Physiological response and microRNA expression profiles in head kidney of genetically improved farmed tilapia (GIFT, Oreochromis niloticus) exposed to acute cold stress

Cold stress has a serious impact on the overwintering survival and yield of genetically improved farmed tilapia (GIFT, Oreochromis niloticus). Understanding the physiological and molecular regulation mechanisms of low-temperature adaptation is necessary to help breed new tolerant strains. The semi-lethal low temperature of juvenile GIFT at 96 h was determined as 9.4 °C. We constructed and sequenced two small RNA libraries from head kidney tissues, one for the control (CO) group and one for the 9.4 °C-stressed (LTS) group, and identified 1736 and 1481 known microRNAs (miRNAs), and 164 and 152 novel miRNAs in the CO and LTS libraries, respectively. We verify the expression of nine up-regulated miRNAs and eight down-regulation miRNAs by qRT-PCR, and found their expression patterns were consistent with the sequencing results. We found that cold stress may have produced dysregulation of free radical and lipid metabolism, decreased superoxide dismutase activity, reduced respiratory burst and phagocytic activity of macrophages, increased malondialdehyde content, and adversely affected the physiological adaptation of GIFT, eventually leading to death. This study revealed interactions among miRNAs and signal regulated pathways in GIFT under cold stress that may help to understand the pathways involved in cold resistance.

Treatment and sampling. A total of 660 experimental fish were used for the actual experiment. The experimental methods and management were as described above.
Experiment one. Sixty experimental fish were placed randomly in six tanks (10 fish per tank); three 96h-LT 50 tanks (LTS group) and three 28 °C control tanks (CO group). The cumulative mortality in each tank was calculated at 0, 2, 6, 12, 24, 48, and 96 h. Experiment two. Six hundred experimental fish were placed randomly in 12 tanks (50 fish per tank); six 96h-LT 50 tanks (LTS group) and six 28 °C control tanks (CO group). Nine samples were collected from three LTS tanks and three CO tanks (three fish from each tank) at each sampling time respectively. The sampling times were 0, 2, 6, 12, 24, 48, and 96 h. Three fish were randomly captured from each tank and rapidly anesthetized with 200 mg L −1 MS-222. The head kidney tissues were removed, divided into two parts, frozen with liquid nitrogen, and stored at −80 °C. One part of the sample was used to measure SOD and LYZ activity and MDA content as follows. The head kidney was homogenized as described by He et al. 2 . SOD activity was determined as described by Beauchamp et al. 27 . MDA content was measured as described by Zhang et al. 28 . LYZ activity was measured as reported by Hultmark et al. 29 . All the required kits were purchased from the Nanjing Jiancheng Biological Engineering Institute (Nanjing, China), and were used according to the manufacturer's instructions. The other part of the sample was used to analyse the expression levels of the miRNAs and their genes at the different sampling times by qRT-PCR.
Another nine samples were collected from the other three LTS tanks and other three CO tanks (three fish from each tank) at each of the sampling times and anesthetized as described above. The head kidney tissues were removed under sterile conditions. Isolation, purification, and culture of the macrophages from the head kidney samples were as described by Chen et al. 30 . We used KOH/DMSO as a blank control. Absorbance was measured at 630 nm using a BioTek Synergy H1 Multi-Mode Reader (Vermont, USA) to determine the RBA of the head kidney macrophages 31 . The method used to measure the PA of the macrophages was as described by Pulsford et al. 32 .
Construction and sequencing of small RNA libraries from head kidney of GIFT exposed to 96h-LT 50 stress by high-throughput sequencing. After 24 h of exposure to 96h-LT 50 stress (LTS group) or to 28 °C (CO group), one fish was randomly captured from each tank; one from each of the six LTS tanks and one from each of the six CO tanks making a total of 12 fish. The head kidney tissues were removed as described above. Total RNA was extracted from the head kidney samples using Trizol reagent (Invitrogen, CA, USA) following the manufacturer's instructions. The quantity and purity of the extracted RNA were measured using a RNA 6000 Nano Lab Chip Kit and a Bioanalyzer 2100 (Agilent, CA, USA), respectively. The samples with an RNA integrity number >7.0 were used for sequencing. Equal amounts of total RNA from the six CO samples or six LTS samples were mixed and pooled separately to build two small RNA libraries. The CO and LTS libraries were sequenced and the reads were assembled following standard procedures 33 .
