Indian Journal of Animal Research

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Full Research Article

Comparative Transcriptomics-based Analysis of Common Carp (Cyprinus carpio) Kidney Tissues Reveals Differential Immune Responses at Two Temperatures

Nishita Chauhan1,2, Vindhya Mohindra1,*, Labrechai Mog Chowdhury1, Alisha Paul1, Rajesh Kumar1, Mohammad Imran2, Ajay Kumar Singh1, Joykrushna Jena3
1ICAR-National Bureau of Fish Genetic Resources, Lucknow-226 002, Uttar Pradesh, India.
2Institute of Bio-sciences and Technology, Shri Ramswaroop Memorial University, Barabanki-225 003, Uttar Pradesh, India.
3Indian Council of Agricultural Research, New Delhi-110 012, India.

ABSTRACT

Background: Common carp is the third most widely farmed fish, globally. It is tolerant to cold stress and resistant to EUS  pathogenic infections, when it faces lower temperatures. In the present study, common carp was used as the research model to examine the interplay of temperature and immunity.

Methods: High-throughput transcriptome sequencing was used to study the differential gene expression profiles of kidney tissues, reared at two different temperatures; 30oC (control) and 20oC (experimental).

Result: Out of 588 differential expressed genes (DEGs), genes under GO terms for abiotic stress were categorised under oxidative stress, response to heat, mechanical stimulus and cold. Fifteen DEGs were found under the immune category, which out of which twelve were under the innate immune category, included 5 lectin family members and 1 tumor necrosis factor. The information generated in common carp would form a transcriptome resource for tolerance to abiotic stress. At the lower temperature studied, modulatory immune response, through the cytokine-cytokine interaction pathway, seems to be a major player, besides higher energy production, up-regulated stress responses, cell growth and survival. These results have the potential to be developed into bio-markers for temperature stress tolerance and modulatory immune responses in aquaculture pond production during the lower temperature conditions.

INTRODUCTION

Fishes frequently encounter changes in environmental conditions, which disrupt the balance between fish physiology and their surroundings and trigger a stress response. Temperature has a critical role in biological processes of fish (Gonen et al., 2024) and due to the stress of regular and/or seasonal temperature changes, modulatory immune responses have evolved in fish occupying specific temperature niches (Goldspink et al., 1995).
       
Common carp (Cyprinus carpio) is cultured across the globe in Asian and some European countries, ranging from temperate to tropical regions. And unlike other cyprinids carps, the common carp exhibits optimal growth between 20 and 30oC, making it well-suited for aquaculture due to their adaptable characteristics (Billard, 1995). It was observed that during the winter season, when the water temperature falls between 18 and 22oC, Epizootic Ulcerative Syndrome (EUS) infection is common in many fish species, as the host immunity gets suppressed in the susceptible hosts (Kumar et al., 2020). However, common carp is recognised as a species naturally resistant to this disease (OIE, 2019). Pradhan et al. (2014) also reported that EUS pathogen was not found in common carp fry and larvae, even in the presence of other fish species getting infected. To study the innate immunity mechanism in common carp at this range of temperature, 20oC was chosen as one of the temperatures for the present study. The kidney, particularly the head kidney/anterior kidney, acts as the principal immune organ, involved in various immune functions (Deivasigamani, 2006; Sivasanker et al., 2020).
       
Advancements in high-throughput transcriptome sequencing technology have led to the studies of global gene expression profiles in various fish species, e.g. in cold susceptible species (Tilapia) for immune and metabolic responses (OuYang et al., 2023) and immunity related gene expression and in cold resistant species for the role of cold-adaptive responses (common carp) (Long et al., 2020). Since the ability of change in mRNA expression levels determines the immunological response to environmental stress, RNA sequencing has been frequently used for relevant investigations in fish (Wang et al., 2020, Xu et al., 2023) and other animals (Tianjiao et al., 2023).
       
The present study aimed to identify comparative large-scale transcriptome responses, especially those involved in immune system in C. carpio, during low temperature acclimation (20oC) as compared to that at 30oC, which would be useful to reveal the mechanism of disease avoidance/resistance, at the varying temperature conditions.

MATERIALS AND METHODS

Experiment design
 
C. carpio specimens (16-19 cm, 91-133 g) were collected from the fish germplasm resource centre of ICAR-NBFGR, Lucknow, India from the progeny of commercial breeding of the same brood stocks, in July month. Fish were randomly divided into two groups in circulatory water fibre-reinforced plastic tanks, each with 500 L of freshwater, with continuous aeration and photoperiod of ten hours’ light, pH: 7.0±1, DO 4.5 to 5 mg/L. After the acclimation period at room temperature, step-cooling/heating (1±0.2oC/day) was carried out to 20±1oC (experimental)/30±1oC (control) and maintained for 45 days, in three replicates of 10 fishes each, with the same feeding regime and husbandry conditions. One day before sample collection, feeding was stopped.
 
