of nuclei (G) in the liver of C. fuscus. HN: he­
patocyte nuclei, VD: vacuolar degeneration, II: inflammatory infiltration.

temperature treatment stage (Fig. 4C-D). It was observed that there were
fewer DEGs both in NT and HT groups in the two comparison groups.
The expression patterns of DEGs in the three treatment stages of the NT
group and HT group were visualized using a heat map (Fig. 4E). The heat
map revealed that the expression patterns of DEGs in the NT group and
HT group differed during acute high temperature stress and temperature
recovery.
3.3.3. Functional classification of DEGs
To gain further insights into the biological significance of the DEGs,
GO enrichment analysis was conducted. In the T72 vs. C group of the NT
condition, the DEGs were significantly enriched in iron ion binding,
unfolded protein binding, protein folding, oxidoreductase activity
(acting on paired donors, with incorporation or reduction of molecular
oxygen), lipid transport, lipid localization and heme binding. The DEGs
in R72 vs. T72 were significantly enriched in unfolded protein binding
and protein folding. Additionally, the DEGs in R72 vs. C were signifi­
cantly enriched in lipid transport, lipid localization, extracellular region
and lipid transporter activity (Fig. S1A–C). For the HT group, the DEGs
in the T72 vs. C group were significantly enriched in unfolded protein
binding, endopeptidase activity, proteolysis, protein folding and other
terms. The DEGs in the R72 vs. T72 group were significantly enriched in
unfolded protein binding and protein folding, while the DEGs in the R72
vs. C did not exhibit significant enrichment (Fig. S1D–F).

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C. Duan et al.
Fig. 3. The levels of biochemical indexes in serum and liver of NT group and HT group at different treatment stages were mean ± SD (n = 6). An asterisk (*) indicates
a statistical difference between the HT group and NT group at the same time point (P < 0.05), two asterisks (**) indicate that there was a significant statistical
difference (P < 0.01), and three asterisks (***) indicate a very significant statistical difference (P < 0.001).
KEGG enrichment analysis of DEGs between different treatment
groups in the NT group showed that DEGs in the T72 vs. C group was
significantly enriched in the PPAR signaling pathway, protein process­
ing in endoplasmic reticulum, steroid biosynthesis and biosynthesis of
nucleotide sugars. The DEGs in the R72 vs. T72 or R72 vs. C groups were
significantly enriched in protein processing in endoplasmic reticulum
(Fig. 5A-C). In the HT group, only the DEGs in T72 vs. C were signifi­
cantly enriched in steroid biosynthesis (Fig. 5D-F). Furthermore, the
enrichment analysis of DEGs between HT group and NT group at the
same treatment stage showed no significant enrichment pathway before
acute high temperature treatment. However, after acute high tempera­
ture stress, the pathways protein processing in endoplasmic reticulum,
steroid biosynthesis, ferroptosis and biosynthesis of nucleotide sugars
were significantly enriched. After temperature recovery, the DEGs were
significantly enriched in the pathways protein processing in endo­
plasmic reticulum and so on (Fig. 4G-I).All of these pathways were
associated with down-regulated genes.
3.3.4. Analysis of temporal expression trends
Trend analysis was performed on the expression patterns of 1104
DEGs in the NT group and 372 DEGs in the HT group. In the NT group,
the 1104 DEGs were significantly clustered into three clusters: profile 10
(p = 1.6e− 15, 131DEGs), profile 9 (p = 9.1e− 14, 102DEGs) and profile 5
(p = 1.7e− 7, 69DEGs) (Fig. 6A). Similarly, in the HT group, the 372 DEGs
were significantly clustered into profile 10 (p = 8.3e− 9, 53 DEGs)
(Fig. 6B). In profile 9 and 10, the expression of these genes showed an
upward trend under acute high temperature stress and a downward
trend after temperature recovery, while profile 5 showed an opposite
trend. Further KEGG enrichment analysis of the DEGs in the NT and HT
groups showed that DEGs in profile 10 of the NT group were signifi­
cantly enriched in protein processing in endoplasmic reticulum and
arginine and proline metabolism (Fig. 6C-F). The DEGs clustered into
6
Aquaculture 590 (2024) 741041
profile 9 were significantly enriched in protein processing in endo­
plasmic reticulum, cytosolic DNA-sensing pathway, herpes simplex virus
1 infection and RIG-I-like receptor signaling pathway. The DEGs clus­
tered into profile 5 were significantly enriched in PPAR signaling
pathway and retinol metabolism. There was no significant enrichment
pathway for DEGs clustered into profile 10 in HT group.
3.3.5. Protein processing in endoplasmic reticulum
The expression levels of genes involved in the protein processing in
endoplasmic reticulum pathway showed significant difference not only
between different treatment stages, but also between the NT and HT
groups at the same treatment stage. To further investigate the effects of
different culture temperatures on the mechanism of acute heat stress
response in C. fuscus, the expression profiles of the key differentially
expressed genes in this pathway were analyzed. The expression levels of
all DEGs in the pathway of protein processing in endoplasmic reticulum
were analyzed during the three treatment periods for both the NT group
and the HT group (Fig. 7A-B). It was found that genes involved in protein
correct folding, inhibition of misfolded protein aggregation and mis­
folded protein degradation were up-regulated after acute high temper­
ature treatment in the NT group.These genes included molecular
chaperones such as pdia3, hyou1, calr3b, hspa5, pdia4, pdia6, as well as
heat shock proteins such as hspa8 and hsp90aa1. Conversely, the
expression level of eif2ak4 was down-regulated. Most genes showd re­
covery in expression level after temperature recovery, with the excep­
tion of genes encoding molecular chaperone proteins related to protein
correct folding and quality control (calr3b, hyou1, hspa5, hsp90b1,
dnajc3a, dnajb11), which exhibited lower expression levels compared to
before acute high temperature stress. The only geneshowing a signifi­
cant increase in expression was dnajb1b. In the HT group, only a few
genes (pdia3 and calr3b) involved in protein correct folding and mis­
folding protein degradation were up-regulated after acute high
C. Duan et al. Aquaculture 590 (2024) 741041

