Genome-Wide Identification and Expression Analysis of the STAT Family in Reeve’s Turtle (Mauremys reevesii)

TL;DR

This study identifies and analyzes the STAT gene family in Reeve’s Turtle, finding that Stat5b is involved in immune responses.

Contribution

The study reports the genome-wide identification and immune-related expression analysis of the STAT family in a reptile species.

Findings

Eight putative Stat genes were identified in the genome of Mauremys reevesii.

Stat5b showed the most significant upregulation in spleen tissues after LPS administration.

Immunofluorescence confirmed Stat5b expression in spleen tissue cells.

Abstract

The Stat (signal transducer and activator of transcription) gene family plays a vital role in regulating immunity and the processes of cellular proliferation, differentiation, and apoptosis across diverse organisms. Although the functions of Stat genes in immunity have been extensively documented in many mammals, limited data are available for reptiles. We used phylogenetic analysis to identify eight putative members of the Stat family (Stat1-1, Stat1-2, Stat2, Stat3, Stat4, Stat5b, Stat6-1, and Stat6-2) within the genome of M.
reevesii, a freshwater turtle found in East Asia. Sequence analysis showed that the Stat genes contain four conserved structural domains protein interaction domain, coiled-coil domain, DNA-binding domain, and Src homology domain 2. In addition, Stat1, Stat2, and Stat6 contain TAZ2bind, Apolipo_F, and TALPID3 structural domains. The mRNA levels of Stat genes were upregulated in spleen tissues at 4, 8, 12, and 16 h after administration of lipopolysaccharide, a potent activator of the immune system. Stat5b expression at 12-h LPS post-injection exhibited the most substantial difference from the control. The expression of Stat5b in spleen tissue cellular was verified by immunofluorescence. These results suggest that Stat5b plays a role in the immune response of M.
reevesii and may prove to be as a positive marker of an immune response in future studies.

The online version contains supplementary material available at 10.1007/s10528-024-10820-7.

Figures

Schematic of the conserved domains in STATs proteins and motifs in STATs genes. All motifs were identified using the Multiple Expectation Maximization for Motif Elicitation database with the complete nucleotide sequences of STATs genes. Gene structure analyses of *Stat* genes in *M. reevesii*. A chromosomal locations of *Stat* genes in *M. reevesii* (B)

Protein structure of the *Stat* gene from *M. reevesii*. Different colors mark conserved domainsFig. 4Phylogenetic analysis and classification of STATs homologs from *M. reevesii* and 16 other species. Red circles mark the STATs proteins in *M. reevesii*. Red lines represent the close relationship between STATs and the corresponding homologs

Expression of STAT genes in spleen of post-injection and control turtles at different time points in *M. reevesii*. The whole experiment was repeated three times to yield a total of 90 samples. The experiment was conducted in triplicate and repeated three times. The results were expressed as mean±standard error, *indicates statistical significance at *P* < *0.05* and indicates statistical significance at *P* < *0.01*