Sample collection and RNA isolation
 
After the temperature exposures, fishes were euthanized with MS222 (Sigma Aldrich, USA) and kidney samples of five random biological replicates of each group were collected in RNAlater (Ambion, USA), snapped frozen in LN2 and stored at -80oC. Total RNA from kidney tissues were extracted based on the TRI-Reagent method (Rio et al., 2010). RNA Integrity Number (RIN) of purified RNA was determined by Agilent Tape station (Agilent Technologies, USA) and libraries were prepared from RNA from five biological replicates of each group, using an Illumina Paired-end library sequencing kit (Illumina Inc., USA).
 
Transcriptome sequencing and assembly
 
Libraries were sequenced, through Illumina HiSeq2500 (Illumina Inc., USA) and the raw fastq reads were preprocessed and quality assessed using FastQC and MultiQC (Ewels et al., 2016). The trimmed fastp reads were filtered for rRNA reads using bowtie2 v2.4.2 (Langmead and Salzberg, 2012) and de-novo assembled by Trinity v2.13.2 (Grabherr et al., 2011). The assembled transcripts were further clustered using CD-HIT-EST (Huang et al., 2010) and filtered by CPAT (Wang et al., 2013) for protein-coding sequences extraction and long non-coding RNAs (lncRNAs) removal. Completeness of final transcriptome was assessed against Actinopterygii, Vertebrate and Eukaryote core genes databases, through BUSCO Orthology pipeline, embedded in gVolante (https://gvolante.riken.jp/).

Differential gene expression (DGE) estimation and their functional annotations
 
The final set of transcripts was quantified using RSEM v1.3.3.4 (Li and Dewey, 2011). The resulting abundance tables were imported into R using the bioconductor package ‘tximport’. The ‘regularized log’ transformation in DESeq2 (Bray et al., 2016) was used for principal component and clustering analysis. Differential expression analysis and normalizations were performed using DESeq2, p-value<0.05 with Log2FoldChange (Log2FC) >2.
       
The DEGs identified were analyzed for gene ontology (GO) terms associated with the annotated genes and molecular pathways for the mapped genes using DAVID v6.8 (https://david.ncifcrf.gov/). The genes under GO terms responses to abiotic stress (GO:0009628), metabolic process (GO:0008152) and signal transduction (GO:0007165) were identified using QuickGO: Annotation List (https://www.ebi.ac.uk/QuickGO/ annotations). The deciphering of network of immune genes and visualization of the functional grouping was according to the GO term and KEGG database (http://www.genome.jp/kegg/kaas/) with reference to zebrafish (p<0.05).
 
Validation of immune genes expression through quantitative real time PCR (qRT-PCR) analysis
 
The gene-specific PCR primers of housekeeping and target genes were designed for qRT-PCR analysis from the present study (Table 1). For cDNA preparation from the RNA samples, first strand cDNA was synthesized using Revert Aid H Minus First strand cDNA Synthesis Kit (Thermo Scientific, USA) using 1 mg of RNA, following standard protocols and qPCR analysis according to Mohindra et al. (2022). Primer efficiency (E) for these genes ranges from 1.9-2.5 and the specificities of the PCR amplifications were confirmed by melting curve analysis. The Ct values were derived using Light Cycler (TM)480 (Roche Diagnostic International Holding AG, Switzerland) software. Reference genes expression was evaluated with RefFinder (Xie et al., 2023) and fold changes of gene expression at 20oC in comparison to control (30oC) were calculated using the 2DDCt method, through Excel.

Table 1: Primers list of target and housekeeping genes validated through qRT-PCR identified in differentially expressed genes of Cyprinus carpio kidney tissue exposed to 20oC and 30oC temperatures.

RESULTS AND DISCUSSION

Transcriptome assembly and functional annotation
 
In the present study, kidney transcriptome generated from two different temperature conditions represented a complete complement of expressed genes, including the major immune genes as well as the important pathways. A total of 84.47 Gb reads were generated and after trimming and removal of contaminants, high-quality paired-end reads and clustering resulted in 1.275 million transcripts, which confirmed 98.60% completeness of the transcriptome.
 
DEGs and analysis for functional enrichment
 
Differential gene expression analysis of common carp kidney tissues from 20oC experimental temperature versus 30oC water temperature (control) conditions revealed 588 DEGs and 253 and 335 genes were found upregulated and downregulated, respectively. Under biological processes, most significant GO terms (FDR<0.05) were DNA integration (GO:0015074), followed by transmembrane transport (GO:0055085) and intracellular signal transduction (GO:0035556), while under molecular functions, GO:0003676-nucleic acid binding followed by GO:0005509-calcium ion binding and GO:0005085-guanyl-nucleotide exchange factor activity and under Cellular component, GO:0016020-membranes and GO:0005886 plasma membrane.
       