Fig. 4. (A) The number of DEGs was compared between NT group and HT group at three treatment times. (B) The same treatment time (C, T72, R72) compared the
number of DEGs between HT group and NT group. The vertical axis represents the number of genes, and red and blue represent up-regulated and down-regulated
DEGs, respectively. (C) Venn diagram shows the distribution of the number of DEGs in T72 vs. C of NT group and HT group. (D) Venn diagram shows the distribution
of the number of DEGs in R72 vs. C in NT group and HT group, and the overlapping part represents the common DEGs. (E) Heatmap analysis of all DEGs hierarchical
clustering in NT group and HT group at three processing times. In the figure, the abscissa is the different treatment time of NT group and HT group, and the ordinate
is each DEGs. The log2 (FPKM +1) value was normalized and transformed (scale number) and clustered. Red indicated high gene expression and blue indicated low
gene expression. The color is from red to blue, indicating that log2 (FPKM +1) is from large to small. (For interpretation of the references to color in this figure legend,
the reader is referred to the web version of this article.)

temperature treatment, and their expression levels returned to normal
after temperature recovery. The expression levels of all genes were not
significantly different from those before acute high temperature stress.
The expression of related genes in the NT group and HT group was
analyzed during the same treatment period (Fig. 7B). Prior to acute high
temperature stress, it was found that the expression of hspa5 and calr3b
was lower in the HT group compared to the NT group while the
expression of hspa8 was up-regulated. Additionally, after 72 h of acute
high temperature stress, only a few genes in the HT group were up-
regulated, and the degree of up-regulation was still lower than that of
the NT group. After temperature recovery, most genes in both groups
exhibited no significant differences in expression, except for dnajb1b and
hsp70.
3.4. qRT-PCR validation of transcriptome data
Nine genes were randomly selected for qRT-PCR verification: hyou1,
hspa8, fabp1, calr3b, canx, fads2, hsp90b1, fabp4 and pdia4. The
expression patterns of all selected genes after different treatments were
consistent in RNA-seq and qRT-PCR (Fig. 8), indicating the accuracy and

