Transcriptomic insights into the immunological similarities and differences among the spleen, head kidney, and trunk kidney in Hemibarbus Labeo

Yaoping Lv; Ling Zhu; Xinming Gao; Qingmin Dai; Zehui Hu; Junkai Lu; Jianhui Tu; Yang Zhao; Yanqin Wu

doi:10.1186/s12864-026-12603-3

. 2026 Feb 4;27:254. doi: 10.1186/s12864-026-12603-3

Transcriptomic insights into the immunological similarities and differences among the spleen, head kidney, and trunk kidney in Hemibarbus Labeo

Yaoping Lv ^1,^#, Ling Zhu ^1,^#, Xinming Gao ^1,^✉, Qingmin Dai ¹, Zehui Hu ², Junkai Lu ³, Jianhui Tu ⁴, Yang Zhao ¹, Yanqin Wu ⁵

PMCID: PMC12964698 PMID: 41639763

Abstract

The spleen and head kidney are important immune organs in fish, and the trunk kidney, while primarily a urinary organ, also plays a significant role in immunity. However, the immunological similarities and differences among these organs have not yet been systematically elucidated in fish. This study investigates the composition of immune cells and gene expression characteristics in the spleen, head kidney, and trunk kidney of Hemibarbus labeo to reveal similarities and differences in their immune functions at both cellular and molecular levels. All three organs contain abundant red blood cells, lymphocytes, and monocytes, with granulocytes particularly prevalent in the head kidney and trunk kidney. A total of 255,871 transcripts and 112,051 unigenes were identified, including 13,739 differentially expressed genes. Genes expressed in the spleen, head kidney, and trunk kidney of H. labeo are enriched in pathways related to pathogen phagocytosis, antigen presentation, and cytokine secretion. However, the main immune-related pathways enriched in each organ differ. Compared to the head kidney and trunk kidney, the spleen shows higher expression levels of genes involved in cytokine-cytokine receptor interactions. In contrast, the head kidney exhibits higher expression of genes related to the hematopoietic cell lineage, the B cell receptor signaling pathway, and Fc gamma R-mediated phagocytosis. The trunk kidney expresses higher levels of genes involved in the leukocyte transendothelial migration pathway. Additionally, neutrophil marker genes such as crp, cxcr2, and ncf1 are more highly expressed in the head kidney and trunk kidney than in the spleen, suggesting that neutrophil-mediated immune functions are more pronounced in these organs. This study provides a molecular foundation for future immunological research on H. labeo and offers valuable insights into the functional similarities and differences among these key immune organs.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12864-026-12603-3.

Keywords: Hemibarbus labeo, Immune organs, Immune cells, Gene expression, Differentially expressed genes, Immune-related signaling pathways

Background

The immune system of fish consists of immune organs, immune cells and immune factors in body fluids [1, 2]. The spleen and kidney are important immune organs in fish and participate in the body’s immune defense response. The spleen is the last immune organ to form in the lymphatic system, and it filters and removes exogenous substances, cellular debris, and senescent blood cells by producing immunoglobulins and various lytic enzymes [3]. The head kidney is the main site of proliferation and differentiation of immune cells. Immune cells in the head kidney can mount a rapid immune defense response to pathogen invasion [4]. As important immune organs in fish, the spleen and head kidney are target organs for disease detection and core organs for fish immunological studies. In studies of pathology and immune responses in diseased fish, the histology, biochemical indices, and transcriptomics of the spleen and head kidney have been a major research focus [5, 6]. Some studies have often analyzed the immunological role of the spleen and head kidney by analyzing the histology of the two organs to identify immune cell types or the biochemical parameters and transcriptomic changes of the two organs in the presence of pathogenic bacterial infections [6–8]. Furthermore, some studies have also analyzed the histological and cell type differences between the two organs [9]. Although the spleen and head kidney are known to play key roles in fish immunity, their immunological similarities and differences have rarely been reported.

In addition to the spleen and head kidney, the immunological role of the trunk kidney in fish has received attention in recent years [10, 11]. The trunk kidney is an important urinary organ in adult fish. It consists of glomeruli, tubules, collecting ducts and other structures involved in the reabsorption of water, salt ions, glucose [12]. The trunk kidney is an important target organ for the study of osmotic pressure regulation mechanisms in fish [12]. It is noteworthy that, histologic studies of the trunk kidney have shown that it contains a variety of immune cells, such as neutrophils and melanin macrophages, suggesting the potential of the trunk kidney to participate in the body’s immunity. Recently, Zhong and Gao [10] found that a large number of immune-related genes were expressed at high levels in the trunk kidney of Pelteobagrus fulvidraco using transcriptomics, and the immune genes Tlr5, Psme1, and Ccl19 were significantly up-regulated in the trunk kidney after stimulation by Aeromonas hydrophila [10]. Sun et al. [13] found that immunological factors such as antiviral genes and pro-inflammatory cytokine genes were up-regulated in the head kidney and trunk kidney of Oncorhynchus mykiss infected with IHNV, and that the expression of TNFα was significantly higher in the trunk kidney than in the head kidney [13]. These studies imply that the trunk kidney plays a significant role in pathogen defense, and it is an important supplementary immune organ in fish, in addition to the spleen and head kidney. Therefore, the role of the trunk kidney in fish immunology and its immunological similarities and differences with the spleen and head kidney deserve to be studied in depth. Research on the immunological functions of these organs can provide critical evidence for preventing and controlling fish diseases, elucidating vaccine response mechanisms, and optimizing immunization strategies [14, 15].

Hemibarbus labeo is a member of the order Cypriniformes, family Cyprinidae, and the genus Hemibarbus. Its meat is delicious and rich in nutrients [16], making it popular among consumers and giving it high economic value and market prospect. Research on the immunology of H. labeo involves antimicrobial peptides and the characterization of their immune functions [17, 18]. However, studies on the function of the main immune organs of H. labeo have rarely been reported. Zhu et al. [9] conducted histological studies on the spleen, head kidney, and trunk kidney of the H. labeo and found that all three organs contained abundant immune cells, such as lymphocytes, granulocytes, and melanin-containing macrophages. Notably, granulocytes were more prevalent in the head kidney and trunk kidney, whereas the spleen featured distinct elliptical structures that may serve as an important blood-spleen barrier. The spleen, head kidney, and trunk kidney of H. labeo exhibit both similarities and differences in cellular composition. Histological features of the trunk kidney suggest that it is an important immune organ in H. labeo. However, the similarities and differences in immunological functions among these three organs require further investigation.

