Evolutionary, comparative, and functional analyses of STATs and regulation of the JAK-STAT pathway in lumpfish upon bacterial and poly(I:C) exposure

Background The Janus kinase/signal transducers and activators of transcription (JAK-STAT) system regulates several biological processes by affecting transcription of genes as a response to cytokines and growth factors. In the present study, we have characterized the STAT genes in lumpfish (Cyclopterus lumpus L.), belonging to the order Perciformes, and investigated regulation of the JAK-STAT signaling pathway upon exposure to bacteria (Vibrio anguillarum) and poly(I:C), the latter mimicking antiviral responses. Methods Characterization and evolutionary analyses of the STATs were performed by phylogeny, protein domain, homology similarity and synteny analyses. Antibacterial and antiviral responses were investigated by performing KEGG pathway analysis. Results We observed that lumpfish have stat1a, 2, 3, 4, 5a, 5b, and 6. Transcriptome-wide analyses showed that most components of the JAK-STAT pathway were present in lumpfish. il-6, il-10, il-21, iκBα and stat3 were upregulated 6 hours post exposure (hpe) against bacteria while type I interferons (IFNs), irf1, irf3, irf10, stat1 and 2 were upregulated 24 hpe against poly(I:C). Conclusions Our findings shed light on the diversity and evolution of the STATs and the data show that the STAT genes are highly conserved among fish, including lumpfish. The transcriptome-wide analyses lay the groundwork for future research into the functional significance of these genes in regulating critical biological processes and make an important basis for development of prophylactic measure such as vaccination, which is highly needed for lumpfish since it is vulnerable for both bacterial and viral diseases.