Full text

Introduction

Signal transducer and activator of transcription (STAT) proteins are intracellular transcription factors involved in regulating the expression of immune-related genes and regulating growth, apoptosis, and differentiation (Villarino et al. 2017). The Stat gene family in vertebrates typically has 5–7 members that are generally divided into six major subfamilies: Stat1, Stat2, Stat3, Stat4, Stat5, and Stat6. Stat genes play an important role in immunity and their molecular structures and biological roles have been extensively studied in mammals, such as humans and mice (Nguyen-Jackson et al. 2010). Stat1 acts as a major regulator and is involved in antiviral immunity. For example, mice lacking Stat1 are susceptible to mucosal fungal infections (Jung et al. 2020). Stat3 is involved in the release of mature neutrophils from the bone marrow to the circulatory system. Stat3 knockout mice do not efficiently mobilize the release of mature granulocytes compared to normal mice (Pencik et al. 2016; Zhao et al. 2016). Stat5A and Stat5B are susceptible to activation by various cytokines, such as interleukins (Hennighausen and Robinson 2008). In humans, Stat6 is involved in the development of lymphoma and leukemia (Bruns and Kaplan 2006). In reptiles, there have been studies on individual members of the STAT family play a crucial role during gonadal differentiation and antiviral responses. In the red-eared slider turtle (Trachemys scripta elegans), calcium concentration affects the phosphorylation status of STAT3, thereby regulating the transcriptional activity of other genes, influencing male gonadal differentiation (Weber et al. 2020). Additionally, the identification of interferon-gamma (IFN-γ) and its receptor (IFN-γR) in the green anole lizard (Anolis carolinensis) not only reveals the significance of the interferon system in the immune response of reptiles but also highlights the central role of the STAT family in regulating these processes, as IFN-γ signaling typically proceeds through the STAT1 pathway (Ghorai and Priyam 2018). Thus, in reptiles such as turtles there is a lack of information on STAT family members as a whole.

In aquaculture, turtles often suffer from infection-based diseases and inflammatory reactions. Current research focuses on pathogenesis, disease diagnostic methods, therapeutic means, and preventive measures (Johnson et al 2005; Di Ianni et al 2015; Ciccarelli et al 2020). Due to the long lifespan and unique physiological characteristics of turtles, the study of their immune response mechanisms is somewhat complex. Lipopolysaccharide (LPS) is a component of the outer membrane of Gram-negative bacteria that elicits a strong immune response in animals. When researchers inject LPS into a model organism, they are not introducing live bacteria or causing an infection in the traditional sense. Instead, they are simulating the effects of an infection by triggering an immune response similar to what might be seen during an actual bacterial infection. This is often done to study the immune system’s response to pathogens or to test the efficacy of drugs and treatments that target inflammatory processes. In recent years, numerous studies have shown that infections that release LPS leads to various diseases in animals (Pinya et al. 2021; Xu et al. 2021; Hashimoto et al. 2020). In LPS-mediated cellular inflammation models, STATs regulate immune cell differentiation through the Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway (Yao et al. 2007). This signaling pathway regulates immunity in zebrafish, and Stat gene expression was detected in the spleen of blunt-mouthed seabream (Zhang et al. 2018). There is considerably less information available about immunity in reptiles (Zimmerman et al. 2010). Stat genes have rarely been studied in freshwater turtles and whether they play a potential role in immune responses requires further exploration.

The Chinese three-keeled pond turtle, Mauremys reevesii, in the family Geoemydidae, is a freshwater turtle distributed across many East Asian country across China, the Korean Peninsula and Japan (Rhodin et al. 2017). M. reevesii holds significant commercial importance in aquaculture and is categorized as “Endangered” on the IUCN Red List of Endangered Species (Rhodin et al. 2017). Notably, this species possesses distinctive characteristics, including a remarkable capacity for hypoxia tolerance, temperature-dependent sex determination, and the ability to produce fertile interspecific and intergeneric hybrids (Xiong et al. 2020). These exceptional traits make it a rare and invaluable model species for evolutionary genetic research. However, the study of gene function in this species, including immunity-related genes, remains unclear and vastly underexplored, resulting in limited information about turtles.

This study characterizes the Stat genes in the freshwater turtle Mauremys reevesii, identifies their location in the genome, and demonstrates that one of the members of this gene family, Stat5b, responds strongly to administration of lipopolysaccharides (LPS), a potent activator of the immune system. Subcellular localization and expression of Stat5b were observed using immunofluorescence (IF). This study provides insight to the potential functional roles of Stat genes in regulating the immune response of M. reevesii.