In this study, we constructed a cellular atlas and gene expression profiles of the spleen, head kidney, and trunk kidney of H. labeo using tissue smears and transcriptome sequencing techniques, thereby laying a cytological and molecular foundation for future immunological research on H. labeo. Furthermore, we analyzed and compared the similarities and differences in transcript expression across these three organs, providing insights into the functional similarities and distinctions of major immune organs in fish, as well as offering cytological and molecular evidence supporting the trunk kidney as a significant immune organ. The organ-specific immune signatures identified in this study not only deepen our fundamental understanding of fish immunity but also provide valuable insights for the development of organ- or cell-targeted vaccines. Furthermore, the identified immune-related pathways and genes can serve as molecular markers for disease-resistant selective breeding and early disease detection in H. labeo and other economically important aquaculture species, thereby advancing health management in sustainable aquaculture.

Materials and methods

Sample collection

The experimental fish were collected from the Jinman aquatic seedling farm in Lishui, China. The experimental fish’s body length, total length and body weight were 20.5 ± 0.5 cm, 24.5 ± 0.6 cm, and 133.7 ± 12.8 g, respectively. Prior to the experiment, eight healthy and vigorous adult H. labeo of the same age were selected from the same culture pond and promptly transported to the laboratory. The retrieved experimental fish were temporarily housed in culture buckets in the laboratory and anesthetized with MS-222 (50 mg/L) before dissection. After measurement of total length, body length and weight, the fish were dissected, and the spleen, head kidney and trunk kidney tissues were excised separately. Part of each tissue was used for tissue smear analysis (Fig. 1), while another part was immediately placed in liquid nitrogen and subsequently transferred to a -80 °C freezer for storage, intended for bioinformatics analysis. The schematic workflow of the study is shown in Fig. 1.

Fig. 1 — Schematic workflow of the study

Tissue smear

After removing the spleen, head kidney, and trunk kidney, the organ surfaces were rinsed with PBS buffer to remove blood. The spleen, head kidney, and trunk kidney were then sliced open, and the freshly cut surfaces were gently smeared onto glass slides. The slides were air-dried, stained with Wright-Giemsa stain solution (Solarbio, Beijing, China ), air-dried again, mounted with neutral balsam, and observed and photographed under a NI-U light microscope (Nikon, Tokyo, Japan).

RNA extraction, library construction and sequencing

The total RNA was extract from spleen, head kidney and trunk kidney tissues using TRIzol reagent (Invitrogen, CA, USA) according to the manufacturer’s instructions. The RNA integrity and concentration were analyzed using the Bioanalyzer 2100 system (Agilent Technologies, CA, USA). Qualified RNA was selected for mRNA enrichment using poly-T oligo-attached magnetic beads, followed by fragmentation. Then, cDNA was synthesized using random hexamer primers, and subsequently undergoes end repair, A-tailing, adapter ligation, fragment selection, amplification, and purification. Following library construction, the libraries were sequenced using an Illumina Novaseq 6000 platform to produce 150 bp paired-end reads. Four samples per organ (each from an individual fish) were analyzed, yielding a total of 12 sequenced libraries. The raw sequencing data have been uploaded to the National Center for Biotechnology Information (NCBI; https://www.ncbi.nlm.nih.gov) with the following accession number: SAMN43202401-SAMN43202412.

Quality control and transcript assembly

To obtain clean reads, the data were processed using FASTP software to exclude jointed reads, ploy-N containing reads, and low-quality reads from the raw data. At the same time, Q20, Q30 and GC content were calculated from the clean data. All samples were spliced using Trinity software. On the basis of Trinity stitching, using Corset to polymerize redundant transcript, and improve the detection rate of differentially expressed genes. Using BUSCO (Benchmarking Universal Single-Copy Orthologs) software to evaluate the splicing quality of Trinity.fasta, unigene.fa and cluster.fasta obtained by splicing, and evaluated the accuracy and completeness of the splicing results according to the proportion and completeness of the comparison.

Gene function annotation and identification of Differentially Expressed Genes (DEGs)

With public databases such as Nr, Nt, Pfam, KOG, Swiss-Prot, KEGG, and GO perform gene function annotation. Differential expression analysis of the comparable groups was performed using the DESeq2 R package (1.20.0). The false discovery rate (FDR)-adjusted P-value was obtained after multiple testing correction using the Benjamini-Hochberg method. The threshold for differential expression was set at adjusted P-value ≤ 0.05 and |log₂ (fold change)| ≥ 1.

GO and KEGG enrichment analysis and clustering of DEGs

All DEGs in each comparison were analyzed for GO and KEGG enrichment analysis using GOseq [19] and KOBAS [20], respectively. Cluster analysis of DEGs was performed using an online tool (https://magic.novogene.com/) to identify changes in the expression of DEGs in the spleen, head kidney, and trunk kidney.

Hierarchical clustering analysis of neutrophil marker genes

Neutrophil marker genes were retrieved from the PanglaoDB database (https://panglaodb.se/). The retrieved marker genes were screened and counted in the transcriptome database of H. labeo. And analyzed by hierarchical clustering through the online tool https://magic.novogene.com/ .

Quantitative real-time PCR (qPCR) validation of DEGs

Primers for these genes were designed using Primer 5.0 software (Supplementary Table 1). The eef1a was used as the internal reference gene [21]. Firstly, the RNA was reverse-transcribed into cDNA using the PrimeScript FAST RT Reagent Kit with gDNA Eraser (Takara, Dalian, China) to serve as the template for qPCR analysis. Subsequently, qPCR experiments were performed on a LightCycler96 system (Roche, Basel, Switzerland) using 2 × RealStar Fast SYBR qPCR Mix (GenStar, Beijing, China). The total volume of the reaction was 20 µL, which contained 5 µL of 1: 30 diluted cDNA, 0.5 µL (10 µM) of each primer, 10 µL of 2 × RealStar Fast SYBR qPCR Mix, and 4 µL of ddH2O. The PCR reaction mixture was pre-denatured at 95 °C for 10 min, followed by 40 cycles of 95 °C for 10 s, 60 °C for 10 s, and 72 °C for 10 s. The relative expression levels of genes were calculated using the 2^−∆∆Ct method with four biological replicates and three technical replicates per group. Data are expressed as mean ± standard deviation.