Introduction
Cytokines promote communication and are important for a range of biological processes, including formation and regulation of immune cells and hematopoietic cells (Zou and Secombes, 2016).The cytokines bind to specific receptors on target cells, triggering a chain reaction of intracellular signaling processes controlled by the Janus kinase/signal transducers and activators of transcription (JAK-STAT) pathway (Morris et al., 2018).JAKs are a family of intracellular non-receptor tyrosine kinases, consisting of four members, jak1, jak2, jak3 and tyk2 (Bousoik and Montazeri Aliabadi, 2018).Activation of the JAKs causes phosphorylation and activation of STATs, which then translocate to the nucleus and bind to DNA regulatory sites to control gene expression (Seif et al., 2017).The JAK-STAT system is a flexible signaling pathway that permits different signaling molecules, such as cytokines, colonystimulating factors, and hormones, to control gene expression and have impact on a variety of cellular functions (O'Shea et al., 2015).While this pathway was initially identified during research on how interferons (IFNs) regulate gene expression (Levy et al., 1988;Schindler et al., 1992), it is now known also to be important for proper functioning of multicellular organisms, involved in various processes such as maintaining homeostasis, host defense, and cell growth both in humans and fish (Ward and Thompson, 2012;Jin et al., 2018).
Each STAT gene has an N-terminal domain (NTD), which takes role in dimerization, nuclear translocation, and protein interaction.They also have a coiled-coil domain, which aids in protein-protein interactions and receptor binding, a DNA binding domain (DBD), a linker region, an SH2 domain needed for receptor binding and dimerization, and a C-terminal transactivation domain (TAD) that regulates STAT transcriptional activity (Langenfeld et al., 2015).A conserved tyrosine residue is shared by all members of the family and is phosphorylated prior to dimerization (Hu et al., 2021), and most STATs have a second phosphorylation site, conserved serine residue, within a P(M)SP motif in the TAD domain (Decker and Kovarik, 2000).The STAT domains emerged at different stages of evolution, with the coiledcoil domain being observed more prominently in the phylum Chordata (Surkont and Pereira-Leal, 2015).
While different cytokines activate specific STATs, the degree of interactive promiscuity between cytokines and any given STAT varies with vertebrates (Skjesol et al., 2010), including fish.Scientists have made substantial progress in characterizing members of the STAT family, which include STAT1-6 (Xu et al., 2022).Fish also possess two homologues of the STAT1 gene, named stat1a and stat1b.The origin of this duplication event can be traced back approximately 35 million years, arising from a genome duplication event in fish.Notably, certain fish species, including salmonids, display an even greater number of stat1 gene copies, ranging from 2 to 5 copies (Skjesol et al., 2010).This highlights the diverse genetic landscape and complexity within the STAT genes across fish species (Volff, 2005;Skjesol et al., 2010;Song et al., 2011;Xu et al., 2022).Similarly, the STAT5 gene is comprised of two homologues, STAT5A and STAT5B, exhibiting 90% similarity at the amino acid level (Maurer et al., 2019).In mammals, these homologues are located adjacently on the same chromosome (Moll et al., 2019), while in fish, stat5a and stat5b are situated on separate chromosomes, despite high sequence similarity (Gorissen et al., 2011).
Cytokine binding to its cognate receptor induces conformational changes that activate the associated JAKs (Bousoik and Montazeri Aliabadi, 2018).Activated JAKs undergo autophosphorylation and/or transphosphorylation, and subsequently phosphorylating the cytokine receptors (Beirer and Hofer, 2006).The resulting phosphorylated receptor tyrosine motifs act as docking sites for the SH2 domains of STATs (Skjesol et al., 2014).Upon binding, the STATs become tyrosine phosphorylated, dissociate from the receptor, dimerize, translocate to the nucleus, bind to specific DNA elements in gene promoters, and activate transcription (Schindler and Darnell, 1995).The activation of the JAK-STAT pathway in zebrafish occurs when IFNs bind to their respective receptors (IFNRs), resulting in the transcription of IFNstimulated genes (ISGs) (Langevin et al., 2013b).
The induction of stat1 and stat2 genes has been observed in diverse fish species in response to viral infections, suggesting their participation in antiviral defense mechanisms, mirroring their mammalian counterparts (Gorissen et al., 2011;Dehler et al., 2019).In all cases the genes linked to the JAK-STAT pathway, including stat1, stat2, IFNs, and IFN regulatory factors (IRFs), show increased expression after poly(I:C) treatment.Other STATs participate in antibacterial responses.Investigations in common carp and Nile tilapia have revealed that the interleukin IL-22 is important in regulating inflammatory reactions by way of the JAK-STAT signaling pathway through STAT3 (Wang JX. et al., 2022;Wang X. et al., 2022).STAT3 can also be activated by IL-6 (Wang JX. et al., 2022).IL-10 is an anti-inflammatory cytokine, mediating the response through STAT3 (Cevey et al., 2019).IL-4 and IL-13 are regulators of T helper 2 (Th2) cell development signaling through STAT6 (Ranasinghe et al., 2018).Teleosts have cytokines related to both and are termed IL-4/IL-13 (Wang and Secombes, 2015;Stocchi et al., 2017).Although the subsets of STATs are conserved within vertebrates (Liongue et al., 2012), it is important to note that fish, particularly salmonids, have several paralogues of the different STATs (Sobhkhez et al., 2014), likely due to the third round of whole genome duplication (Glasauer and Neuhauss, 2014).
Model systems like zebrafish will continue to offer valuable insights into the JAK-STAT pathway and other immunological signaling pathways.However, to fully understand immunological responses in lumpfish, it is crucial to unveil their genetic distinctions in comparison to other fish species (Eggestøl et al., 2020a;Eggestøl et al., 2020b).In our previous studies in lumpfish, early immune responses upon exposure to bacteria and poly(I:C) have been described (Eggestøl et al., 2018;Rao et al., 2023).Upon bacterial encounter, the soluble version of toll like receptor 5 (TLR5s) and proinflammatory cytokines were among the most highly upregulated genes (Eggestøl et al., 2018), while upon poly (I:C) stimulation, the RLR family members MDA5 and LGP2, as well as type I IFNs were highly upregulated (Rao et al., 2023).However, characterization of STATs has not been addressed so far.This research article presents a comprehensive analysis of previously published data to identify and characterize STATs and the JAK-STAT signaling pathway in lumpfish (Eggestøl et al., 2018;Rao et al., 2023), unveil their immunological importance, as well as shed light on the evolution of STATs.