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Fig. 5. (A) KEGG enrichment bubble diagram of DEGs in T72 vs. C of NT group. (B) KEGG enrichment bubble diagram of DEGs in R72 vs. C of NT group. (C) KEGG
enrichment bubble diagram of DEGs in R72 vs. T72 of NT group. (D) KEGG enrichment bubble diagram of DEGs in T72 vs. C of HT group. (E) KEGG enrichment
bubble diagram of DEGs in R72 vs. C of HT group. (F) KEGG enrichment bubble diagram of DEGs in R72 vs. T72 of HT group. (G) KEGG enrichment bubble diagram
of DEGs between HT group and NT group before acute high temperature treatment. (H) KEGG enrichment bubble diagram of DEGs between HT group and NT group
at 72 h after acute high temperature treatment. (I) KEGG enrichment bubble diagram of DEGs between HT group and NT group at 72 h after temperature recovery
treatment. The abscissa is the ratio of the number of DEGs annotated to the KEGG pathway to the total number of DEGs, and the ordinate is the KEGG pathway.

reliability of RNA-seq. leading to various physiological disorders, including endocrine disor­

4. Discussion
Temperature is a critical external factor that impacts fish survival.
Frequent changes in temperature can induce stress responses in fish,
ders and immune system damage, and ultimately posing a threat to their
survival (Li et al., 2023). This study investigated the capacity of C. fuscus
to respond to acute high temperature stress following a 90-day period of
continuous high temperature stress. We observed the histological
changes of the liver after acute temperature changes and compared the

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Fig. 6. All DEGs in NT group (A) and HT group (B) were clustered by STEM according to the change trend of log2 (FPKM +1) value of genes, and the trend map was
arranged according to the number of genes in each profile, from high to low, and the color was significantly clustered (P < 0.05). (C) KEGG enrichment bubble
diagram of NT group clustering genes in profile 10. (D) NT group clustering KEGG enrichment bubble diagram of genes in profile 9. (E) KEGG enrichment bubble
diagram of NT group clustering genes in profile 5. (F) KEGG enrichment bubble diagram of HT group clustering genes in profile 10.

9
C. Duan et al. Aquaculture 590 (2024) 741041

Fig. 7. (A) Protein processing pathways in endoplasmic reticulum. Orange is the correct folding process of the protein, green is the endoplasmic reticulum-related
degradation process, and blue is the unfolded protein reaction process. (B) The expression of all DEGs in the pathway of protein processing in endoplasmic reticulum
was compared between the NT group and the HT group during the three treatments. In the figure, the abscissa is the different treatment groups of NT group and HT
group, and the ordinate is the gene name. The log2 (FPKM +1) values were normalized and transformed (scale number) and clustered. Red indicated high gene
expression and blue indicated low gene expression. The color is from red to blue, indicating that log2 (FPKM +1) is from large to small (the results can only be
compared horizontally rather than vertically). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of
this article.)