Result

Cell types identified through tissue smear analysis

In tissue smears of the spleen, head kidney, and trunk kidney of H. labeo, various cell types were identified, including red blood cells (RBCs), lymphocytes, neutrophils, basophils, monocytes, and thrombocytes. RBCs are oval with a central nucleus, staining deep blue in the nucleus and light red in the cytoplasm (Fig. 2A). Lymphocytes are classified into large and small types based on size; both are oval-shaped, with light blue cytoplasm and a nucleus that is relatively large compared to the cytoplasmic volume (Fig. 2B). Neutrophils are round or oval, with a nucleus displaced to one side and light red cytoplasm (Fig. 2C). Basophils resemble neutrophils in morphology but have light blue cytoplasm (Fig. 2D). Monocytes are slightly larger, round or oval, with blue cytoplasm and nuclei that are either oval or kidney-shaped and often eccentrically positioned (Fig. 2E). Thrombocytes are smaller, round or oval cells with deeply stained purple nuclei (Fig. 2F).

Fig. 2 — Tissue smear of *H. labeo* spleen, head kidney and trunk kidney. A Red blood cell. B Lymphocyte. C Neutrophilic granulocyte. D Basophilic granulocyte. E Monocyte. F Thrombocytes. G Smear of the spleen. H Smear of the head kidney. I Smear of the trunk kidney. BGC: basophilic granulocyte; MC: monocyte; ML: macrolymphocyte; NGC: neutrophilic granulocyte; RBC: red blood cell; SL: small lymphocyte; TC: thrombocytes

Microscopic examination revealed distinct cellular compositions among the organs. In the spleen, RBCs, lymphocytes, monocytes, and thrombocytes were observed. RBCs were abundant and densely packed, while granulocytes were difficult to identify (Fig. 2G). The head kidney contained all six cell types, with RBCs more sparsely distributed. Neutrophils were numerous and densely aggregated, while basophils were rare (Fig. 2H). The trunk kidney smear characteristics were similar to those of the head kidney, featuring fewer RBCs, monocytes, and both large and small lymphocytes. Neutrophils were abundant and clustered, whereas basophils were very scarce (Fig. 2I).

Sequencing data statistics and transcript splicing

A total of 254,181,290 raw reads were obtained from the 12 cDNA libraries. After filtering out sequences containing junctions, ploy-N and low quality, a total of 248,238,403 clean reads were obtained, with a total base number of 74.48G, and an average base number of 6.21G for each sample. Among them, the GC content of 12 samples ranged from 46.31% to 47.39%, which basically conformed to the normal distribution (Supplementary Table 2). This indicates that the transcriptome sequencing results are good and can meet the requirements for subsequent analysis.

A total of 255,871 transcripts and 112,051 unigene were obtained. The length of these transcripts ranged from 301 bp to 27,277 bp, with an average length of 1,323 bp (Supplementary Fig. 1A). While the average length of unigene is 1055 bp (Supplementary Fig. 1B). After that, we used the software BUSCO to evaluate the effect of transcriptome assembly of the spleen, head kidney and trunk kidney of the adult H. labeo, and the results showed that the integrity of the unigene reached 83.0%, which indicated that the result of transcript splicing and assembly was good (Supplementary Fig. 1C). Principal component analysis of the transcriptome samples showed good correlation between four parallels (Supplementary Fig. 1D).

Gene function annotation

To obtain comprehensive gene function information, the spliced unigene was compared with sequences in public databases such as Nr, Nt, Pfam, KOG, Swiss-prot, KEGG and GO for similarity comparison. All 112,051 unigenes could be matched to at least one of the databases, and 7429 unigenes (6.63%) were successfully matched to seven databases. The fewest unigenes were annotated to the KOG database (9.26%) and the most unigenes were annotated to the Nt database (98.19%) (Supplementary Fig. 1E). In addition, the species with the highest similarity among the annotated sequences were Ctenopharyngodon idella, followed by Megalobrama amblycephala, Anabarilius grahami, Rhinichthys klamathensis goyatoka and Labeo rohita among other carp species (Supplementary Fig. 1F).

KEGG enrichment analysis of individual tissue genes in spleen, head kidney and trunk kidney of H. labeo

In the spleen, a total of 96,846 Unigenes were obtained, and the top 20 pathways enriched by KEGG enrichment analysis for these genes included four common immune-related pathways, including chemokine signaling pathway, antigen processing and presentation, T cell receptor signaling pathway and Fc gamma R-mediated phagocytosis (Fig. 3A).

Fig. 3 — KEGG enrichment analysis of individual tissue genes in spleen, head kidney and trunk kidney of *H. labeo*. A Top 20 pathway dot plots significantly enriched for genes in spleen. B Top 20 pathway dot plots significantly enriched for genes in head kidney. C Top 20 pathway dot plots significantly enriched for genes in trunk kidney. The vertical axis indicates pathway categories; the horizontal axis indicates enrichment factors; the dot sizes indicate the number of DEGs associated with the pathways, and the dot colors show different P values. Red underlines denote commonly recognized immune pathways

In the head kidney, a total of 87,507 Unigenes were obtained. KEGG enrichment analysis of genes expressed in the head kidney revealed that common immune-related pathways among the top 20 pathways include T cell receptor signaling pathway, natural killer cell mediated cytotoxicity, chemokine signaling pathway, endocytosis and lysosome (Fig. 3B).

In the trunk kidney, a total of 81,990 Unigene were obtained. Genes were enriched in multiple pathways, including aldosterone-regulated sodium reabsorption, endocrine and other factor-regulated calcium reabsorption, and proximal tubule bicarbonate reclamation (Supplementary Table 3). Additionally, KEGG enrichment analysis of these genes revealed that common immune-related pathways among the top 20 pathways included Fc gamma R-mediated phagocytosis, leukocyte transendothelial migration, cytokine-cytokine receptor interaction, and endocytosis (Fig. 3C).

In the top 20 enriched pathways, the chemokine signaling pathway and T cell receptor signaling pathway were enriched in both spleen and head kidney. The Fc gamma R-mediated phagocytosis was enriched in the spleen, head kidney, and trunk kidney. The antigen processing and presentation were enriched in the spleen. The endocytosis and lysosome pathways were enriched in the head kidney. The leukocyte transendothelial migration pathway was significantly enriched in the trunk kidney. These results suggest both similarities and differences in the major immunoregulatory pathways among the three organs.