Fish and rearing conditions
Unvaccinated, farmed lumpfish were provided by Fjord Forsk Sogn AS (a commercial breeder in Sogn & Fjordane County, Norway), and kept in a 500 L tank in the rearing facilities at the Industrial and Aquatic Laboratory (ILAB) at Bergen High Technology Centre under normal rearing conditions, with optimum temperature oxygen level and salinity.The fish were fed with dry commercial feed.In accordance with Norwegian law, the fish were sacrificed by receiving a sharp blow to the head.All experiments were conducted in accordance with the necessary regulations and guidelines.Under Norwegian law, raising fish in normal, ideal conditions is not subject to ethical review (FOR 1996-01-15 no. 23).

Isolation of leukocytes and in vitro stimulation
The head kidney was dissected aseptically from lumpfish and leukocytes were isolated using discontinuous Percoll gradient as described earlier (Haugland et al., 2012), (n=15 fish).The quality and quantity of leukocytes were determined using a CASY cell counter, and 5 x 10 6 leukocytes were added per well in 24-well plates.For bacteria exposure, leukocytes were exposed to V. anguillarum serotype O1, multiplicity of infection (MOI) of 1:10, for 6 and 24 hours (hrs) as described in Eggestøl et al (Eggestøl et al., 2018).To monitor anti-viral responses, 100 µg mL-1 poly(I:C) were added to 5 x 10 6 leukocytes in 24-well plates and incubated for 6 and 24 hrs as described in Rao et al. (2023).For negative controls, leukocytes with L-15+medium were used instead of bacteria (V.anguillarum) or poly(I:C).

RNA isolation and RNA sequencing
RNA was isolated using GeneElute Mammalian Total RNA miniprep kit (Sigma).The quality and quantity were determined by bioanalyzer.Prior to RNA sequencing, an equal amount (1 µg) RNA from leukocytes isolated from five fish were pooled (Eggestøl et al., 2018;Rao et al., 2023), and in total there were three parallels in the RNA sequencing.

Differential expression analysis
Normalized read counts analyses which make the basis for the differential expression analysis are described in Eggestøl et al. (2018) and Rao et al. (2023), and the read count files has been submitted to Array express, accession numbers E-MTAB-6388 (V.anguillarum) and E-MTAB-12884 (poly(I:C)), respectively.The set of differentially expressed genes (DEGs) comprised those genes that demonstrated statistical significance, with a p-value below 0.05, as determined by our analysis (Eggestøl et al., 2018;Rao et al., 2023).

Multiple sequence alignment and phylogenetic analyses
The lumpfish transcriptomes from leukocytes exposed to V. anguillarum (E-MTAB-6388 (Eggestøl et al., 2018)) and poly(I:C) (E- MTAB-12884 (Rao et al., 2023)) was utilized to isolate the sequences of the STAT candidate genes, encompassing the open reading frame (ORF) as well as the untranslated regions.The sequence alignment of the STATs was performed in MEGA 11 (Tamura et al., 2021) using the MUSCLE program with default parameters.The best-fitting models of sequence evolution were determined with complete deletion of gaps.The maximum likelihood method was used to construct the phylogenetic tree using IQ-TREE with automatic model selection and 100,000 bootstraps (Nguyen et al., 2015).

Synteny analysis
Synteny analysis was performed to compare the genomic arrangements of lumpfish STAT genes with those of human (Homo sapiens), Atlantic salmon (Salmo salar), zebrafish (Danio rerio), and stickleback (Gasterosteus aculeatus) to identify orthologous genes across species.To visualize the genomic arrangement of the STAT genes in other model organisms, the lumpfish sequences were used as queries in the ENSEMBL (https:// www.ensembl.org)database.The chromosomal localization, transcriptional orientation, and neighborhood genes of the orthologous STAT genes in all species were obtained using the genome data viewer of NCBI (https://www.ncbi.nlm.nih.gov) and comparative genomics alignment viewer of genomicus v.109 (https://www.genomicus.bio.ens.psl.eu/genomicus-109.01).

Results
Comprehensive bioinformatic analyses were performed to identify and characterize the STAT family members in lumpfish, to gain insight into the evolution of the STAT family and regulation of the JAK-STAT signaling pathway upon exposure to bacteria and poly(I:C).