Molecular pathway analysis of these DEGs identified 118 genes in Organismal Systems, with a maximum number of 41 genes under immune system; 76 in environmental information processing with 57 genes under signal transduction, 29 in genetic information processing with 9 genes under folding, sorting and degradation and 59 genes under metabolism with 16 genes under amino acid metabolism. A total of 56 genes were classified under cellular processes, with 26 genes under transport and catabolism.
       
Under pathway analysis, maximum DEGs were observed in signal transduction (57) and signalling molecules and interaction (28) under environmental information processing, followed by endocrine system (47) and immune system (41) under organismal systems. Under signal transduction and signaling molecules and interaction, maximum number of DEGs was observed in Calcium signaling pathway [PATH: ko04020], MAPK signaling pathway [PATH: ko04010], Apelin signaling pathway [PATH: ko04371], cAMP signaling pathway [PATH: ko04024], PI3K-Akt signaling pathway [PATH:ko04151]  with DEGs 13, 13, 8, 8 and 8, respectively. Under metabolism, pathways of Arginine and proline metabolism [PATH: ko00330] and Purine metabolism [PATH: ko00230]   contained 5 and 4 DEGs, respectively; and under cell growth and death category, apoptosis [path: ko04210] 6 DEGs. Thus, the present study revealed an increase in purine, arginine and proline metabolisms, which may play a vital role in countering cold stress. These pathways were reported to be involved in energy production and mitigating the effects of cold stress (Melis et al., 2017, Xu et al., 2018) and the results are in line with the earlier studies that fish tolerate cold temperature stress by regulating its metabolism in response to increased energy demand (Schleger et al., 2021). Since the current study found that the cold temperature had no effect on the glycolysis or glycogenesis pathways, the genes involved in the purine, arginine and proline metabolism pathways appear to be important for energy production and to combat cold stress in common carp.
       
Molecular pathways identified under immune system category of organismal systems identified 40 DEGs, out of which 7 genes were under complement and coagulation cascades pathway, Chemokine signalling pathway and Platelet activation followed by 5 genes each under B cell receptor signaling, C-type lectin receptor signaling and T cell receptor signaling pathways and 5 genes under IL-17 signaling pathway (Table 2).

Table 2: Molecular pathways identified under Immune system category of total differentially expressed genes in kidney tissue transcriptome of Cyprinus carpio during exposure to two temperatures, 20oC vs 30oC (p<0.05).


 
DEGs classification under abiotic stress
 
With reference to Danio rerio database,  5 GO terms with 22 DEGs were identified, with 10 DEGs under response to oxidative stress (GO:0001666, GO:0055093, GO:0071456), followed by 4 under response to heat (GO:0009408, GO:0034605), 5 under response to mechanical stimulus (GO:0009612, GO:0071260) and 2 under response to cold (GO:0009409, GO:0070417) (Fig 1). The most significant pathways identified were MAPK signalling, Focal adhesion and necroptosis for these genes. The stress-regulated  MAPK signaling pathway mainly involves kinases, which are reported to be mainly the mitogen-activated protein kinase (MAPK) family members (Cross et al., 2000),  that is involved in numerous significant cellular functions, including proliferation, differentiation and migration of cells (https://www.kegg.jp/pathway/hsa04010).. Interesting points of concern are significant upregulation of genes: Mitogen-activated protein kinase 4 (MAP4K4), cytosolic phospholipase A2 (cPLA2a) and Thrombospondin-1 (tsp-1). Up-regulation of MAP4K4 gene is indicative of positive stress response (Singh et al., 2023) andcPLA2a activation in renal epithelium has been suggested to promote renal repair by initiating partial de-differentiation, proliferation and migration (Montford et al., 2016). and Third up-regulated gene under abiotic stress under the present study, that is tsp-1 under colder conditions, which is a component of the PI3K-Akt signaling pathway,  has been reported with a role in the immune system regulation (Bao et al., 2022) and in adaptation to varying temperatures (Zhang et al., 2021).

Fig 1: Temperature related DEG’s identified under Gene Ontology term (GO:0009628) responses to abiotic stress of Cyprinus carpio kidney tissue exposed to 20oC and 30oC temperatures.