Materials and Methods

Sample Collection

Healthy adult turtles (101.02 ± 6.21 g) were sourced from the East China Turtle Breeding Facility (Wuhu, Anhui Province, China) and were maintained in water at a temperature of 28 °C. The LPS injection experiment followed methods described in a previous study (do Amaral et al. 2002). The turtles were randomly divided into two groups: the treatment group and the control group. In the treatment group, turtles were administered LPS (Sigma, St. Louis, MO, USA) at a dose of 2.5 mg per 100 g of body weight, which was dissolved in a 0.75% NaCl solution. Turtles in the control group were administered the same volume of a 0.75% NaCl solution. Spleen tissues were collected from a total of 30 turtles spread across five time periods – 0-h, 4-h, 8-h, 12-h, and 16-h post-injection (3 infected turtles as a group, n = 3)—representing control and treatment classes. The whole experiment was repeated three times to yield a total of 90 samples which yielded sufficient statistical power. All samples were immediately snap frozen in liquid nitrogen and subsequently stored at − 80 °C. All experiments complied with the guidelines and regulations of the Institutional Animal Care and Use Committee of Wannan Medical College in Wuhu, China.

Characterization of Stat family Genes

The Stat gene sequences from the Chinese softshell turtle (Pelodiscus sinensis), Zebrafish (Danio rerio), and Chinese crocodile lizard (Shinisaurus crocodilurus) (Table S1) were used as reference for screening Stat family candidate genes within the genome assembly of M. reevesii via BLASTN analysis (E-value threshold < 1e–5). Homologous sequences of the Stat genes of M. reevesii were aligned using DNAMAN (https://www.lynnon.com/download/, v6.0), and structural domains of the STAT proteins were predicted using Pfam (http://pfam-legacy.xfam.org/, v36.0) (E-value threshold < 1e–5) (Liu and Yu 2011). The conserved regions of STAT proteins were predicted by HMMER 3.3.2 software using default parameters and then validated by SMART analysis. MEME (https://meme-suite.org/meme/doc/meme.html, v5.5.4) and InterProScan (https://www.ebi.ac.uk/interpro/about/interproscan/, V 5.65–97.0) with the default parameters were used to identify conserved motifs of STAT genes (Bailey et al. 2009). The gene structure of M. reevesii Stat genes was analyzed using TBtools (https://github.com/CJ-Chen/TBtools/releases, v1.045) (Chen et al. 2020). Stat genes promoter regions (2000 bp from the start codon, supplementary Table 1) were used in JASPAR database (https://jaspar.elixir.no/) as described previously (Wu et al. 2021). Genomicus (https://www.genomicus.bio.ens.psl.eu/genomicus-110.01/cgi-bin/search.pl, v110.01) was used to determine the location of Stat genes on chromosomes.

STAT protein sequences for 16 species (Homo sapiens, Mus musculus, Bos taurus, Gallus gallus, Meleagris gallopavo, Danio rerio, Xenopus laevis, Gasterosteus aculeatus, Oreochromis mossambicu, Chrysemys picta, Terrapene mexicana, Pelodiscus sinensis, Chelonia mydas, Thamnophis elegans, Alligator mississippiensis, and Anolis carolinensis) were downloaded from NCBI (Table S2) to which the STAT amino acid sequence for M. reevesii was added. Phylogenetic analysis (MEGA 11.0 software (https://megasoftware.net/, v11.0) (Edgar 2004)) was used to assign the Stat genes of M. reevesii standard vertebrate classes (Stat1-6). ExPASy (https://web.expasy.org/protparam/) and SnapeGene (https://www.snapgene.cn/, v7.0) were used to assess the physiological and biochemical properties of STAT protein sequences. Prediction of 3D protein structure was conducted using the Phyre2 protein structure homology model building program (http://www.sbg.bio.ic.ac.uk/phyre2, v2.0) (Wass et al. 2010).