Identification and phylogenetic analyses of STATs in lumpfish.
Seven STAT genes were identified in our transcriptomic data (E-MTAB-6388 and E-MTAB-12884) and STATs candidates in lumpfish were also searched for in Genomicus, Ensembl and NCBI Genbank databases.A phylogenetic tree was constructed to determine the subgroups of lumpfish STATs and the phylogenetic relationship among the groups.Two main clades were made, one with stat1-4 sequences (STAT-A) and the other clade with stat5 and 6 (STAT-B).The phylogenetic analyses unveiled that lumpfish have stat1a, 2, 3, 4, 5a, 5b and 6 (Figure 1A), and that the lumpfish and stickleback STATs had the highest level of similarity.stat1b was not identified in lumpfish.The stat1b of stickleback grouped together with stat1b sequences from zebrafish and salmon but was distantly related.
All the STATs identified in lumpfish had conserved domains which include N-terminal domain, coiled-coil domain, DNA-linker domain, a linker region, SH2 domain and a C-terminal transactivation domain (Figure 1B).The conserved amino acids known to be involved in phosphorylation, S, S and Y, in the Cterminal were present in all the lumpfish sequences (Supplementary Figure 1).A multiple sequence alignment of the different STATs showed high similarity within the different domains (Supplementary Figure 1).A distance matrix analysis showed the same grouping into STAT-A and STAT-B as the phylogenetic tree (Figure 1C).The different STAT members in lumpfish contained conserved domains and phosphorylation sites shared by other STATs from different phyla, highlighting the proteins' vital role in signal transduction.However, the lack of stat1b in lumpfish unveils species specific adaptation to prevent infections.
3.2 Synteny analyses show that the STAT loci are conserved within teleosts.
Synteny analyses were performed for the STATs (Figure 2).In humans STAT1 and STAT4 are located next to each other on chromosome 2.In fish, most species have two copies of stat1 (a and b), except lumpfish which lack stat1b, and it is the stat1b that is located next to stat4 in proximity to wdr75 and slc40a1 (Figure 2).The lumpfish stat1a was found on chromosome 2 in proximity to mstn and nab1a, like in the other fish species.Both STAT2 and STAT6 had conserved synteny across human and fish (Figure 2).In humans, STAT3, STAT5A, and STAT5B are adjacent genes, whereas in fish, they are divided into separate genomic regions.Interestingly, in salmon, the conserved stat3 gene is duplicated on both chromosomes.However, in zebrafish, stickleback, and lumpfish, the chromosome containing stat5b lacks the stat3 gene, with only a single copy of stat3 gene present (Figure 2).Synteny analyses revealed that the STAT loci were conserved, but in some cases divergent from humans (STAT1 and STAT5A/B), which is not unexpected as fish have gone through an extra round of WGD.
3.3 Description of the JAK-STAT pathway and regulation upon V. anguillarum and poly(I:C) exposure.
Transcriptome and genome data mining for genes belonging to the JAK-STAT pathway, revealed that most genes were present including jak1-3 and tyk2, all STATs except stat1b (Figure 1), suppressor of cytokine signaling (SOCS)1a/b, 3b, 5b/9, 6a and other components of the pathway (Figure 3A; Table 1).DEG analyses of lumpfish leukocytes exposed to V. anguillarum and poly(I:C) were performed in two separate experiments.Notably, exposure to bacteria resulted in high upregulation of il-6, and moderate upregulation of il-10, il-4/13, and il-21 (Figure 3B).stat1a and 3 were weakly upregulated at both 6 and 24 hpe to bacteria, and opposite to stat4, stat6 was slightly upregulated at 6 hpe, but not at 24 hpe.Upon exposure to poly(I:C), the highest upregulations were seen for ifnphi1 (5.4 and 6.7 log2fold upregulated at 6 and 24 hpe, respectively) and ifnphi3 (6.5 log2fold upregulation).stat1 and 2 were both upregulated at 24 hpe, as also irf3 (Figure 3B).Based on the DEG analyses, we have suggested a model of how JAK and STAT family members are involved in antibacterial and antiviral responses in lumpfish (Figure 4) IL-2 might have a dual role promoting IL-4 receptor expression directly, and/or it might also activate STAT5A and STAT5B, which then bind to the IL-4 promoter, resulting in its overexpression.Over expression of STAT3 with JAK1, JAK3 and STAT6 can have a Synteny analyses of human, Atlantic salmon, zebrafish, stickleback, and lumpfish STAT genes.Genomic loci maps comparing STAT related genes were constructed using the Genomicus Browser and NCBI database.The bar lengths are not proportional to the distances between genes.The white boxes represent non-conserved genes on the chromosomes/scaffolds.The direction of the arrows indicates the gene orientation.

