The channel catfish exhibits a remarkable degree of adaptability and is extensively cultivated on a global scale. Its genome has been meticulously sequenced, assembled, and analyzed by Liu et al. [20, 21]. Further, Ju et al. have conducted analyses on the cerebral transcriptomic data of this species [22], while Zeng et al. have explored the differential transcriptomic profiles in gonadal gene expression between male and female channel catfish at 90, 100, and 110 days post-fertilization [23]. By employing RNA sequencing (RNA-seq) technology, we scrutinized the transcriptomic disparities in hepatic tissue, cephalic renal tissue, and splenic tissue between infected and sham-infected groups of channel catfish. This endeavor facilitated the identification of an extensive array of differentially expressed genes. The findings from this comparative analysis provide a foundational basis for an intricate comprehension of the molecular dynamics underpinning antimicrobial activity in channel catfish.
In the present study, 4,400, 6,339 and 4,615 DEGS genes were identified in liver, spleen and head kidney treated with A. hydrophila infection, respectively. The number of up-regulated and down-regulated genes in liver was 2329 and 2071, respectively. The number of up-regulated and down-regulated genes in spleen was 3293 and 3046, respectively. The number of up-regulated and down-regulated genes in the head kidney was 2539 and 2076, respectively. The number of up-regulated genes was greater than the number of down-regulated genes in all three experimental groups. Hybrid sturgeon infected with A. hydrophila had 2,723 differentially expressed genes detected in the spleen, including 1,420 up-regulated genes and 1,303 down-regulated genes[24]. The differentially expressed genes in the head kidney of goldfish infected with A. hydrophila for 3 and 7 days were 4638 and 2580, respectively[25]. 4413 differentially expressed genes were identified in Pacu's liver after A.hydrophila infection, including 2000 up-regulated expressions and 2413 genes down-regulated expressions[26]. In this study, the number of differentially expressed genes in the liver, spleen, head and kidney differed significantly from that of other fish species.
The transcriptome of multiple tissues of channel catfish infected with A. hydrophila was studied for the first time.We focused on the expression of genes associated with inflammation and immunity. KEGG metabolic pathway analyses showed that DEGs related to inflammation and immunity in the liver, spleen and head kidney was mainly enriched in metabolic pathways such as MAPK signaling pathway, Cytokine-cytokine receptor interaction, Salmonella infection, NOD-like receptor signaling pathway, Neuroactive ligand-receptor interaction, and Endocytosis.
JNK and p38
The mitogen-activated protein kinase (MAPK) signaling cascade mediates a plethora of cellular physiological processes including proliferation, ontogeny, differentiation, and programmed cell death [27]. Distinct loci within the MAPK pathway can precisely detect and respond to pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs), thereby initiating a MAPK cascade reaction that bolsters immune defenses against bacterial encroachment [28]. This signaling network predominantly comprises four principal branches: extracellular signal-regulated kinases (ERK), c-Jun N-terminal kinases (JNK), p38/MAPK, and ERK5. Notably, JNK and p38 pathways share functional similarities, being implicated in inflammation, apoptosis, and cellular growth. The activation of the JNK pathway can be triggered by diverse stimuli including cytokines, endotoxins, free fatty acids, and metabolic perturbations. In Penaeus vannamei, the JNK gene contributes to the immunological counter-response to white spot syndrome virus (WSSV) through the modulation of antimicrobial peptide synthesis [29]. In Crassostrea hongkongensis tissues subjected to infection by Vibrio alginolyticus or Staphylococcus haemolyticus, ChJNK mRNA levels were significantly upregulated [30]. Additionally, p38 mRNA expression was notably enhanced in peritoneal cells of sea cucumbers upon in vivo infection with Vibrio splendens [31]. Furthermore, over expression of the p38α gene in Epinephelus punctatus infected with Singapore grouper iridovirus (SGIV) has been demonstrated to repress viral gene transcription and protein biosynthesis [32]. In this study, more DEGs were found in the liver, head kidney and spleen of channel catfish after A. hydrophila infection, and the number of up-regulated expressed genes was greater than the number of down-regulated expressed genes (Table 4). In the present investigation, tumor necrosis factor (TNF), interleukin-1 (IL1), TNF receptor (TNFR), and p38 exhibited upregulation in the hepatic, cephalic renal, and splenic tissues of channel catfish following A. hydrophila infection. Conversely, c-Jun N-terminal kinase (JNK) expression was elevated in the cephalic kidney but remained unaltered in both hepatic and splenic tissues. These findings imply that the MAPK signaling cascade, along with its downstream pro-inflammatory mediators, may play a pivotal role in orchestrating the immune response to A. hydrophila infection within channel catfish. The specific immunomodulatory mechanisms elicited by the MAPK pathway across varied tissue types warrant further elucidation.