The basic sequencing data and identification of conserved and novel miRNAs, as well as differentially expressed miRNAs were analysed according to Qiang et al. 34 . Briefly, the raw data were filtered by removing common RNA families (rRNA, tRNA, snRNA, snoRNA), adapter sequences, and sequences with low complexity and repeats. Then, the remaining small RNA sequences of 18-30 nt that matched the Nile tilapia reference genome (http://www.ncbi.nlm.nih.gov/genome/?term=Oreochromis%20niloticus) were compared against all the known mature miRNA sequences of animals in miRbase Release 21.0 (http://www.mirbase.org/) by BLAST searches. Small RNAs with less than two mismatches outside the seed region were considered as conserved miRNA. The remaining small RNAs that did not match known miRNA sequences were mapped to the reference genome. Their flanking sequences were obtained and used to predict the stem-loop structures of the candidate miRNA precursor sequences. Sequences that formed a stable stem-loop structure were considered to be novel 5p-or 3p-derived miRNAs.
Validation of differentially expressed miRNAs in GIFT head kidney after 24 h of 96h-LT 50 stress by qRT-PCR. Total RNA was extracted from head kidney tissue samples from GIFT exposed to 96h-LT 50 cold stress (LTS group) or to 28 °C (CO group) for 24 h; two samples from each of the six CO and six LTS tanks, using Trizol reagent (Invitrogen, CA, USA). A Mir-X ™ miRNA First-Strand Synthesis kit and a SYBR ® PrimeScript TM miRNA RT-PCR kit (Takara, Dalian, China) were used for the RT reaction and qRT-PCRs of the miRNAs as described previously 34 . The relative fold changes in the expression levels of the miRNAs relative to the expression of U6 (used as the reference gene) were calculated by the 2 −ΔΔCt method. The miRNA specific primers were synthesized by Genewiz, Inc. (Genewiz, Suzhou, China).
Expression profiles of some miRNAs and their target genes in head kidney of GIFT under 96h-LT 50 stress. Head kidney tissue samples were taken from the GIFT in Experiment two. Total RNA extraction, and the RT reaction and qRT-PCRs of the miRNAs were as described above. We used the miRanda v3.3a toolbox (http://www.microrna.org/microrna/home. do) and TargetScan 5.2 (http://www.targetscan.org/ vert_50/) to predict the potential target genes of selected differentially expressed miRNAs using a procedure based on Qiang et al. 34 . The RT reaction and qRT-PCRs of the predicted target mRNAs were measured using PrimeScript ™ RT Master Mix and SYBR ® Premix Ex Taq kits (Takara, Dalian, China), as described in our previous study 21 . 18 S rRNA was used as a reference. The mRNA and 18 S rRNA primers were synthesized by Shanghai GeneCore Bio Technologies Co., Ltd. (Shanghai, China) ( Table 1). The measurement of miRNA expression was as described above. The results were used to analyse the expression levels of the miRNAs and their potential target genes in GIFT under 96h-LT 50 cold stress. Data analysis. Data were expressed as mean ± standard error of mean (SEM). The data were analysed by one-way analysis of variance (ANOVA) using the SPSS 17.0 software. P values < 0.05 were considered statistically significant.
Data availability. All data supporting the conclusions of this article are included within the article.

Results
Assessment of 96h-LT 50 cold stress in GIFT. The stress temperatures used in Experiment one had significant effects on GIFT survival ( Fig. 1). At 12 °C, there were no GIFT deaths at 96 h. At 11 °C, GIFT began to die at 12 h and the cumulative mortality was 20% at 96 h. At 9 °C and 8 °C, the cumulative mortalities at 12 h were 6.7% and 20%, respectively; at 24 h, they were 23.3% and 33.3%, respectively; and at 48 h and 96 h, the cumulative mortalities were 56.7% and 83.3%, respectively. Thus, after 96 h of exposure to cold stress, the cumulative mortality of the GIFT exposed to 8 °C stress was significantly higher than the cumulative mortality of the GIFT in the other cold-exposed groups (P < 0.05). The 96h-LT 50 Table 1. Primer design of miRNA and mRNA. and when the abdomen of the fish was touched gently, the fish would swim quickly; such fish soon died. At 24 h, GIFT began to die, and at 48 h and 96 h, the mortality rose sharply ( Fig. 2A). LYZ activity in the head kidney of GIFT in the LTS group was significantly higher than in the CO group at 6 h post-9.4 °C stress (P < 0.05), but there was no significant difference in LYZ activity between the LTS and CO groups at 12 h post-9.4 °C stress (P > 0.05) (Fig. 2B). At 24 h post-9.4 °C stress, the activities of LYZ and SOD decreased significantly (Fig. 2C). At 48 h and 96 h post-9.4 °C stress, the activities of SOD and LYZ were significantly lower in the LTS group compared with the CO group (P < 0.05). The MDA contents in the GIFT head kidney was significantly higher in the LTS group compared with the CO group at 24 h post-9.4 °C stress (P < 0.05) (Fig. 2D). 50 stress. At 6 h post-9.4 °C stress, no significant differences were detected in the RBA and PA of the macrophages between the CO and LTS groups ( Fig. 3). At 12 h post-9.4 °C stress, the RBA and PA decreased significantly (P < 0.05). This observation combined with the changes in SOD and LYZ activities and MDA content, indicated that 24 h under 96h-LT 50 stress may be an important threshold for the stress response of GIFT. Therefore, we used the head kidney tissues of GIFT at 24 h post-9.4 °C stress to construct the small RNA libraries and analyse the miRNA expression profiles.