Establishment of evolutionary model of STAT genes
Our comparative genomics analysis reveals that STAT genes in fish have undergone multiple duplications throughout evolutionary history (Figure 5).The origin of the STAT genes, represented by STAT-A and STAT-B, can be traced back to early chordate to vertebrate evolution.This likely resulted from two whole genome duplication (1R) events after the divergence of cephalochordates (Glasauer and Neuhauss, 2014), but before the emergence of Chondrichthyes.Two rounds (2R) of whole genome duplication during early vertebrate evolution led to the emergence of multiple STAT genes with distinct functions (Boudinot et al., 2021).For instance, the precursor gene STAT-A underwent independent chromosomal duplications, giving rise to STAT1 with STAT4 in one chromosome and STAT2 in another.Similarly, STAT-B underwent independent chromosomal duplications, leading to the formation of STAT3, two copies of STAT5 in one chromosome, and STAT6 in another.In Salmon and Zebra fish species, a third round of whole genome duplication occurred, resulting in the presence of multiple pseudo-genes.Investigating the lumpfish genome, we observed unique gene distribution compared to other fish species.Notably, the lumpfish genome contained stat1a, stat2, stat6, stat5b, and stat4 genes in separate chromosomes, while stat3 along with stat5a was found on a single chromosome (Figure 5).Some variations are seen in fish species, e.g., two copies of stat1a in salmon and lack of stat1b in lumpfish.Several gene duplications in fish STAT genes, dating back to early vertebrate evolution, become apparent in our comparative genomic analyses, highlighting the consequences of WGD events.
A proposed mechanism for STAT evolution based on 2R hypothesis as a basis of the radiation of the STAT repertoire, the fish-specific whole genome duplication (3R), and subsequent (partial) loss of paralogous stat1b genes.Animal diagrams from left to right: human, Atlantic salmon, zebrafish, Nile tilapia, Japanese medaka, stickleback and lumpfish.