In addition to the top 20 pathways, the spleen, head kidney, and trunk kidney can all accumulate common immune-related signaling pathways such as the B cell receptor signaling pathway, complement and coagulation cascades, and chemokine signaling pathway (Table 2), reflecting their important roles in the immune system of the H. labeo.

Table 2.

Immune pathways significantly enriched in KEGG enrichment analysis of DEGs in different comparison groups

Groups	Major immune pathways	Pathway ID	GeneRatio	up or down
SP vs. HK	PI3K-Akt signaling pathway	ko04151	111\|1005	up
	Cytokine-cytokine receptor interaction	ko04060	53\|1005	up
	MAPK signaling pathway	ko04010	87\|1005	up
	Fc gamma R-mediated phagocytosis	ko04666	14\|198	down
	Complement and coagulation cascades	ko04610	21\|564	down
	B cell receptor signaling pathway	ko04662	20\|564	down
	Hematopoietic cell lineage	ko04640	25\|814	down
SP vs. TK	Cytokine-cytokine receptor interaction	ko04060	53\|814	up
	PI3K-Akt signaling pathway	ko04151	83\|814	up
	Platelet activation	ko04611	43\|814	up
	Hematopoietic cell lineage	ko04640	25\|814	up
	Leukocyte transendothelial migration	ko04670	47\|1427	down
HK vs. TK	B cell receptor signaling pathway	ko04662	17\|198	up
	Intestinal immune network for IgA production	ko04672	9\|198	up
	C-type lectin receptor signaling pathway	ko04625	14\|198	up
	Fc gamma R-mediated phagocytosis	ko04666	14\|198	up

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Analysis of DEGs

A total of 13,739 DEGs were screened in the three comparison groups SP vs. HK, SP vs. TK, and HK vs. TK. There were 3883, 3498, and 1072 up-regulated DEGs and 3035, 6464 and 5409 down-regulated genes in the three comparison groups, respectively. The highest number of DEGs was detected in the SP vs. TK comparison, while the fewest number of DEGs was found in the HK vs. TK comparison (Fig. 4).

Fig. 4 — Differentially expressed gene analysis. A Volcano plots of DEGs in the SP vs. HK comparison. B Volcano plots of DEGs in the SP vs. TK comparison. C Volcano plots of DEGs in the HK vs. TK comparison. Red dots indicate up-regulated genes, green dots indicate down-regulated genes, and blue dots indicate genes with no significant differences. SP: spleen. HK: head kidney. TK: trunk kidney

GO enrichment analysis and KEGG enrichment analysis of DEGs

To better investigate the function of these DEGs, we performed GO enrichment analysis of all DEGs. The DEGs were significantly enriched in single-organism process, single-organism cellular process and membrane, and other terms (Supplementary Fig. 4). Terms enriched for DEGs in the SP vs. HK comparison group include signaling, molecular transducer activity, extracellular region, and other factors (Supplementary Fig. 5A). Terms enriched for DEGs in the SP vs. TK comparison group included signaling, molecular transducer activity, extracellular region (Supplementary Fig. 5B). Terms enriched for DEGs in the HK vs. TK comparison group included oxidoreductase activity, transmembrane transport, vitamin metabolic process, and other terms (Supplementary Fig. 5C).

KEGG enrichment analysis was performed on all DEGs, identifying 39 significantly enriched pathways (Supplementary Table 4), including common immune-related signaling pathways such as cytokine-cytokine receptor interactions, hematopoietic cell lineage, leukocyte transendothelial migration, and Fc gamma R-mediated phagocytosis, indicating that the expression of immune-related genes in the spleen, head kidney, and trunk kidney differs, suggesting that the three immune organs may exhibit certain differences in immune function regulated by related immune pathways.

Perform KEGG enrichment analysis on each gene that is upregulated or downregulated in each comparison group. There are 41 up- or down-regulated significantly enriched KEGG pathways in the SP vs. HK comparison group (Fig. 5A and B), among which the up-regulated genes were significantly enriched in three common immune-related pathways: the PI3K-Akt signaling pathway, cytokine-cytokine receptor interactions, and MAPK signaling pathway; Downregulated genes were significantly enriched in four common immune-related pathways: Fc gamma R mediated phagocytosis, complement and coagulation cascades, B cell receptor signaling pathways, and hematopoietic cell lineage (Tables 1 and 2). In addition, among the top 20 pathways with downregulated gene enrichment, there were also common immune-related signaling pathways such as leukocyte transendothelial migration, but the enrichment was not significant. These results suggest that immune functions related to the PI3K-Akt signaling pathway, cytokine-cytokine receptor interactions, and MAPK signaling pathway may be stronger in the spleen, while immune functions regulated by signaling pathways such as Fc gamma R-mediated phagocytosis, complement and coagulation cascades, and B cell receptor signaling pathways may be stronger in the head kidney.

Fig. 5 — KEGG enrichment analysis of DEGs. A Top 20 down-regulated pathways enriched in SP vs. HK. B Top 20 up-regulated pathways enriched in SP vs. HK. C Top 20 down-regulated pathways enriched in SP vs. TK. D Top 20 up-regulated pathways enriched in SP vs. TK. E Top 20 down-regulated pathways enriched in HK vs. TK. F Top 20 up-regulated pathways enriched in HK vs. TK. Asterisks indicate significant enrichment. Red underlines denote commonly recognized immune pathways

Table 1.

Comparative results of enrichment of major immune pathways in spleen, head kidney and trunk kidney

Immune-related pathways	Enrichment results by organ
Immune-related pathways	SP	HK	TK
Cytokine-cytokine receptor interaction	+++	+	+
PI3K-Akt signaling pathway	+++	+	+
MAPK signaling pathway	+++	+	++
Antigen processing and presentation	+*	+	+
Platelet activation	++ (SP vs. TK)	+	+
Complement and coagulation cascades	+	++ (HK vs. SP)	+
Hematopoietic cell lineage	++	+++	+
B cell receptor signaling pathway	+	+++	+
Leukocyte transendothelial migration	+	+	++* (TK vs. SP)
Fc epsilon RI signaling pathway	+	+	+
Fc gamma R-mediated phagocytosis	+*	+++*	+*
Toll-like receptor signaling pathway	+	+	+
Natural killer cell mediated cytotoxicity	+	+*	+
Chemokine signaling pathway	+*	+*	+
T cell receptor signaling pathway	+*	+*	+
Intestinal immune network for IgA production	+	++ (HK vs. TK)	+