The expression profiling of the miRNAs between the CO and LTS libraries revealed 394 miRNAs that were significantly down-regulated and 189 miRNAs that were significantly up-regulated at 24 h post-9.4 °C stress. Based on transcripts with at least 100 reads in each of the libraries 35 , we identified 17 differentially expressed miRNAs, of which nine were up-regulated and eight were down-regulated (Table 3). We used qRT-PCR to validate these 17 miRNAs and found that their expression patterns were consistent with the results obtained by high-throughput sequencing (Fig. 6).  50 stress. *Indicates significant differences (P < 0.05) between values obtained pre-and post-9.4 °C stress, by paired-samples t-test. Diverse lowercase letters show significant differences (P < 0.05) between the control group and 9.4 °C-stressed group at each sampling point, by Duncan's multiple range test.    Prediction of target genes and associated regulated pathways. To determine the possible functions of the differentially expressed miRNAs associated with cold stress, we first predicted their target genes using TargetScan 5.2 and the miRanda v3.3a toolbox. We used the gene ontology (GO) database to annotate the predicted target genes 34 . Then, we constructed a heat map based on the 12 most enriched biological pathways at 24 h under 96h-LT 50 stress, which showed that most of the known differentially expressed miRNAs were predicted to be regulate one or more genes involved in the cold-stress response, including mainly lipid metabolic processes, response to temperature stimulus, response to oxidative stress, and cellular response to light stimulus (Fig. 7). 50 stress by qRT-PCR. The miR-10, miR-106a, and miR-143 expression levels first increased and then decreased under 96h-LT 50 stress (Fig. 8). In the LTS group, the miR-10 expression level reached a peak value at 48 h and, and was significantly lower at 96 h than at 24 h and 48 h (P < 0.05). The miR-106a expression levels were significantly higher for the LTS group than for the CO group from 2 h to 24 h under 96h-LT 50 Table 3. Differentially expressed miRNAs in GIFT head kidney between the control group (CO group) and 9.4 °C-stressed group (LTS group). These 17 miRNAs had transcripts with at least 100 reads in each of the libraries. no significant difference between the LTS and CO groups (P > 0.05). Also, the miR-122 levels in the LTS group were significantly decreased at 12 h, 24 h and 96 h compared with the CO group (P < 0.05). Based on the O. niloticus transcriptome sequence data, the GO and KEGG annotations, and the miRNA and mRNA expression patterns 36 , we screened and identified potential target genes of miR-10/106a/143/29a/192/462/122, which were related with cold stress. We detected negative expression levels between the following miRNA-mRNA pairs: miR-10-sirtuin 1 (SIRT1), miR-106a-thiopurine S-methyltransferase (TPMT), miR-143-protein phosphatase 1 regulatory subunit 12B (PPP1R12B), miR-192/462-muscleblind-like protein 1 (MBNL1), and miR-29a/122stearoyl-CoA desaturase (SCD). The expression levels of SIRT1, TPMT, and PPP1R12B were significantly lower in the LTS group compared with the CO group at 12 h and 24 h, whereas the expression levels of MBNL1 and SCD were significantly higher in the LTS group compared with the CO group (Fig. 9). However, at 96 h, the expression levels of SIRT1 and SCD showed no significant differences between the LTS and CO groups, whereas the expression level of MBNL1 was significantly lower than its expression level in the LTS group at 12 h and 24 h.