enzyme activity and transcriptome of the the NT group and the HT
group. Our findings revealed that both continuous high temperature
treatment and acute high temperature treatment could cause liver cell
damage and affect the activities of enzymes related to immune and
metabolic pathways in fish. However, the continuous high temperature
culture group exhibited relatively mild heat stress. The results of tran­
scriptome analysis confirmed these findings and elucidated the molec­
ular level response mechanism of catfish to heat stress.
4.1. High temperature treatment induces liver histopathological damage
Numerous studies have demonstrated that exposure to high tem­
perature stress commonly results in liver damage in various fish species.
Similar to previous studies on hybrid catfish (♂Clarias gariepinus
(Burchell, 1822) × ♀C. macrocephalus (Günther, 1864)) (Khieokha­
jonkhet et al., 2022), M. salmoides (Zhao et al., 2022) and Japanese
flounder (Paralichthys olivaceus) (Han et al., 2023), hepatocytes in the
NT group showed vacuolization, inflammatory infiltration, decreased
number and size shrinkage of nuclei after acute high temperature
treatment, while hepatocytes in the HT group exhibited a weak
response. These pathological phenomena in liver tissue indicate that
long-term high temperature culture could lead to histological damage of
hepatocytes, and the liver tissue of high temperature cultured C. fuscus
did not show additional damage after acute high temperature stress. It is
hypothesized that acute high temperature treatment may have less
histopathological impact on long-term high temperature-cultured
C. fuscus. This could be attributed to the body’s adaptive mechanism
in response to liver cell injury, which helps in maintaining internal
homeostasis and reducing the adverse effects of heat stress (Luo et al.,
2022).
4.2. High temperature treatment can deepen the body’s degree of
peroxidation and change the oxidation balance
In addition to histological changes, biochemical indicators can also
reflect the body’s state. ALP occupies an important position in the
regulation of calcium and phosphorus balance, as well as immune and
metabolic processes in fish (Dalvi et al., 2017). The activity of ALT and
AST in the serum can serve as indicators to assess the liver’s physio­
logical state (Li et al., 2019). Moreover, the accumulation of ALT and
AST in the serum can lead to metabolic disorders, resulting in the
accumulation of T-BIL. In this study, there were significant differences in
ALP and ALT levels between the HT group and the NT group after 90
days of high temperature culture. These findings suggest that the liver
cells were damaged and the immune and metabolism balance of the fish
was altered in the HT group (Ahmad et al., 2011). Following acute high
temperature treatment, the changes in ALP, ALT, and AST levels in the
serum were consistent with previous studies on rainbow trout (Onco­
rhynchus mykiss) (Li et al., 2022). However, the TBIL content in the NT
group decreased, possibly due to insufficient bilirubin production
caused by liver dysfunction. The activity of AST in HT group was
affected by acute high temperature treatment and increased faster than
that in NT group. This could be attributed to the increased vulnerability
of liver cells in the HT group to damage following high temperature
culture, resulting in reduced stress resistance.
The homeostasis of the internal environment of the fish requires a
delicate balance between oxidation and anti-oxidation. Excessive reac­
tive oxygen species (ROS) decomposed into MDA, which can reduce the
permeability of cell membrane and lead to cell damage, making MDA
tissue content an indirect indicator of oxidative stress and membrane
damage (Chen et al., 2022). The three antioxidant enzymes CAT, SOD
and GSH-Px cooperates to protect cells from peroxide damage, and
maintain various physiological and biochemical functions of the body
(Klein et al., 2017; Chu et al., 2023). In this experiment, HT group’s
MDA content in the liver was significantly different from that of NT