Interleukins and interleukin receptors
Cytokines constitute a category of small molecular weight polypeptides or glycoproteins that are integral to autocrine, paracrine, and endocrine signaling. They serve as pivotal mediators in the coordination of immune system development and activity [33, 34]. Based on receptor architecture, specificity, and molecular composition, cytokines can be categorized into distinct classes including interleukins (ILs), interferons (IFNs), tumor necrosis factors (TNFs), chemokines, and colony-stimulating factors (CSFs)[35]. Interleukins play an important role in regulating cell proliferation and differentiation, maintaining the normal function of the immune system, and participating in the inflammatory response of the body after pathogenic infections. In recent years, there has been an increasing number of studies on fish interleukins. In this study, hepatic interleukins (IL6, IL11, IL12, IL1β) and interleukin receptors (IL12Rβ1, IL12Rβ2, IL1R1, IL1R2, IL2RG, IL4R, IL15RA, IL21R, IL4R, IL13RA1, and IL10RA) were up-regulated and expressed in the liver after A.hydrophila infection. Interleukins (IL6, IL11, IL12, IL27α, IL10, IL20, IL22) and interleukin receptors (IL6R, IL12Rβ1, IL12Rβ2, IL1R, IL1R2, IL2RG, IL4R) were up-regulated and expressed in the spleen. Interleukins (IL6, IL11, IL10, IL20, IL1β) and interleukin receptors (IL6ST, IL12Rβ1, IL10RA, IL1R1, IL1R2, IL17RA, IL17RC) were up-regulated for expression in the head kidney. The results suggest that these interleukins play a role in the resistance of channel catfish to bacterial infection. Channel catfish employed these interleukins to defense against the infection. At the same time there are some differences in the interleukins functioning in different tissues. IL34, a new cytokine originally discovered in humans, has now been identified in fish such as pufferfish, grouper, greater amberjack and Atlantic salmon[36]. Research has shown that IL-34 is involved in the regulation of many serious health disorders such as metabolic diseases, heart disease, infections and even cancer[37].The use of IL-34 as an immune adjuvant had a positive effect on improving the relative survival of largemouth bass[36]. In this study, IL34 was upregulated in liver, approved which, and head kidney. The mechanism of IL34 regulation in response to bacterial infection in the Channel catfish needs to be further investigated.
Pattern recognition receptor
Pattern recognition receptors (PRRs) are important components of the body's innate immune system and are widely distributed in cell membranes, endosomal membranes, lysosomal membranes and cytoplasm.The PRR consists mainly of Toll-like receptors (TLRs), retinoic acid-inducible gene-I(RIG-I)-like receptors (RLRs), nucleotide oligomerization domain(NOD)-like receptors(NLRs), C-type lectin receptors(CLRs) and intracellular nucleic acid receptors[38]. In this study, 55, 67 and 54 differentially expressed genes of NLRs were found in liver, spleen and head kidney, respectively, and the number of up-regulated expressed genes was significantly more than the number of down-regulated expressed genes. Up-regulated expression of NOD1, NOD2, NLR-C3 and NLR-C5 in the intestines of the channel catfish after infection with Edwardsiella tarda, A. hydrophila and Streptococcus spp[39]. In this study, NOD1 and NLRP3 were upregulated in liver. NOD2 upregulated in spleen. NLRP12 upregulated in head kidney. It is likely that NLRs in different tissues respond to external stimuli through different immune modalities[40].
More than 20 Toll-like receptors have been identified in scleractinian fish, of which TLR(5s, 14, 18, 19, 20, 25, 27, 28) are scleractinian-specific TLRs[41]. TLR4 could recognises Aeromonas hydrophila and LPS stimuli, prompting relevant immune responses in some Cyprinidae fish[42]. TLR5 could recognises bacterial flagella[43]. As detected in this study, TLR5 was up-regulated and TLR7/8 was down-regulated in the liver. TLR2, TLR4, TLR5, TLR6 were upregulated and TLR7/8 and TLR9 were downregulated in spleen. TLR5 and TLR6 were upregulated and TLR7/8 and TLR9 were downregulated in the head kidney.Based on the results we hypothesised that the TLRs of channel catfish are able to receive bacterial stimulation signals to activate NF-kb.