Discussion
The JAK-STAT pathway is a vital signal transduction cascade responsible for regulating diverse biological processes such as immunity, development, and oncogenesis (Seif et al., 2017).The JAK-STAT pathway in fish, particularly the lumpfish system, has been the subject of interest for this study.Lumpfish are widely distributed from the Bay of Biscay in France to the frigid waters of the North Atlantic and Arctic oceans (Whittaker et al., 2018).In recent years, it has become a valuable aquaculture species in Europe and Canada, as they are used as cleaner fish to control sea lice on farmed salmon (Powell et al., 2018;Haugland et al., 2020).The identification of STATs and the JAK-STAT signaling pathway in lumpfish is a major step towards understanding the species' immunological responses and is important for developing effective disease management strategies for this species.Our findings have provided insights into the activation of this pathway during the immune response to bacterial and viral infections.Due to the complexity and interaction of cytokine signaling pathways, the increasing understanding of the roles of the STATs is important for the development of effective disease therapeutics.
The evolution process describes how organism populations evolve throughout time (Liongue et al., 2012;Liongue et al., 2016).Changes in the heritable traits of biological populations result in genetic variations (Sunyer and Lambris, 1998).Because of recurrent stress followed by reproduction, generations of a single organism can diverge from an ancestral population.Population changes can be so extreme that a new species with a varied range of gene allies is assumed to have developed on rare occasions.Understanding the degree of heterozygosity in neutral evolution is critical for explaining how variations in gene expression over time emerge from divergence accumulation in the absence of selective pressure (Roberts et al., 1998).A crucial stage in the development of the JAK-STAT pathway was the assembly of these STAT domains to produce each individual component.sequence divergence can alter these domains.Instead of creating a domain from scratch the analysis of the individual STAT supported these processes as the primary mechanism for protein domain variation in eukaryotes.
The generation of novel genes in eukaryotes primarily occurs through gene duplications, domain shuffling, and related mechanisms, rather than the creation of entirely new genes (Boudinot et al., 2021).Prior to the first whole genome duplications (WGD), the first generation of the STAT genes underwent tandem duplication (Liongue et al., 2012), giving rise to STAT1/2/3/4 and STAT5/6 (Liongue and Ward, 2013).During the second phase of WGD, three gene clusters were created: STAT1 and STAT4, STAT2 and STAT6, and STAT3 and STAT5 (Boudinot et al., 2021), Gene loss following duplication occurrences may also restrict the gene growth.The STATs are divided into the STAT-A and STAT-B groups, with STAT1 to STAT4 being a part of STAT-A, and STAT5 and STAT6 being a part of STAT-B (Cao et al., 2021).In the present study, we observed that apart from the absence of stat1b, seven STAT genes have been identified in lumpfish.The host's defense systems are greatly influenced by these genes, which are preferentially expressed in distinct cell types.
JAK proteins are crucial components of the signaling pathways of various cytokines (Bousoik and Montazeri Aliabadi, 2018), which regulate immune responses, inflammation, and intercellular communication (Morris et al., 2018).The DEG analyses from the present study (Figure 3B), made a basis for our model in lumpfish, where cytokines like IL-4/13, IL-2, IL-21, IL-6, IL-10, and IFN activate specific Janus Kinase (JAK) family receptors to transmit signals, leading to the activation of distinct STAT proteins (Figure 4).Among the JAK family members, TYK2 holds significance in mediating IL-10 dependent signaling as antiinflammatory responses during bacterial infections.However, the mechanism by which IL-10 triggers anti-inflammatory gene expression differs markedly from the ability of IL-6 to induce inflammatory gene activity.While JAK3 is activated in response to cytokine stimulation, including IL-2 family members such as IL-4 and IL-21, it is primarily expressed in hematopoietic and immunologic cells.On the other hand, JAK2, which is more widely distributed, serves as the predominant kinase activated by approximately two-thirds of ligands (Seif et al., 2017).The significance of STAT3 in regulating immune reactions and maintaining immune homeostasis is underscored by its crucial role in both pro-inflammatory and anti-inflammatory responses (Hillmer et al., 2016).In fish, IL-6 is essential for pro-inflammatory immune responses against bacterial infections, enhancing antibody production and activating macrophages (Wang JX. et al., 2022).The IL-6/STAT3 signaling pathway potentially contributes to the expression of antimicrobial genes in infected cells (Ching et al., 2018;Wang JX. et al., 2022).This vital role of STAT3 extends to lumpfish, reaffirming its importance in immune regulation across species.