Discussion
MiRNAs play important roles in regulating gene function, and the systematic identification of miRNAs and their abundance is a basis for their functional analysis. Until now, there are no reports of miRNA analysis in tilapia exposed to cold stress. Our small RNA sequencing data revealed that the 21-23 nt long reads accounted for more than 65% of the total small RNAs in the CO and LTS libraries. The proportion of common reads between the two libraries was relatively high, indicating good overall consistency in both libraries. We identified a large number of conserved miRNAs (1736 and 1481 in the CO and LTS libraries, respectively) and some novel miRNAs (164 and 152 in the CO and LTS libraries, respectively) in the head kidney of GIFT, suggesting that different species have their own sets of specific miRNAs and conserved miRNAs, and confirming that miRNAs have a faster rate of evolution than other RNA genes 37 .
Aquatic animals depend on immune regulation and cellular response to adapt to changes in environmental temperature within a certain range 5 . The miR-10 family, and miR-181a, miR-181a-5p, and let-7 are essential in mediating cell proliferation and differentiation. We found that the expression levels of members of the miR-10 family were high in the CO and LTS libraries. Several studies have shown that the miR-10 family is highly conserved among different species. Members of the miR-10 family play important regulatory roles in the development of some tumours. For example, the expression levels of miR-10b-5p and miR-10b-3p were significantly higher in the brain tissue of Huntington chorea compared with normal brain tissue 38 . The expression levels of miR-10a and miR-10b were significantly increased in invasive pituitary adenomas and were closely related to the invading degree 39 . The expression of miR-10b was positively correlated with tumour diameter of pituitary adenomas 39 . However, the role of the miR-10 family has been little reported in fish. Our high-throughput sequencing and qRT-PCR results show that, at 24 h post-9.4 °C stress, there were significant effects on the miR-10, miR-10d, and miR-10c expression levels in GIFT head kidney, indicating that these miRNAs may be involved in cellular immunity and differentiation of the head kidney post-stress. We used bioinformatics software to predict the potential target gene for miR-10, and found that the up-regulation of miR-10 may inhibit the expression of SIRT1 mRNA. SIRT1 is a representative member of the histone deacetylase family and is involved in regulating cell metabolism, aging and apoptosis, and catalysing the deacetylation of histone, nuclear factor kappa B, and activating protein 1, thereby altering chromatin conformation, reducing transcription factor activity, and down-regulating the transcription of inflammatory genes 40 . RBA and PA have been used as the main indicators of a non-specific defence response in fish cells. In this study, the RBA and PA in head kidney macrophages of GIFT were significantly inhibited at 12 h post-9.4 °C stress. The immunosuppression induced by inhibition of SIRT1   Figure 8. Expression levels of selected miRNAs in the control group (CO group) and 9.4 °C-stressed group (LTS group) in GIFT head kidney for 96 h. *Indicates significant differences (P < 0.05) between values obtained pre-and post-9.4 °C stress, by paired-samples t-test. Diverse lowercase letters show significant differences (P < 0.05) between the CO and LTS groups at each sampling point, by Duncan's multiple range test. expression levels may have been caused by changes in cellular membrane mobility and stability of head kidney, as well as disrupting the physiological balance, which inhibited the phagocytic function of macrophages 41 .
Both miR-181a and miR-181a-5p were abundant in the two libraries. Li et al. 42 showed that miR-181a played an important role in T cell differentiation and T cell receptor signalling pathways. In the serum of dairy cows under heat stress, miR-181a was significantly down-regulated 43 . The predicted target gene of miR-181a was highly enriched in the T cell and B cell receptor signalling pathway, suggesting that miR-181a may regulate tumour necrosis factor and CD38, thereby intervening in the immunity of dairy cows. Post-9.4 °C stress, the up-regulation of miR-181a-3p may be related to the cellular inflammatory response and immunoregulation of head kidney, causing the immune levels of the GIFT to decrease at 24 h post-9.4 °C stress, and leading to the observed increase in the mortality of the GIFT at 48 h and 96 h post-9.4 °C stress.