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C. Duan et al.
Fig. 8. Comparison of gene expression levels determined by RNA-seq and qRT-PCR methods in C. fuscus liver.
group, while the activity of the three antioxidant enzymes was slightly
higher in the HT group compared to the NT group, suggesting the
establishment of a new oxidative balance in the HT group following high
temperature culture. The content of MDA increased after acute high
temperature stress, indicating increased lipid peroxidation in the body.
However, the content decreased in the later stages of acute high tem­
perature stress and during temperature recovery, possibly due to in­
duction and activation of the antioxidant system, leading to a reduction
in lipid peroxidation. The activities of CAT, SOD and GPX decreased
slightly in the early stage of acute high temperature stress, likely due to
inhibition of the in vivo antioxidant system by high temperatures. As the
body gradually adapted to high temperature, the activity of the three
enzymes gradually increased. During the early stage of rewarming,the
activity of the three enzymes continued to increase in order to eliminate
excess ROS in the body, and then decreased as ROS levels decreased.
Thess results are consistent with studies on pufferfish (Takifugu obscurus)
(Cheng et al., 2018) and S. lucioperca (Li et al., 2019), indicating that
increased antioxidant enzymes levels can contribute to alleviate the
oxidative damage caused by heat stress, and temperature recovery
treatment can mitigate the adverse effects of acute heat stress on fish
(Liu et al., 2022b).
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Aquaculture 590 (2024) 741041
4.3. High temperature treatment can change the mechanism and degree of
response to heat stress
Transcriptome analysis is a widely used method for studying the
physiological response of organisms to environmental changes. In recent
years, it has provided crucial information for understanding the
response of aquatic organisms to global warming (Zhao et al., 2021). In
this study, we utilized transcriptome analysis to explore the key mo­
lecular mechanism of liver response to acute high temperature stress.
Previous studies on turbot (Scophthalmus maximus), small yellow croaker
(Larimichthys polyactis), G.maculatum have shown that temperature
fluctuations can lead to changes in gene expression (Huang et al., 2020;
Chu et al., 2020; He et al., 2023b). The differential gene analysis in this
study revealed that the number of DEGs in the high temperature (HT)
group was lower than that in the normal temperature (NT) group. This
suggests that the liver of C. fuscus, after 90 days of high temperature
culture, exhibits reduced responsiveness to acute high temperature
treatment (Sessions et al., 2021). Further analysis using Venn diagrams
and heat maps showed significant difference in number, category and
response degree of genes between the two groups.
GO functional and KEGG enrichment analysis revealed that high
C. Duan et al.
temperature stress in the NT group affected the expression of genes
related to amino acid metabolism and lipid metabolism. The results were
consistent with those of M. salmoides, spotted seabass (Lateolabrax
maculatus) and O. mykiss (Zhao et al., 2022; Cai et al., 2020; Quan et al.,
2021b). In contrast, there were limited pathways activated in the HT
group in response to acute heat stress. Compared with NT group, the
most significant difference in response was the protein processing in
endoplasmic reticulum pathway, which was most closely related to heat
stress. This suggests that after undergoing long-term culture in high
temperatures, the pathway may have achieved a new equilibrium in
related functions (Costábile et al., 2022), thereby weakening the
response to acute high temperature treatment. STEM analysis further
showed that the key molecular mechanisms underlying temperature
response in the two catfish groups are divergent.
ER is an important place for the synthesis and processing of proteins.
Its oxidative environment is conducive to the correct folding of proteins
and plays an important role in cell function and survival (Das et al.,
2023). Misfolded proteins can be degraded by ERAD, otherwise it will
cause UPR. The heat shock protein (HSP) family occupies an important
position in response to heat stress. The family can maintain cellular
protein homeostasis under stress conditions by renaturation of heat-
damaged proteins and removal of unfolded damaged proteins (Duan
et al., 2023). In this study, Hspa5 was down-regulated and Hspa8 was
up-regulated in Hsp70 of HT group compared with NT group before
acute high temperature treatment. This result is consistent with the
study of Russian sturgeon (Acipenser gueldenstaedtii) (Costábile et al.,
2022). Hspa8 plays a key role in protein folding, renaturation, trans­
membrane transport and targeted protein degradation (Silva et al.,
2021; Stricher et al., 2013). The down-regulation of Hspa5 expression
may be due to the UPR, while the high expression of Hspa8 regulates
endoplasmic reticulum homeostasis. It is speculated that Hspa8 plays an
important role in endoplasmic reticulum response to acute heat stress.
The Hsp70 family proteins (Hyou1, Hspa5 and Hspa8) were up-
regulated after acute hyperthermia in the NT group, indicating an
increased proportion of misfolded proteins (Huang et al., 2018). The
three enhance the folding ability of proteins in the endoplasmic reticu­
lum by shielding the unfolded regions of surrounding proteins, while
promoting the degradation of misfolded or unfolded proteins and
maintaining the dynamic balance of protein folding (Sun et al., 2023; Li
et al., 2008; Lee, 2005; Schroӧder and Kaufman, 2005; Woehlbier and
Hetz, 2011). In addition, the up-regulated proteins also include Hsp90
family proteins (Hsp90aa1), which is a universal marker gene in fish
heat stress research (Swirplies et al., 2019). The up-regulation of the
above genes ensures the correct folding of proteins and the degradation
of misfolded/ unfolded proteins under heat stress conditions, ensuring