Interferons (IFNs) are essential for coordinating vigorous and efficient antiviral responses in vertebrates (Bonjardim, 2005), including fish (Zhang and Gui, 2012).STAT1 is especially important for innate immunity and downstream interferon (IFN) function (Skjesol et al., 2010).Both STAT1 in humans and its fish counterpart, stat1a, exhibit similar functions and play crucial roles in antiviral defense mechanisms.The critical role of STAT1 is believed to be conserved in fish, as demonstrated by the successful restoration of IFN-induced signaling in zebrafish stat1 (Song et al., 2011).Upon activation, both STAT1 and stat1a form complexes with transcription factors like IRF9, resulting in the activation of interferon-stimulated gene (ISG) expression (Lu et al., 2020;Tolomeo et al., 2022).This well-studied pathway has been extensively explored in the context of dangerous viruses such as Ebola and Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in humans as host (Tolomeo et al., 2022;Escudero-Perez et al., 2023).These results provide important information on the conservation and variety of the JAK-STAT pathway in fish and set the stage for future studies on the functional involvement of STATrelated genes in controlling important biological processes.It is critical to comprehend the JAK-STAT pathway's regulatory mechanisms in fish since this reveals the pathway's capacity to control a variety of biological activities.
Poly(I:C) is a synthetic compound that is a dsRNA mimicking viral infection and is often used to generate a type I IFN response in fish (Zhang and Gui, 2012), and other animals.In a study utilizing minipig kidney cell line, it was discovered that stat1a and stat1b were both highly upregulated after poly(I:C) stimulation, demonstrating that they had different reactions to stimulation (Wu et al., 2019).Exposure of lumpfish leukocytes to poly(I:C) led to the upregulation of stat1a and stat2.However, more research is needed to ascertain whether the underlying mechanisms for immune regulation in fish is like that in mammals, and to investigate the functional specialization of each STAT family member in fish.In chinook salmon cell line EC, stat1b1 and stat2 were induced with a FC > 1.5 following stimulation by salmonid recombinant type I IFN, while stat1a1 was also induced to some extent.However, stat1b2 was not up regulated (Dehler et al., 2019).The expression of stat1a paralogs was higher than stat1b under steady state conditions, consistent with the functional constitutive expression of stat1a genes observed in zebrafish (Langevin et al., 2013a;Levraud et al., 2019).
It has been observed that stat1a and stat1b expressions are linked to different tissues in fish (Xu et al., 2022).E.g., after A. hydrophila infection in the spleen, different expression patterns of the STAT genes were observed.At the early stages of infection, three genes, including stat1b, stat5a, and stat6, were markedly regulated, and two genes, stat1a and stat2, were significantly induced.Notably, the relative expression of stat1b in the liver, spleen, and kidney was at least 10-fold higher at the early stage of infection (Xu et al., 2022).A study found that both zebrafish and salmonid showed robust upregulation of genes stat1b and stat2, but not of stat1a, in response to type I IFN induction (Langevin et al., 2013a;Levraud et al., 2019).Similarly, in a similar study involving channel catfish infected with Edwardsiella, stat1b was significantly up-regulated in the liver and intestine at 72 hpi, but it was significantly down-regulated in the intestine at 24 hpi (Jin et al., 2018).Further research is needed to confirm the consistency of fish's regulation patterns with those of mammals and to explore the functional differentiation of each STAT family member in fish.
Different STATs have been identified in various fish species, and they play essential roles in regulating immune responses.For example, STAT1 is involved in the induction of type I IFNs and the activation of macrophages and natural killer cells.STAT3 is involved in the regulation of T cell differentiation and the production of inflammatory cytokines, while STAT5 is involved in the development of lymphoid cells and the regulation of antibody production.Additionally, STAT6 is involved in the differentiation of Th2 cells and the production of anti-inflammatory cytokines.Overall, the expression and function of different STATs in fish suggest that they play critical roles in modulating both innate and adaptive immune responses.
The identification of specific STAT-related genes in lumpfish, along with the comparison of transcriptional targets across the genome, can aid in comprehending the pathway's regulatory mechanisms.Further studies on the functional roles of these genes in regulating vital biological processes are necessary to gain a comprehensive understanding of the JAK-STAT pathway's significance in fish.In conclusion, this study emphasizes the importance of the JAK-STAT pathway in fish and highlights the need to gain a comprehensive understanding of its regulatory mechanisms.The study's findings provide significant information on the conservation and diversity of the JAK-STAT pathway in fish, specifically in the lumpfish system.This research can aid in developing a foundation for further research on the functional roles of specific STAT-related genes in regulating critical biological processes in fish.
FIGURE 3 Transcriptome-wide analyses of the JAK-STAT pathway in lumpfish.(A) Identified of the JAK-STAT pathway are shown in grey boxes.The figure has been modified from hsa04630.(B) DEG analyses of the JAK-STAT pathway upon exposure to bacteria and poly(I:C) after 6 and 24hpe.

FIGURE 4
FIGURE 4Predicted model for involvement of the different JAKs and STATs family members in intracellular signaling in lumpfish upon bacterial and viral (mimicked by poly(I:C)) encounter.In bacterial responses, il-4/13, il-21, il-6, and il-10 bind to their cognate receptors, recruit JAK family members (jak1/2/3/tyk2) to interact with the cytokine receptor and thereafter STAT (stat3/6 in bacteria responses).In viral responses, IFNs bind to receptors, recruit jak1/2 and stat1/2.Predicted immune processes initiated are listed.

TABLE 1
Verified genes belonging to the JAK/STAT pathway (KEGG map04630) in lumpfish.