Transcriptomics

Total RNA extraction from spleen tissues was done using TRIzol reagent (Invitrogen, Waltham, MA, USA). RNA concentration and purity were determined utilizing a NanoDrop 2000 system (Thermo Fisher, Waltham, MA, USA). Reverse transcription to cDNA was performed with the PrimeScript™ RT Kit (Takara, Shiga, Japan) (Ijaz et al 2022). To assess the relative expression of Stat mRNA within spleen tissue samples, quantitative real-time polymerase chain reaction (qRT-PCR) was conducted using the StepOnePlus™ Real-Time PCR System (Thermo Fisher) in conjunction with SYBR Green I Master Mix (Krishnaveni et al 2023). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as the internal control. The qRT-PCR amplification conditions consisted of 40 cycles, including initial denaturation at 95 °C for 30 s, followed by denaturation at 95 °C for 5 s, annealing at 60 °C for 20 s, and extension at 72 °C for 20 s.

Relative expression levels of Stat genes were computed using the 2^–ΔΔCt^ method (Schmittgen et al. 2008; Huan et al 2022; Li et al 2023). Single-factor analysis of variance (ANOVA, SPSS 24.0) was used to assess the relative expression levels of Stat genes. Additionally, data from all experiments, presented as mean ± standard deviation (SD), were analyzed using single-factor ANOVA via GraphPad Prism9. Results are expressed as mean ± standard error (SE). Statistical significance was determined at α = 0.05.

Immunofluorescence

Stat5b expression was examined through immunofluorescence (IF) analysis (Donaldson 2001) for samples taken 12 h following LPS post-infection. Spleen tissues were categorized into three groups: the treatment group (12 h after LPS/NaCl post-infection), the positive control group (NaCl only), and the negative control group (background check, not antibody applied). The tissues were fixed in 4% paraformaldehyde, dehydrated in gradient alcohol. Paraffin-embedded sections were cut using a microtome (5 μm). Sections were subjected to a heat treatment at 65 °C for 6 h, followed by dewaxing and hydration in graded ethanol. They were then rinsed with PBST (phosphate-buffered saline containing 0.3% Triton X-100 detergent; BSW-005, Biotyscience) and subjected to antigen retrieval with sodium citrate before cooling (Abbas and Alkheraije 2023). After three 5 min washes with PBST, the sections were blocked with 5% bovine serum albumin for 30 min. The treatment and positive control sections were then incubated overnight at 4 °C with STAT5B antibody (A12356, 1:100; Abclonal, Wuhan, China), washed again with PBST, and subsequently exposed to a secondary antibody (AlexaFluor 488 donkey anti-rabbit IgG, Invitrogen) at room temperature for 2 h. The negative control sections were treated the same, but without antibody. Nuclei were stained using 4ʹ,6-diamidino-2-phenylindole (286 nmol/L, Sigma) and fluorescence signals were observed using a laser confocal microscope (SP8, Leica, Wetzlar, Germany).

Results

Characterization of Stat family Genes

We identified a total of eight Stat genes for M. reevesii (Table 1). Among these genes, Stat6-1 had the longest amino acid sequence, spanning 861 base pairs, while Stat1-2 possessed the shortest amino acid sequence at 715 base pairs. In terms of intron count, Stat1, Stat3, and Stat4 had the highest number of introns (23 introns), followed by Stat2 and Stat6 (21 introns) and then Stat5b (18 introns) (Fig. 1A). These genes were distributed across three chromosomes within the M. reevesii genome—chromosomes 11, 25, and 27. Notably, several pairs of Stat genes were found on the same chromosomes, such as Stat1 and Stat4 on chromosome 11, Stat2 and Stat6 on chromosome 25, and Stat3 and Stat5 on chromosome 27. Additionally, some duplicated genes were arranged on the same assembly scaffold, including Stat1-1/Stat1-2 and Stat6-1/Stat6-2 (Fig. 1B).Table 1. The STAT family genes identified in M. reevesiiGene symbolAccession numbermRNAProteinCDS length (bp)Protein length (aa)Chromosome locationPIInstability indexHydropathicitySTAT1-1120,374,631XM_039494596.1XP_039350530.12268756Chromosome 115.5448.10− 0.415STAT1-2120,374,631XM_039494600.1XP_039350534.12145715Chromosome 116.1948.15− 0.445STAT2120,391,254XM_039514731.1XP_039370665.12556852Chromosome 255.5447.37− 0.488STAT3120,392,259XM_039516943.1XP_039372877.12316772Chromosome 275.9488.25− 0.354STAT4120,374,670XM_039494691.1XP_039350625.12250750Chromosome 115.9751.33− 0.265STAT5B120,392,261XM_039516947.1XP_039372881.12349783Chromosome 255.9254.66− 0.472STAT6-1120,391,151XM_039514557.1XP_039370491.12583861Chromosome 255.9594.33− 0.242STAT6-2120,391,151XM_039514558.1XP_039370492.12490830Chromosome 256.0291.32− 0.238Fig. 1A Gene structure analyses of STAT genes in M. reevesii. The exons are indicated by yellow rectangles and introns by lines. Yellow rectangles: The sizes of exons number. UTR, untranslated region; CDS, coding sequence (exons). B Chromosomal locations of STAT genes in M. reevesii