Let-7 is highly conserved in vertebrate and invertebrates, including molluscs, foci, and zebrafish 44 . Let-7 regulates brain and nervous system development in mouse 45 , and plays an important role in heart development in vertebrates. Dicer is essential for the synthesis of let-7, and Dicer knockout mice exhibit cardiac hypertrophy and dysplasia, and die from 12.5 d to 14.5 d during embryonic development 46,47 . In zebrafish embryos, Dicer knockout impaired blood circulation 46 . Johnson et al. 48 found that let-7 was highly expressed in normal lung tissue, and that inhibition of let-7 increased the division of A549 lung cancer cells, whereas overexpression of let-7 in lung cancer cells affected cell-cycle progression and reduced cell division. These results suggested that the target genes of let-7 may be directly or indirectly related with cell cycle and division, thereby regulating the development of lungs. The up-regulation of let-7 in GIFT head kidney post-9.4 °C stress may alter the cellular stress response, resulting in some degree of adaptive regulation.
In mammals, miR-21, miR-139, and miR-106a are important biomarkers for cancer development [49][50][51] . Increased expression of miR-21 inhibited the tumour suppressor gene and increased the proliferation and invasion of prostate cancer cells 49 . Lu et al. 52 and Opstad et al. 53 found that miR-21 was an important regulator of delayed-type hypersensitivity and coronary artery disease, thereby regulating the interleukin-12/interferon-γ proinflammatory loop. Up-regulation of miR-139 inhibited chondrocyte proliferation and migration by regulating the mRNAs encoding eukaryotic translation initiation factor 4 gamma 2 and insulin-like growth factor 1 receptor, making miR-139 a possible therapeutic target for osteoarthritis 54 . The up-regulation of miR-21 and down-regulation of miR-139 may be associated with immunosuppressive and cell inflammatory responses in the GIFT head kidney cells after cold stress. miR-106a was found to modulate multidrug resistance in gastric, ovarian, and non-small cell lung cancers by regulating its respective target genes 51,55 . In the present study, up-regulation of miR-106a may inhibit the expression of its predicted target TMPT. TMPT is an intracellular enzyme that specifically catalyses the mercaptomethylation of heterocyclic and aromatic ring compounds 56 . TPMT is widely present in various tissues of the human body and has the highest activity in liver and kidney, and is also involved mainly in acute leukaemia and in the regulation of some immune diseases 56 . LYZ is an important component of the lymphocyte enzyme system in aquatic animals, and its activity reflects changes in the natural immune level of fish 57 . In this study, at 12 h post-9.4 °C stress, the down-regulation of TMPT mRNA levels may have an immunosuppressive effect on the adaptive adjustment of GIFT, and reduce the LYZ activity in head kidney, thereby resulting in increased mortalities.
Chu and Xu 58 reported that increased miR-192 levels reduced the abundance of interleukin 1 receptor type I, which was involved in inflammatory and immune responses in miiuy croakers (Miichthys miiuy) exposed to Vibrio anguillarum. miR-462 played a vital role in the signal pathway of hypoxia-responsive elements in blunt snout bream (Megalobrama amblycephala) under hypoxia stress 59 . In this study, the down-regulation of miR-192 and miR-462 may up-regulate the expression of MBNL1. MBNL1 can inhibit breast cancer metastasis, which is associated with reduced clinical metastatic relfront outcomes 60 . In addition, the loss of MBNL1 function was considered to have a key role in disease, and localized adeno-associated virus delivery of MBNL1, as well as MBNL1 overexpression partially rescued skeletal muscle pathology in mice with myotonic dystrophy 61 . Increased levels of MBNL1 mRNA may help to enhance the immunization ability of GIFT head kidney and improve survival at 24 h post-9.4 °C stress.
The head kidney of GIFT may be involved in "lipid metabolic process", and miR-130c, miR-29a, and miR-122, which were down-regulated in the LTS library, may be involved in the adaptive regulation of metabolic levels post-cold stress. When GIFT are exposed to water temperatures below the suitable temperature, internal body cold may inhibit the metabolic rate and peripheral blood flow, which could affect the integrity of tissue structure 62 . miR-130a/b were proposed as predictive biomarkers for metabolic disorders and could reflect the physiological status of cells and organs 63 . Wang et al. 64 suggested that miR-130b may be involved in the pathogenesis of metabolic diseases in mouse models of obesity and in human. In addition, up-regulation of miR-130a/b was found to suppress peroxisome proliferator-activated receptor expression in the rat liver fibrosis model, and it increased the expression of extracellular matrix mRNA 64 . miR-29a and miR-122 were predicted to be involved in regulating SCD expression by bioinformatics analysis. In our previous study, inhibition of miR-29a stimulated SCD expression in GIFT fed a saturated fatty acid diet, and increased the conversion of 16:0 and 18:0 to 16:1 and 18:1 21 . After silencing of miR-122 in mouse liver, plasma triglycerides and cholesterol were significantly decreased and fat deposition was reduced 65 . Post-9.4 °C stress, down-regulation of miR-122 and miR-29a may promote the expression of SCD mRNA and increase the conversion from saturated fatty acids to monounsaturated and polyunsaturated fatty acids, which would help to maintain cell membrane fluidity in head kidney macrophages, and mitigate cell damage caused by the risk of rigidity 2 .