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+ indicates that total gene enrichment analysis of a single tissue type shows the presence of this pathway. ++ indicates that the pathway was significantly up-regulated in one comparison group. +++ indicates that the pathway was significant enrichment in two comparison groups. * indicates that the total gene enrichment analysis of a single tissue type showed pathways in the top 20

SP spleen, HK head kidney, TK trunk kidney

There were 75 up- or down-regulated significantly enriched KEGG pathways in the SP vs. TK comparison group (Fig. 5C and D, Supplementary Table 5). Among the downregulated genes, those significantly enriched in the leukocyte transendothelial migration pathway were identified, while the upregulated genes were significantly enriched in four common immune-related pathways: cytokine-cytokine receptor interaction, PI3K-Akt signaling pathway, platelet activation, and hematopoietic cell lineage (Table 2). This suggests that immune functions related to leukocyte transendothelial migration may be stronger in the trunk kidney. while the spleen may exhibit stronger immune functions regulated by signaling pathways such as cytokine-cytokine receptor interactions, the PI3K-Akt signaling pathway, platelet activation, and the hematopoietic cell lineage.

There were 56 up- or down-regulated significantly enriched KEGG pathways in the HK vs. TK comparison group (Fig. 5E and F, Supplementary Table 5). Among the downregulated genes, there was no significant enrichment in common immune-related pathways. In contrast, the upregulated genes were significantly enriched in pathways such as the B cell receptor signaling pathway, the intestinal immune network for IgA production, the C-type lectin receptor signaling pathway, Fc gamma R-mediated phagocytosis, and other common immune-related pathways (Table 2), suggesting that these pathways may play a stronger role in regulating immune functions in the head kidney. Additionally, among the top 20 enriched pathways for the upregulated genes, common immune-related signaling pathways such as the Toll-like receptor signaling pathway, cytokine-cytokine receptor interactions, T cell receptor signaling pathway, chemokine signaling pathway, and natural killer cell-mediated cytotoxicity were also present, though the enrichment was not significant (Tables 1 and 2).

Cluster analysis of DEGs and KEGG enrichment analysis of genes in related cluster subgroups

Cluster analysis was performed on all DEGs, which were classified into 8 different gene subclusters (Fig. 6). The subcluster 1 includes 2853 genes with the lowest expression in SP samples, the highest expression in HK samples, and relatively lower expression in TK samples (Fig. 6). KEGG enrichment analysis of these genes identified 36 significantly enriched pathways, and 10 immune-related pathways, including Fc gamma R-mediated phagocytosis, B-cell receptor signaling pathway and Leukocyte transendothelial migration (Fig. 7A). The subcluster 2 included 547 genes whose expression was significantly higher in HK and TK samples than in SP samples (Fig. 6). KEGG enrichment analysis of these genes was significantly enriched to complement and coagulation cascades and hematopoietic cell lineage (Fig. 7B).

Fig. 7 — KEGG enrichment analysis of eight different subclusters of DEGs. A-H The top 20 pathways enriched across subclusters 1 to 8. Asterisks indicate significant enrichment. Red underlines denote commonly recognized immune pathways

The subcluster 3 included 3679 genes; subcluster 4 included 1667 genes; subcluster 7 included 319 genes. The genes of these subclusters had the highest expression at TK (Fig. 6). The genes in subpopulations 3 and 4 were significantly enriched in 11 and 41 pathways, respectively, but neither was significantly enriched in immune-related pathways (Fig. 7C and D); the genes in subpopulation 7 were significantly enriched in 19 pathways, including immune-related cell pathways such as leukocyte transendothelial migration (Fig. 7G).

The subcluster 5 included 2061 genes; subcluster 6 included 2147 genes; subcluster 8 included 466 genes. The genes of these subclusters had the highest expression in SP samples. The genes of the subcluster 5 were significantly enriched to common immune-related pathways cytokine-cytokine receptor interactions and PI3K-Akt signaling pathway (Fig. 7E). The genes in subcluster 6 were not significantly enriched in immune-related pathways, while the genes in subcluster 8 were significantly enriched in common immune-related signaling pathways such as hematopoietic cell lineage, cytokine-cytokine receptor interactions, and PI3K-Akt (Fig. 7F and H).

Hierarchical clustering (heatmap) analysis of neutrophil marker gene expression

Expression of 24 neutrophil marker genes was analyzed in the spleen, head kidney and trunk kidney. These marker genes are all DEGs. The results showed that il1b, itgax, csf3r, mmp9, c5ar1, s100a4, cfp, pstpip1, sorl1, crp, tlr2, serpinb1, snx20, cxcr2, ncf1, adam8, and itgam were highly expressed in the head kidney and trunk kidney, whereas their expression was relatively low in the spleen. Three genes, slc1a5, ptgs2, and lyst, had relatively high expression in the spleen and trunk kidney, and low expression in the head kidney. Four genes, pecam1, mylk, mme, and adpgk, had high expression in the spleen, and relatively low expression in the head kidney and trunk kidney (Fig. 8).

Fig. 8 — Heatmap analysis of marker gene expression in neutrophils from spleen, head kidney and trunk kidney. SP: spleen. HK: head kidney. TK: trunk kidney

Validation of RNA-Seq results via qPCR

To confirm the reliability of the RNA-seq results, qPCR experiments were conducted to analyze the expression levels of nine DEGs. As shown in Fig. 9, the qPCR results were compared with the RNA-seq results, and the gene expression levels detected by qPCR were consistent with those from the RNA-seq data, indicating that the transcriptome data were highly reliable.