The coding sequences of Stat genes ranged in length from 2145 to 2583 base pairs, encoding proteins ranged in size from 715 to 861 amino acids. Additionally, the isoelectric points of these proteins ranged from 5.54 to 6.19. The sequence analysis revealed that all of the Stat genes shared four conserved structural domains: the protein interaction domain (INT), coiled-coil domain (CCD), DNA-binding domain (DBD), and Src homology domain 2 (SH2). Sequence analysis showed that STATs contain four conserved structural domains, INT, CCD, DBD, and SH2, in addition to STAT1, STAT2, and STAT6, which contain TAZ2bind, Apolipo_F, and TALPID3 structural domains. Some conserved domains might have special biological functions (Fig. 2A). Furthermore, among these genes, Stat1, Stat2 and Stat6 contained additional structural domains, namely TAZ2bind, Apolipo_F, and TALPID3.Fig. 2. Schematic of the conserved domains in STATs proteins and motifs in STATs genes. All motifs were identified using the Multiple Expectation Maximization for Motif Elicitation database with the complete nucleotide sequences of STATs genes. Gene structure analyses of Stat genes in M. reevesii. A chromosomal locations of Stat genes in M. reevesii (B)

Ten motifs were identified in Stat1, Stat1-2, Stat2, Stat3, and Stat4. Eight motifs were identified in Stat5b, Stat6-1, and Stat6-2; motifs 7 and 10 were missing. The analysis of motifs showed that motifs 2, 6, and 10 were related to the DBD domain, which combines with the transcriptional initiation region of downstream genes to regulate gene expression. However, motif 10 was not found in the DBD domain of the Stat5b and Stat6. Motif 3 was related to the CCD domain, which consists of the coiled-coil (alpha) domain of the Stat proteins. This domain is a region that interacts with other proteins. Motifs 1, 4, and 9 were related to the SH2 domain, which is involved in signal transduction. Motifs 5 and 8 corresponds to the INT domain, which is a protein interaction domain. Motifs 3 and 7 appeared twice in Stat3 and Stat5b, respectively (Fig. 2B). The potential binding sites for transcription factors (TFs) in the stat genes promoter were investigated by JASPAR database (supplementary Table 3). Among these TFs, TATA-box, and MYC have been reported as TFs that regulate the stat genes expression (Gergely et al 2024).