 
Differentially expressed immune and related genes
 
Molecular pathways identified under immune system category of organismal systems identified 40 DEGs, out of which 7 genes were under complement and coagulation cascades pathway, Chemokine signaling pathway and Platelet activation followed by 5 genes each under B cell receptor signaling, C-type lectin receptor signaling and T cell receptor signaling pathways and 5 genes under IL-17 signaling pathway (Table 2). Out of 40 DEGs, 15 were immune genes, majority (12) under innate immune categorywhile only 3 adaptive immune DEG were identified  (Table 3). These genes fall under 9 GO terms, of which under biological process, immune response (GO:0006955); under cellular component under membrane (GO:0016020); under molecular function signaling receptor activity (GO:0038023), carbohydrate binding (GO:0030246) and GTP binding (GO:0005525) (Fig 2).  Three DEGs were found under under cytokine-cytokine receptor interaction (CCRI) pathway and 2 each under Chemokine signaling pathway and Viral-protein interaction with CCR (Fig 3). In the current study, these results involving DE immune genes point to highly significant groups, with the largest number of genes are under Response to stimulus and CCRI pathways (Fig 4), which are interlinked and share most of the associated DEGs. CCRI, one of the vital immunity pathways, has a crucial role in the inflammatory and defense process of the host (Cheng et al., 2017).  In an earlier study in transgenic zebrafish under cold stress (Wang et al., 2014), where CCRI genes were mostly down-regulated. However, in the current study, up-regulated CCRI genes (ccr1, 2) at 20oC might be providing the innate immune response in common carp. Verma et al., (2021) also reported the chemokine signaling pathway to be a critical pathway, with the innate immune gene ccr2 up-regulation in the early and middle stage infection of oomycete, A. invadans in common carp. It is fascinating to find in the present study, a very high expression of another innate immune gene, complement component 7b, identified under C-C chemokine binding, which performs function in host defence against pathogens and promotion of inflammation through complement and coagulation pathway (Bossi et al., 2009). Thus, the relative higher expression of the immune genes indicates enhanced pathogen defense (Philominal et al., 2025).

Table 3: Significant Immune differentially expressed genes list in kidney tissue of Cyprinus carpio during exposure to two temperatures, 20oC vs 30o (p<0.05).



​

Fig 2: Gene Ontology terms identified in differentially expressed immune related genes in kidney tissue of Cyprinus carpio during two temperatures, 20oC and 30oC.



​

Fig 3: Significant molecular pathways identified in immune and immune related differentially expressed genes of Cyprinus carpio kidney tissue exposed to 20oC and 30oC temperatures.



​

Fig 4: Molecular pathway Cytokine-cytokine receptor interaction identified in differentially expressed immune related genes of Cyprinus carpio kidney tissue exposed to 20oC and 30oC temperatures [20±1oC (experimental group)/30±1oC (control group)].


 
Validation of expression pattern of immune genes
 
Out of the 11 genes, rps20, hprt, eif5a, rlp7, b-actin, 18s-1, myb-2, anxa1a, errif1a, kat2b-1 and ep300a-1 tested for their stability in expression, eif5a was found to show most stable expression, so it was utilized as the house keeping gene, for normalization of Ct values of target genes. The target genes, ccr2, si:dkey-241l7.3, cc7 and ifi44/loc795887 were tested and all these genes showed similar trend in the expression patterns for transcriptome and qRT-PCR experiments, except ccr7 at 20oC.
       
Thus, the present transcriptomic study points out that cold resistant common carp can modulate the cold stress positively and also its immune responses, which may facilitate the resistant responses to the disease, to which cold susceptible species succumbed to. However, deep studies are required for the combinations of these factors and processes, to reveal overall modulatory effects.

CONCLUSION

The information generated in common carp, a cold resistant species, in the present study, would form a transcriptome resource for tolerance to abiotic stress, along with information on possible modulatory mechanisms, when it encounters lower temperature conditions. These strategies include arginine and proline metabolism essential for energy production and thus mitigating the effects of cold stress. Other important mechanisms to overcome temperature stress response were by MAP4K4 signal transduction and PI3K-Akt signaling pathways. At a low temperature, upregulation of most genes of innate immune response in the cytokine-cytokine interaction seem to be a major player. These results have the potential to be developed into biomarkers for temperature stress tolerance and modulatory immune responses in common carp in aquaculture pond production during the lower temperature conditions. However, in-depth protein studies are needed to support the results obtained through present transcriptome studies.

ACKNOWLEDGEMENT

Authors are grateful to the Director, ICAR-NBFGR, Lucknow for providing facilities for this work. This work was carried out under ICAR-NBFGR component of ICAR-Consortium Research Platform (CRP) on Genomics, New Delhi, India and financial assistance by ICAR-CRP Genomics is duly acknowledged. Dr. Rajeev K. Singh and Dr. B. Kushwaha are acknowledged for their support.
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
 
Informed consent
 
All the protocols followed were approved by the Institute Animal Ethics Committee (IAEC), ICAR-NBFGR, Lucknow, India, vide G/IAEC/2022/6, Dated 25-05-2022. All the methods were performed in accordance with the relevant guidelines and regulations.

CONFLICT OF INTEREST

The authors declare that there is no conflict of interest.

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