Discussion

Functions of the spleen and its transcript characterization

The spleen, as a secondary lymphoid organ of the fish immune system, is capable of recognizing and removing pathogens [22]. It also performs physiological functions such as hematopoiesis, blood storage, blood filtration, and the renewal of RBCs and platelets [23]. According to research, the spleen contains a large number of immune cells such as lymphocytes and macrophages, as well as elliptical structures, which play an important role in the body’s immune system [9]. This study established a tissue smear atlas and gene expression profile of the H. labeo spleen. The spleen was found to contain RBCs, lymphocytes, monocytes, and other cell types. Genes involved in immune-related signaling pathways, such including chemokine signaling pathway, cytokine-cytokine receptor interactions, antigen processing and presentation, and Fc gamma R-mediated phagocytosis, are abundant, consistent with the spleen’s role as an important immune organ. When foreign antigens enter the body, antigen-presenting cells (APCs) process them and present mature antigen peptides to T lymphocytes, thereby initiating a specific adaptive immune response [24]. During this process, various cells release cytokines, low-molecular-weight soluble proteins induced by immunogens, mitogens, or other stimuli. Cytokines play a pivotal role in regulating both innate and adaptive immunity [25–27]. Among these, the key subclass of chemokines, as specialized cytokines, guides leukocyte migration to sites of infection or inflammation, thereby coordinating cellular immune responses [28, 29]. Aggregated and activated immune cells eliminate pathogens through effector mechanisms such as phagocytosis. Among these, Fc gamma receptor-mediated phagocytosis represents a critical pathway for the engulfment and clearance of opsonized foreign substances. After Fc gamma R-mediated stimulatory signals enter the cell, they can drive actin polymerization and phagosome formation, thereby efficiently clearing pathogens and other harmful substances [30]. In this study, we found that chemokine signaling pathway and cytokine-cytokine receptor interaction were significantly enriched in the spleen, indicating it, as a central hub of cytokines, necessitates an immune response. Significant enrichment of the Fc gamma R-mediated phagocytosis signaling pathway implies the presence of splenic cells capable of phagocytosis of processed pathogenic bacteria, which is consistent with the presence of a large number of melanophagic macrophages in the spleen [9]. Moreover, studies have shown that fish RBCs are not only capable of expressing various inflammatory cytokines but can also secrete tumor necrosis factor alpha and interferon gamma, and possess phagocytic activity [31, 32]. Our study found a significant accumulation of RBCs in the spleen, further supporting the spleen’s critical role in cytokine-mediated immune regulation and the phagocytic clearance of pathogens. All of these results suggest that the H. labeo spleen plays an important role in pathogen phagocytosis, antigen presentation, and cytokine secretion, strongly reinforcing its functional identity as a secondary lymphoid organ, the key site where adaptive immune responses are initiated and “immune training” occurs. In the inflammatory response, the spleen may be able to kill pathogenic bacteria by secreting cytokines, and at the same time stimulate T lymphocytes to generate an immune response by processing antigen through the antigen presenting cells in H. labeo.

Functions of the head kidney and its transcript characterization

The head kidney is an important hematopoietic and immune organ in fish, and there are abundant immune cells in the tissue, including lymphocytes, neutrophils and macrophages [4, 9]. Lymphocytes secrete cytokines and participate in antibody production [27, 33–36]. Macrophages and neutrophils can phagocytose pathogens [37]. This study revealed that the head kidney contains RBCs, lymphocytes, neutrophils, basophils, monocytes. Genes in the head kidney are enriched in pathways including Fc gamma R-mediated phagocytosis, endocytosis, and lysosome, highlighting the important role of the H. labeo head kidney in pathogen phagocytosis. This finding aligns with Zhu et al. [9], who reported abundant melanin-containing macrophages and neutrophils in the H. labeo head kidney. The transcriptomic information in this study also showed that a large number of genes related to the T cell receptor signaling pathway and chemokine signaling pathway in the head kidney, indicating the presence of abundant T lymphocytes and chemokines in this organ. Therefore, the H. labeo head kidney may perform important immune functions through pathogen phagocytosis and chemokine secretion under the stimulation of pathogens. These findings support the head kidney’s dual function as both a hematopoietic organ and a primary and secondary lymphoid organ in H. labeo.

Function of the trunk kidney and its transcript characterization

The trunk kidney is the main excretory organs of fish, responsible for filtering blood, removing waste products and regulating the water and electrolyte balance of the body [12]. Histological studies have shown that the trunk kidney has a structure consisting of renal tubules, collecting ducts, and renal corpuscles, with the lumens containing abundant brush borders that promote the reabsorption of water and salt ions [38]. This study found that genes in the trunk kidney were enriched in multiple pathways, including aldosterone-regulated sodium reabsorption, endocrine and other factor-regulated calcium reabsorption, and proximal tubule bicarbonate reclamation, which are related to ion reabsorption and osmotic pressure balance. The identification of these pathways and related genes provides a molecular basis for studying renal osmoregulation and reabsorption in the H. labeo.

According to research, the trunk kidney plays an important role in the body’s immune system. For example, The trunk kidney of the H. labeo contains a large number of lymphocytes and phagocytes [9]. After fish are infected with pathogens, the expression of immune-related genes in the trunk kidney is upregulated [8, 13]. This study identified lymphocytes, neutrophils, basophils, monocytes, and other cell types in the trunk kidney using tissue smears, further supporting this view. In addition, we found that genes expressed in trunk kidney were enriched to numerous immune-related pathways, including Fc gamma R-mediated phagocytosis, leukocyte transendothelial migration, B cell receptor signaling pathway, and cytokine-cytokine receptor interaction. Among them, Fc gamma R-mediated phagocytosis and leukocyte transendothelial migration were among the top 20 enriched pathways. Fc gamma R activates phagocytic vesicles in macrophages to phagocytose the recognized substance into the macrophage interior. Macrophages release a number of digestive enzymes, such as proteases and nucleases, to digest the phagocytosed material [39]. Leukocyte transendothelial migration plays an important role in the body’s immune surveillance and inflammatory response and enrichment of the signaling pathway implies an important role for the trunk kidney in these response [40]. The trunk kidney is close to the ventral surface of the spine, usually reaching the full length of the body cavity. The enrichment of the leukocyte trans-endothelial migration signaling pathway may be more conducive to a rapid response by the organism. These results suggest the important role of the trunk kidney in pathogen phagocytosis, providing cellular and molecular biological evidence for the important immunological role of the trunk kidney in H. labeo and supporting its role as a secondary lymphoid organ in fish.

Transcriptomic insights into the immunological similarities and differences among the spleen, head kidney, and trunk kidney in H. labeo

Our previous study found that the spleen, head kidney, and trunk kidney of H. labeo are rich in immune cells, including melanin macrophages, granulocytes, and lymphocytes [9]. The similar results have also been reported in other fish species, such as Ctenopharyngodon Idella [41]. In addition to having a similar composition of immune cells, they also have some differences. For instance, we found that the spleen of H. labeo is not as rich in granulocytes as the head and trunk kidneys, but has some specific immunologically relevant structures, such as ellipsoids [9]. These histologic similarities and differences suggest that the three organs may have synergistic and complementary roles in fish immunity. Elucidating the organ-specific functions in fish will greatly benefit future research on vaccine response mechanisms and the optimization of immunization strategies [14, 15]. This study compared the similarities and differences in transcript expression among the three organs to provide insights into immune synergy and complementary roles of the spleen, head kidney and trunk kidney.