The protein sequences of Stat genes were employed for predicting protein structures (Fig. 3). Furthermore, an unrooted phylogenetic tree was constructed using STAT protein sequences from a total of 17 different species (Fig. 4). These homologous sequences were categorized into distinct subfamilies, namely Stat1, Stat2, Stat3, Stat4, Stat5, and Stat6. M. reevesii demonstrated its closest genetic affinity with other turtle species, including C. picta, T. mexicana, C. mydas, and P. sinensis. The subfamilies were organized into two primary cluster groups: Group I encompassed fish Stats, while Group II comprised reptile, mammalian, and avian Stats. The comparative analysis of Stat gene locations is presented in Supplementary Table 2.Fig. 3. Protein structure of the Stat gene from M. reevesii. Different colors mark conserved domainsFig. 4Phylogenetic analysis and classification of STATs homologs from M. reevesii and 16 other species. Red circles mark the STATs proteins in M. reevesii. Red lines represent the close relationship between STATs and the corresponding homologs

It is noteworthy that the composition of the Stat family members demonstrated significant interspecies variability. Particularly, the Stat1 and Stat6 subfamilies exhibited the highest gene diversity. For instance, species, such as D. rerio, T. mexicana, C. mydas, and among others, featured the coexistence of both Stat1A and Stat1B paralogs. In contrast, within the context of M. reevesii, only the presence of Stat5b was identified.

Expression of Stats After LPS Infection

All of the Stat genes were upregulated compared with the control group (0 h). The expression levels of all genes except Stat2 and Stat3 increased rapidly from 0 to 4 h and peaked at 12 h after infection, with a subsequent gradual decrease until 16-h post-infection. Notably, Stat5b displayed significantly upregulated expression compared to the other genes after LPS infection, gradually increasing up to 12 h after infection and then decreasing at 16 h. However, Stat5b exhibited a significant increase compared to other Stat genes (Fig. 5).Fig. 5. Expression of STAT genes in spleen of post-injection and control turtles at different time points in M. reevesii. The whole experiment was repeated three times to yield a total of 90 samples. The experiment was conducted in triplicate and repeated three times. The results were expressed as mean±standard error, *indicates statistical significance at P  < 0.05 and **indicates statistical significance at P  < 0.01

Validation of Expression of Stat5B by Immunofluorescence

The spleen of M. reevesii exhibited a significant increase in both size and weight following LPS infection (Figs. 6A, B). This phenomenon may be attributed to compensatory hyperplasia of the spleen, triggered by the immune response provoked by LPS. In the control group, Stat5b was localized within the cytoplasm but was not expressed within the nucleus of spleen cells. However, in the LPS-treated group, Stat5b was observed in both the cytoplasm and the nucleus of spleen cells. Conversely, no relevant expression of Stat5b was detected in the negative control group. Additionally, the number of *Stat5b-*positive cells in the LPS-treated group demonstrated a marked increase in comparison to the control group. This observation suggests the involvement of *Stat5b-*positive cells in the LPS-induced immune response (Fig. 6C).Fig. 6A Morphological changes of spleen. B Weight statistical analysis of control spleen tissues and LPS post-injected spleen. The experiment was conducted in triplicate and repeated three times. *indicates statistical significance at P  < 0.05. C Cellular localization and expression of STAT5B in spleen tissue after immune response. The first vertical of line of DAPI was in the cell nucleus. Original magnification × 100. Bar  =  50 μm. PALS, periarterial lymphatic sheath (white arrow). Control means without LPS injection, Model means LPS post-injection 12 h, NC (normal control), treated with antibody diluent solution instead of antibody to determine whether there is non-specific staining in the positive results. Green fluorescence represents the gene signal (STAT5B). Original magnification × 400. Bar = 200 μm. Blue fluorescence represents the DAPI signal. The experiment was conducted in triplicate and repeated three times

Discussion

The findings from our study shed light on the characterization and expression patterns of Stat family genes in the freshwater turtle species M. reevesii. These genes have pivotal roles in orchestrating diverse and fundamental cellular processes in tetrapods, including immunity, cell proliferation, differentiation, and apoptosis (Levy and Darnell 2002). We identified eight Stat genes distributed across three chromosomes. Some genes in the same Stat family reside on the same chromosomes suggesting gene duplication or close evolutionary relationships—a phenomenon observed in various species where Stat duplications have evolved into novel genes through rapid sequence diversification and neofunctionalization (Wang and Levy 2012). Specifically, Stat1-1, Stat1-2, and Stat4 occurred on chromosome 11, Stat2, Stat6-1, and Stat6-2 on chromosome 25, and Stat3 and Stat5b on chromosome 27. This is unusual and sets M. reevesii apart from the conventional distribution of Stat genes seen in other vertebrates.