the quality of protein synthesis. After temperature recovery, most of the
gene expression levels returned to the initial value, while the expression
level of dnajb1b was still significantly higher than the initial value after
temperature recovery. Dnajb1 belongs to the Hsp40 family and is
considered to be an anti-apoptotic factor that plays an important role in
the decay and recovery phases of heat shock response (Cui et al., 2015;
Raeburn et al., 2022). However, only a few genes were up-regulated in
HT group after acute heat stress treatment, and the degree of up-
regulation was lower than that in NT group. It is speculated that
continuous high temperature treatment will lead to weak expression of
protein processing pathway in endoplasmic reticulum of C. fuscus in
response to acute heat stress, which may be due to the damage of protein
quality control related functions (Wang et al., 2019).
Significant differences were observed in the expression of the PPAR
signaling pathway, steroid biosynthesis, and ferroptosis pathway be­
tween the NT group and HT group. Studies have shown that lipid
metabolism pathways can interact with each other and respond to heat
stress by regulating complex regulatory networks related to lipid
metabolism (Huang et al., 2020; Yu et al., 2023). In this study, the down-
regulated expression of fabp4, cpt1a, acsbg1 and acsbg2 in the PPAR
signaling pathway of NT group may be due to heat stress leading to lipid
12
Aquaculture 590 (2024) 741041
peroxidation in the liver, thereby reducing its content to ensure the
homeostasis of lipid metabolism (Zheng et al., 2022). The only up-
regulated gene acsl4 not only plays a key role in lipid biosynthesis and
fatty acid metabolism, but also is closely related to ferroptosis (Kuwata
and Hara, 2019; Quan et al., 2021a; Yuan et al., 2016; Zhao et al., 2023;
Liu et al., 2023). Ferroptosis is an iron-dependent regulatory cell death
(RCD) that can be activated when lipid peroxides accumulate to toxic
levels (Marteau et al., 2022). Acsl4 is a key determinant of ferroptosis.
The increase of Acsl4 content of NT group indicates that there is too
much lipid peroxide in the body, which may be caused by acute heat
stress leading to lipid metabolism disorder, so as to cause liver tissue
damage (Zhao et al., 2023; Liu et al., 2023). Conversely, the higher
GPX4 expression in the HT group compared to the NT group suppressed
ROS production, thus mitigating the adverse effects of ferroptosis (Kang
et al., 2021). In the steroid biosynthesis pathway, the higher expression
of tm7sf2 in NT group indicates that it is required to provide more energy
for the body by up-regulating the biosynthesis of steroids to maintain
metabolic balance in response to heat stress (Zhang et al., 2023).
The research findings above indicate that heat stress has an impact
on the protein processing balance, with a subsequent trend towards
recovery after temperature normalization. In response to long-term high
temperature exposure, the body adjusts the expression of key quality
control genes to adapt to these changes. Additionally, heat stress affects
lipid metabolism by inducing lipid peroxidation and deposition, and
when lipid peroxide reaches toxic levels, it triggers ferroptosis. There­
fore, the ability to maintain lipid metabolism homeostasis following
temperature variations is crucial for the survival of C. fuscus.
5. Conclusion
In summary, long-term exposure to high temperature would cause
damage to the liver of C. fuscus, resulting in reduced sensitivity of related
functions to heat stress. The PPAR signaling pathway, protein processing
in the endoplasmic reticulum and steroid biosynthesis appear to play an
important role in the liver’s response to heat stress in C. fuscus. These
findings enhance our understanding of molecular mechanisms involved
in the heat stress response of C. fuscus under varying culture tempera­
tures. Additionally, these provide a scientific foundation for compre­
hending the tolerance and adaptive strategies of C. fuscus in dealing with
potential heat shocks.
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.aquaculture.2024.741041.
Funding
This research was funded by Guangdong Basic and Applied Basic
Research Foundation (2021A1515010733), the Department of Educa­
tion of Guangdong Province (2023KTSCX042), the Self-financing Proj­
ect of Guangxi Agricultural Science and Technology (Z2019123), the
Modern Seed Industry Park for Whiteleg Shrimp of Guangdong Province
(K22221), and the Undergraduate Innovation Team Project of Guang­
dong Ocean University (CXTD2023003).
Ethics approval
All experimental protocols in this study were approved by the Ani­
mal Research and Ethics Committee of Guangdong Ocean University
(NIH Pub. No.85–23, revised 1996). This study does not involve en­
dangered or protected species. All efforts were made to minimize the
suffering of the animals. All experiments were performed in accordance
with relevant guidelines and regulations.
CRediT authorship contribution statement
Cunyu Duan: Writing – original draft, Methodology, Investigation,
Formal analysis, Data curation. Changxu Tian: Writing – review &
C. Duan et al.
editing, Supervision, Resources, Project administration, Methodology,
Investigation, Funding acquisition, Data curation, Conceptualization.
Yingyi Guan: Investigation, Formal analysis. Hongfei Xu: Resources,
Formal analysis. Yu Chen: Investigation, Formal analysis. Yong Liu:
Investigation, Formal analysis. Yijun Shen: Investigation. Yulei Zhang:
Investigation. Shouxiong Cao: Resources. Yang Huang: Resources.
Guangli Li: Writing – review & editing, Supervision, Funding
acquisition.
Declaration of competing interest
The authors declare that there are no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Data availability
The datasets generated in this study have been uploaded to the NCBI
database with the accession number PRJNA1025152.
Acknowledgements
The authors thank each project for the funding and all the members
of the laboratory for their careful experiments and valuable discussions.
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