This study found several immune-related pathways among the top 20 pathways enriched in gene expression in the three organs. Similar to our histological findings [9], gene enrichment analysis of the spleen, head kidney, and trunk kidney revealed several similar immune-related pathways, including Fc gamma R mediated phagocytosis, chemokine signaling, and T cell receptor signaling. These results suggested that spleen, head kidney and trunk kidney play a role in antigen phagocytosis, antigen presentation, and secretion of cytokines. The three organs may jointly produce phagocytosis reaction to engulf pathogens when the pathogen invades, and at the same time process antigens through phagocytes, present mature antigens to T lymphocytes, stimulate the immune response of the body, resist the invasion, and ensure the health of the body. Genes involved in hematopoietic cell lineage, complement and coagulation cascades are also expressed in the spleen, head kidney and trunk kidney. Hematopoietic cell lineage is a key pathway for the body to generate T-cells, B-cells, and natural killer cells, all of which are important in the regulation of the immune system [42]. Complement is an important molecule associated with nonspecific immunity in fish, and complement molecules can clear pathogenic microorganisms such as bacteria, fungi, and viruses through the complement pathway [43]. These results suggest that the spleen, head kidney, and trunk kidney are capable of producing B cells, T cells, and complement molecules, which work synergistically to defend against invading pathogenic bacteria.

In addition to the aforementioned similarities, we observed differences among the three organs in various immune-related pathways. We observed that the complement and coagulation cascades pathway exhibited higher gene expression levels in the head kidney compared to the spleen. However, a previous study in Squaliobarbus curriculus reported that the complement and coagulation cascade pathway was exclusively enriched in the spleen following grass carp reovirus infection [44], suggesting that this pathway can also be rapidly activated in the spleen during infection. Additionally, we found that gene expression levels involved in the cytokine-cytokine receptor interaction pathway were higher in the spleen than in the head kidney and trunk kidney, and gene expression levels involved in the PI3K-Akt signaling pathway and MAPK signaling pathway were higher in the spleen than in the head kidney. Cytokines include anti-inflammatory factors, pro-inflammatory factors, and chemokines, which can guide white blood cells to migrate to sites of inflammation or damaged tissue, participate in inflammatory responses, and regulate the functions of T cells and B cells to promote antibody production [5]. PI3K-Akt signaling pathway is involved in cell growth, proliferation, apoptosis, and inflammatory responses [45]. MAPKs are a group of serine-threonine protein kinases [46], which are involved in the regulation of a variety of cellular processes such as apoptosis and stress response. MAPK signaling is regulated by various feedback mechanisms and can also collaborate with other pathways to control gene transcription [46]. As a regulatory center of signal transduction [13], it plays an important role in cell proliferation, apoptosis, and immune responses [46]. The high expression of genes involved in these signaling pathways in the spleen suggests that they play a stronger role in the immune responses regulated by these signaling pathways.

In the head kidney, we found higher expression of genes involved in the B-cell receptor signaling pathway, Fc gamma R-mediated phagocytosis and hematopoietic cell lineage. In a comparative transcriptome analysis of the head kidney and trunk kidney of the yellow catfish (Pelteobagrus fulvidraco) [10], the B-cell receptor signaling pathway was found to be significantly enriched in the head kidney, consistent with our findings. B-cell receptors recognize various extracellular antigens during bacterial and viral infections, allowing host defense, and also prevent dysregulated activation of self-reactive B cells, thereby inducing autoimmunity in secondary lymphoid organs [47]. Genes involved in the B cell receptor signaling pathway are expressed at higher levels in the head kidney than in the spleen and trunk kidney, suggesting that the head kidney plays a stronger role than the trunk kidney and spleen in recognizing and binding antigens and transmitting antigenic signals. Phagocytosis is an important innate immune defense. Phagocytes contain monocytes, macrophages, and various granulocytes, which can kill pathogens by secreting active substances [39]. In the study, gene expression involved in Fc gamma R-mediated phagocytosis and the lysosomal pathway is higher in the head kidney than in the trunk kidney and spleen, indicating that the number of phagocytes in the head kidney is greater than in the spleen and trunk kidney, or that its phagocytic capacity is stronger. The head kidney is the production hub for immune cells and can autonomously differentiate into RBCs, lymphocytes, and various granulocytes [41]. Gene expression of hematopoietic cell lineage is higher in the head kidney than in the spleen and trunk kidney, suggesting it can generate more immune cells such as granulocytes and lymphocytes, consistent with its function as a primary lymphoid organ.

In the trunk kidney, this study found that gene expression involved in leukocyte transendothelial migration was higher than that in the spleen. Leukocytes are able to attach to the vessel wall, move locally along the wall to the endothelial border through the blood circulation, cross the endothelium and the subendothelial basement membrane, and migrate through the interstitial tissues to the site of infection, where they come into direct contact with pathogens, phagocytose and kill pathogens [40]. Gene expression involved in the leukocyte transendothelial migration pathway is higher in the trunk kidney than in the spleen, suggesting that the trunk kidney plays a stronger role in leukocyte transendothelial cell reactions. However, no systematic studies have yet been conducted on the crucial process of leukocyte transendothelial migration during immune responses in the trunk kidney.

Tissue smear observations revealed a low number of granulocytes in the spleen, making them difficult to identify, which is consistent with the histological findings of Zhu et al. [9]. This study analyzed the expression characteristics of several neutrophil marker genes and found that most of these markers, including crp, cxcr2, and ncf1, were expressed at lower levels in the spleen than in the head kidney and trunk kidney, providing molecular-level support for the tissue smear observations. Neutrophils are key phagocytes in the innate immune system, capable of responding to chemotactic signals from bacterial peptides and inflammatory cytokines to migrate precisely to sites of infection. They eliminate pathogens through multiple mechanisms including phagocytosis, reactive oxygen species (ROS) production, cytokine release, and degranulation [48, 49]. The release of ROS triggers local oxidative stress [50]. Therefore their abundant presence in spleen can destroy carrier RBCs which can hinder the immunological training of other immune cells like macrophages. These factors may contribute to the relatively low number of neutrophils in the spleen under steady-state conditions. These findings provide insights into the synergistic interactions and functional complementarity among immune organs in maintaining fish health.