Our comparative analysis of Stat gene locations in M. reevesii and other reptilian species, whose chromosome-level assemblies enabled the comparison of gene locations, has unveiled intriguing patterns of genomic organization and evolutionary adaptation. The conservation of specific Stat genes on chromosomes across species hints at shared ancestral characteristics and the preservation of crucial signaling pathways. Notably, both M. reevesii and C. mydas exhibit the co-location of Stat1 and Stat4 on Chromosome 11, suggesting a potential common evolutionary history or functional requirement for these genes in immune responses and cellular regulation in both species. Furthermore, the presence of Stat3 on Chromosome 27 in both M. reevesii and C. mydas raises questions regarding the significance of this genomic arrangement. Stat3 is renowned for its pivotal roles in signal transduction and immune responses across species and its shared location may imply conserved functions in the immune systems of these reptilian species. This shared gene organization could offer insights into their adaptive responses to environmental challenges. However, the comparative analysis also highlights distinct genomic features that set M. reevesii apart from other species. Unlike C. mydas, A. carolinensis, and T. elegans, M. reevesii possesses multiple Stat6 genes (Stat6-1 and Stat6-2) on Chromosome 25. This unique arrangement suggests that M. reevesii may have undergone different genomic adaptations, possibly driven by its ecological niche or environmental pressures. These adaptations could be associated with many immune responses and cellular functions to cater to the species’ unique requirements.

One of the most notable findings from our study was the interspecies variation in the number of Stat family members. Specifically, the Stat1 and Stat6 subfamilies exhibited the highest gene diversity. For instance, species such as D. rerio, T. Mexicana, C. mydas, and others featured both Stat1a and Stat1b paralogs, suggesting potential functional specialization or redundancy within these genes. In contrast, M. reevesii was found to possess only Stat5b in this context, implying a unique genetic composition within this species. Comparing Stat gene distribution across species reveals both shared patterns and unique arrangements. In birds like G. gallus and M. gallopavo, Stat1 and Stat4 genes co-locate on Chromosome 7, potentially signifying their significance in avian immune responses. Conversely, M. reevesii displays a distinct organization with Stat1-1, Stat1-2, and Stat4 on Chromosome 11. When examining mammals, humans and cattle share the Chromosome 2 location for Stat1 and Stat4, while M. musculus differs with Chromosome 1. These differences in genomic organization may be indicative of the species’ distinct evolutionary history or adaptation to specific environmental pressures, leading to such genomic divergence. Continuing our investigation into the functional consequences of these genomic variations will play a pivotal role in unraveling the complex interactions between genomic organization, evolutionary history, and ecological adaptations that underlie the immune responses of these turtle species.

Stat genes following LPS infection were significantly upregulated at various time points when compared to the control group. The most pronounced up-regulation occurred at the 12 h followed by a gradual decline until 16-h post-infection, signifying the involvement of Stat genes in the immune response to LPS infection. Up-regulation was greatest in Stat5b suggesting a central role in the immune response. Our immunofluorescence analysis of the Stat5b gene, coincident with a substantial increase in spleen size and weight following LPS infection, suggests compensatory hyperplasia driven by the immune response. Stat5b, originally localized in the cytoplasm but absent in the nucleus of spleen cells in the control group, translocated to both cellular compartments in the LPS-treated group. This suggests a role also for Stat5b in regulating immune responses at the cellular level.