However, in our study, the expression pattern of SCD mRNA at 96 h post-9.4 °C stress was different from the patterns observed at 12 h and 24 h post-9.4 °C stress. This may be because many miRNAs have a common target gene, and inhibition of one miRNA may be compensated by other miRNAs, resulting in adaptive regulation 66 . In addition, the longer stress time of 96 h caused inhibition of metabolism and immune regulation, and increased cell membrane damage. The loss of cellular homeostasis is generally accompanied by the formation of ROS. Increased amounts of ROS can cause changes in the unsaturated/saturated fatty acids ratio of the phospholipid bilayer of the cell membrane and increase the production of lipid peroxides 2 . This was reflected in the increased MDA content and decreased SOD activity in GIFT post-9.4 °C stress. We found that "regulation of oxidoreductase activity" and "cellular response to oxidative stress" were associated with the important antioxidant regulatory pathway of GIFT post-9.4 °C stress. Zhao et al. 67 found the miR-143 expression level was stimulated during oxidative stress in vascular smooth muscle cells, and up-regulated miR-143 was found in cardiomyocytes treated with H 2 O 2 68 . In this study, the up-regulation of miR-143 may inhibit the expression of PPP1R12B. PPP1R12B has been reported to be an insulin regulating phosphatase subunit 69 . The lipids of head kidney macrophages in fish were rich in phospholipids 70 . Therefore, inhibition of PPP1R12B in head kidney of GIFT may be involved in the regulation of phosphorylase/phosphokinase on cell membranes. Juvenile GIFT under 96h-LT 50 stress may not be able to produce the adaptive regulation response, which could have caused free radical metabolic disorders and lipid metabolic abnormalities, and disrupted oxidation-antioxidation homeostasis. Two miR-126 target sites (miR-126a/b) have been detected in the zebrafish genome, and they act synergistically to regulate angiogenesis and maintain vascular integrity 71 . Head kidney tissue is an important haematopoietic organ in fish, and its integrity may help to maintain homeostasis. At 24 h post-9.4 °C stress, the inflammatory response and lipid peroxidation may damage vascular endothelial cells and vascular integrity, leading to up-regulation of miR-126a expression levels. Olena et al. 72 found that inhibition of miR-216a could up-regulate sorting nexin 5 to modulate Notch signalling during zebrafish eye development. In this study, "response to light stimulus signal pathway" was enriched by cold stress. Down-regulation of miR-216a may interfere with the sensitivity of GIFT to light or cause eye damage and reduce balance capacity, which may explain the accumulation of GIFT at the bottom of the tank after exposure to 96h-LT 50 stress. We could not find any reports about the possible roles of miR-9312 and miR-6596-5p. The heat map analysis showed that these two miRNAs may be associated mainly with "response to light stimulus" and "RNA processing" pathways. In future experiments, we plan to study the signal pathways that involve the target genes of these two miRNAs.
In conclusion, we first analysed the physiological responses and miRNA profiles of GIFT head kidney under 96h-LT 50 cold stress. Seventeen differentially expressed miRNAs were found between the CO and LTS libraries, including nine up-regulated and eight down-regulated miRNAs. Five potential target genes were identified and their functional characteristics showed they were involved mainly in signalling pathways, including "lipid metabolic processes", "response to immune regulation and cellular response", and "response to oxidative stress". The 96h-LT 50 cold stress may have led to free radical metabolic disorders as a result of excessive oxidative stress, increased MDA content, and lipid metabolism dysfunction, resulting in imbalance of the antioxidant system. In addition, the immune responses of GIFT head kidney cells were inhibited, and the LYZ activity, and RBA and PA were significantly decreased under 96h-LT 50 cold stress, eventually leading to increased mortality. In future studies, we will further investigate the relationship among the GIFT miRNAs and their target genes, and use a proteome profiler to better understand the molecular regulatory networks associated with the cold-stress response. Furthermore, the tolerance of fish to cold stress may be different at different developmental stages. Therefore, we will study the biological response characteristics of cold-stressed fish at different developmental stages to obtain more information about the adaptation and regulation mechanisms in teleost.