Although this study systematically elucidates the immunological functional similarities and differences among the spleen, head kidney, and trunk kidney, it primarily relies on tissue smear observations and transcriptomic data, lacking corresponding functional experimental validation. Moreover, the current analysis reflects only the baseline immune status under steady-state conditions and fails to dynamically capture the temporal dynamics and regulatory mechanisms of organ-specific immune responses following pathogen invasion. To gain a deeper understanding of the specific roles of the spleen, head kidney, and trunk kidney in fish immunity and their significance for overall health, more comprehensive comparative studies are urgently needed to profile the dynamic changes in immune responses and gene expression across these organs post-infection. Furthermore, the integration of cutting-edge technologies such as single-cell RNA sequencing holds great promise for revealing cellular heterogeneity and providing high-resolution insights into the organ-specific immune functions at the single-cell level.

Conclusions

This study constructed tissue smear atlases and gene expression profiles of the spleen, head kidney, and trunk kidney in H. labeo. The results revealed that all three organs harbor abundant immune cells and exhibit substantial expression of immune-related genes, although the expression levels of these genes vary across organs. Specifically, the spleen is likely involved in regulating and integrating immune signals, the head kidney appears responsible for the generation and maturation of immune cells, and the trunk kidney may play a key role in mediating their migration. However, these distinct functions are maintained primarily under normal, healthy conditions; upon infection, the organs may operate in a more coordinated or similar manner. Additionally, the spleen was found to contain fewer neutrophils and show lower expression of associated genes, a phenomenon whose biological significance warrants further investigation. These findings lay a foundation for immunological research in H. labeo and help clarify the immunological roles of the spleen, head kidney, and trunk kidney at both cellular and molecular levels. Notably, the immune-related pathways and genes identified in this study can serve as potential molecular markers for disease-resistant selective breeding and early disease detection in H. labeo. To gain deeper insights into the immune functions of these organs and their importance to overall organismal health, further comparative studies on their immune responses and dynamic gene expression profiles following pathogen challenge are clearly warranted.

Supplementary Information

Supplementary Material 1.^{(18.2MB, docx)}

Acknowledgements

The authors would like to express their sincere gratitude to Jinman Mei, Zufei Mei and Junyi Li for their help with sample collection, and to Fei Xu for the technical guidance during the operation of the LightCycler96 system.

Authors’ contributions

Yaoping Lv: Conceptualization, Data curation, Funding acquisition, Investigation, Methodology, Writing – original draft, Writing – review & editing. Ling Zhu: Data curation, Formal analysis, Investigation, Methodology, Software, Visualization, Writing – original draft, Writing – review & editing. Xinming Gao: Conceptualization, Funding acquisition, Investigation, Methodology, Project administration, Software, Writing – original draft, Writing – review & editing. Qinmin Dai: Formal analysis, Methodology, Supervision, Validation, Writing – review & editing. Zehui Hu: Methodology, Validation, Writing – review & editing. Junkai Lu: Conceptualization, Methodology, Validation, Visualization, Writing – review & editing. Jianhui Tu: Methodology, Resources, Writing – review & editing. Yang Zhao: Investigation, Methodology, Writing – review & editing. Yanqin Wu: Methodology, Resources, Writing – review & editing.

Funding

This research was funded by the Joint Fund of Zhejiang Provincial Natural Science Foundation of China (ZCLLSSZ26C1901), the Lishui Science and Technology Plan Project (2024GYX13), the Scientific Research Foundation for Doctor of Lishui University (QD2322), and the Talents Leading Programme of One Hundred Doctors in One Hundred Enterprises in Lishui City.

Data availability

The raw sequencing data have been deposited in the National Center for Biotechnology Information (NCBI; https://www.ncbi.nlm.nih.gov ) under accession numbers SAMN43202401 to SAMN43202412. Additional data are available upon reasonable request from the corresponding author.

Declarations

Ethics approval and consent to participate

The principles and procedures of the sampling methods were in accordance with the requirements of the governing regulation for the use of experimental animals in Zhejiang Province (Zhejiang provincial government order No. 263, released in 17 August, 2009, effective from 1 October, 2010). The animal study protocol was reviewed and approved by the Ethics Committee of Lishui University (Grant No: STD009).

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Yaoping Lv and Ling Zhu contributed equally to this work.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1.^{(18.2MB, docx)}

Data Availability Statement

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PERMALINK

Transcriptomic insights into the immunological similarities and differences among the spleen, head kidney, and trunk kidney in Hemibarbus Labeo

Yaoping Lv

Ling Zhu

Xinming Gao

Qingmin Dai

Zehui Hu

Junkai Lu

Jianhui Tu

Yang Zhao

Yanqin Wu

Abstract

Supplementary Information

Background

Materials and methods

Sample collection

Fig. 1.

Tissue smear

RNA extraction, library construction and sequencing

Quality control and transcript assembly

Gene function annotation and identification of Differentially Expressed Genes (DEGs)

GO and KEGG enrichment analysis and clustering of DEGs

Hierarchical clustering analysis of neutrophil marker genes

Quantitative real-time PCR (qPCR) validation of DEGs

Result

Cell types identified through tissue smear analysis

Fig. 2.

Sequencing data statistics and transcript splicing

Gene function annotation

KEGG enrichment analysis of individual tissue genes in spleen, head kidney and trunk kidney of H. labeo

Fig. 3.

Table 2.

Analysis of DEGs

Fig. 4.

GO enrichment analysis and KEGG enrichment analysis of DEGs

Fig. 5.

Table 1.

Cluster analysis of DEGs and KEGG enrichment analysis of genes in related cluster subgroups

Fig. 6.

Fig. 7.

Hierarchical clustering (heatmap) analysis of neutrophil marker gene expression

Fig. 8.

Validation of RNA-Seq results via qPCR

Fig. 9.

Discussion

Functions of the spleen and its transcript characterization

Functions of the head kidney and its transcript characterization

Function of the trunk kidney and its transcript characterization

Transcriptomic insights into the immunological similarities and differences among the spleen, head kidney, and trunk kidney in H. labeo

Conclusions

Supplementary Information

Acknowledgements

Authors’ contributions

Funding

Data availability

Declarations

Ethics approval and consent to participate

Consent for publication

Competing interests

Footnotes

References

Associated Data

Supplementary Materials

Data Availability Statement

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