Stat5b is a critical component of the Janus kinase (JAK)-signal transducer and activator of STAT pathway, which is pivotal in transmitting signals from cytokine receptors to the nucleus, thus influencing gene expression (Hashimoto et al 2020). The growth hormone (GH)/insulin-like growth factor (IGF) axis may play a role in fish immune regulation and the Tyrosine-protein kinase (JAK2)/STAT5B pathway activated by GH/IGF signaling. Stat5b is predominantly expressed in the liver and responds to growth factor, GH has been shown to stimulate phosphorylation of JAK2 and activation of STAT5B transcriptional activity (Ahmed and Farquharson 2010), and GH-induced expression of hepatic IGF-1 genes is reduced upon loss of Stat5b function (Lau-Corona et al. 2017). In this study, We also enriched the JAK/STAT5B pathway from our previous gonadal transcriptomic data of M. reevesii (Xiong et al 2019) (Supplementary Fig. 1), although this was not the tissue after being injected with LPS, the expression of related genes on this pathway, such as growth hormone receptor (GHR), SOCS2 (Suppressor of cytokine signaling 2), JAK2, and IGF-1, showed significant differential expression (Supplementary Fig. 2) between the ovaries and testes, suggesting that JAK/STAT5B pathway is highly sensitive, and whether it actively participates in the immune response after LPS post-injection deserves further investigation.

The realm of freshwater turtle immunity relatively underexplored due to the non-model status of these organisms, which presents a significant lack of genomic information and coordination in the study of their cellular functions. This inherent challenge has hampered our understanding of their immune systems. However, previous studies have unearthed key components such as polymeric immunoglobulin receptors and Toll-like receptors in the innate immunity of the Chinese soft-shelled turtle (Xu et al. 2021; Hashimoto et al. 2020; Yao et al. 2007).

In our current study, we conducted an in-depth analysis that led to the identification and characterization of eight Stat genes within the genome of M. reevesii. This comprehensive examination allowed us to gain insights into the structural domains and potential binding sites of these Stat genes, ultimately shedding light on their functional roles. Notably, these structural domains have been conserved across species and are likely to carry specific biological functions. For instance, the TAZ2bind domain, identified in Stat genes, has been demonstrated to function as a transcriptional activator capable of initiating the transcription of specific genes, as documented in previous studies (Kim et al. 2022). Additionally, the TALPID3 domain, another conserved feature of these Stat genes, has been associated with the binding of target genes within the Hedgehog pathway, a critical pathway involved in protein anchoring (Beachy et al. 2010; Ben et al. 2011). These findings provide substantial scientific evidence supporting the functional significance of these conserved domains within the Stat genes of M. reevesii.

The substantial up-regulation of Stat5b in LPS-infected cells strongly implies its active participation in the immune response. Nonetheless, the precise cell type responsible for this response remains to be identified. It might be possible to isolate the different immune cell subpopulations by cell sorting techniques and perform functional assays to determine which cell types exhibit up-regulation of Stat5b after LPS stimulation and show altered immune cell function. Nevertheless, Stat5b presents itself as a promising candidate for a potential immune response marker. This research contributes significantly to our expanding comprehension of immune regulation in non-model organisms, such as freshwater turtles, and underscores the importance of further exploration in this domain.

Conclusion

Our study offers comprehensive insights into the characterization of Stat family genes in M. reevesii, emphasizing their structural diversity, potential functions, and evolutionary relationships. The upregulation of Stat genes following LPS infection, with particular emphasis on Stat5b, suggests their pivotal role in orchestrating the immune response in this species. These findings contribute to our understanding of the molecular mechanisms underlying immune responses in turtles and provide a foundation for future research in this field.

Supplementary Information

Below is the link to the electronic supplementary material.Supplementary file1 (TIF 53812 kb)Supplementary file2 (TIF 6882 kb)Supplementary file3 (XLSX 16 kb)Supplementary file4 (XLSX 18 kb)Supplementary file5 (XLSX 11 kb)