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This study investigated the effects of acute ammonia nitrogen (NH3-N) exposure on kidney antioxidant ability and phosphatases and related gene expression in juvenile yellowfin tuna (Thunnus albacares). The 180 juvenile yellowfin tuna (260.39 ± 55.99 g, 22.33 ± 2.28 cm) were exposed to ammonia for 6, 24, and 36 h using natural seawater (0 mg/L) as a control and NH3-N at 5 and 10 mg/L. The lipid peroxidation byproduct malondialdehyde (MDA) and the levels of antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-PX), alkaline phosphatase (AKP), and acid phosphatase (ACP), were measured using the colorimetric method in the trunk kidney to determine changes in antioxidant ability and phosphatase activity of juvenile yellowfin tuna exposed to NH3-N. Results indicated that, at 36 h, MDA, SOD, CAT, and GSH-PX levels rose in the 5 mg/L group versus the control. In the 10 mg/L group, MDA and SOD, CAT, and GSH-PX activities significantly increased after 24 and 36 h exposure compared to the control. Phosphatases play a pivotal role in the immune system. AKP activity significantly increased at 6 h, and ACP activity markedly rose at 36 h in the 5 mg/L group versus the control. Real-time fluorescence quantitative PCR was applied to detect alterations in the antioxidant genes SOD2, CAT, and glutathione peroxidase 1b (GPX1b) and immune cytokines-related genes Interleukin 10 (IL-10) and Interleukin 6 receptor (IL-6r) expression in the head kidney in juvenile tuna. Relative to the control, antioxidant gene expression in the 5 mg/L group significantly rose at 6 and 36 h, and in the 10 mg/L group, SOD2 and GPX1b were significantly elevated at 36 h. Compared to the control group, IL-10 expression in the 5 mg/L group significantly increased at 6 h, whereas IL-6r expression decreased. In the 10 mg/L group, both IL-10 and IL-6r levels were observed to be lower. Low ammonia nitrogen concentrations boost antioxidant defenses, phosphatase activities, and gene expression levels, whereas higher levels may induce suppressive effects. In yellowfin tuna juvenile farming, NH3-N concentration significantly affects the health of the juveniles. When the NH3-N concentration is between 5–10 mg/L, the stress duration should be limited to 24 h; if the concentration is below 5 mg/L, the stress duration can be extended to 36 h. [ABSTRACT FROM AUTHOR]

Long noncoding RNAs (lncRNAs) are crucial regulators of various biological processes. However, it remains unclear how lncRNAs can be involved in the immune response to Nocardia seriolae invasion in largemouth bass (Micropterus salmoides). Here we leveraged the whole transcriptome RNA-seq technique to screen lncRNAs associated with the head kidney infected with Nocardia seriolae. A total of 5,661 lncRNAs were identified, comprising 2,427 known lncRNAs and 3,234 novel lncRNAs. Among these, 531 lncRNAs were found to be differentially expressed, with 213 showing increased expression and 318 showing reduced expression. In these differentially expressed lncRNAs, 176 lncRNAs may have cis-regulatory relationships with 243 protein-coding genes. For example, MSTRG.17818.1, MSTRG.10160.1 target mapk1 and rela, respectively, thereby regulating gene expression. We then exploited Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) to analyze the enrichment of target genes of lncRNAs. These target genes were found to be significantly enriched in immune signaling pathways, including RIG-I-like receptor signaling and Toll-like receptor signaling. Furthermore, we harnessed a total of 12 mRNAs, 33 miRNAs, and 167 lncRNAs to construct two lncRNA-miRNA-mRNA network maps. CeRNA maps revealed that some lncRNAs can competitively bind to the same miRNA and affect the expression of immune-related genes. For instance, MSTRG.13668.1 and MSTRG.5180.1 can simultaneously bind to mir-155–5p to increase irak4 expression. These findings suggest that lncRNAs may participate in the immune response process of infected largemouth bass with Nocardia seriolae. Overall, this work presents a thorough lncRNAs expression profile of Nocardia seriolae-infected largemouth bass and offers insights into the ceRNA regulatory processes that influence fish's immune response to bacterial infections.

The anatomical and histological characteristics of spleen, head kidney and trunk kidney of Hemibarbus labeo were studied by dissection, paraffin sections and H.E staining. The results showed that the spleen is single with dark red. It is applanate and oval, and is separated into many lobes by the connective tissue diaphragm from the capsule. The boundary between red pulp and white pulp in the lobes is not obvious. The spleen contains many of the red blood cells, lymphocytes and melano-macrophages, and the blood vessels are rich. The kidney can be obviously divided into the head kidney and the trunk kidney. The head kidney is located above the diaphragm of the back of the heart at the front end of the body cavity, divided into left and right lobes, and distributed symmetrically. The parenchyma of the head kidney has no nephron, and is mainly composed of lymphoid tissue, including red blood cell centers and white blood cell canters. The head kidney also contains many of red blood cells, lymphocytes, granulocytes, and a certain number of melano-macrophages, which are similar to the spleen. The trunk kidney is located in the thoracic and abdominal segments of the fish body, and close to the two sides of the spine on the back of the body cavity. Its parenchymal part is mainly composed of nephron, collecting segment and lymphoid tissue. Many of red blood cells, lymphocytes, granulocytes, and melano-macrophages can be observed in the lymphatic tissue. These results indicated that the spleen and head kidney are important immune and hematopoietic organs, and the trunk kidney also plays an important role in immunity and hematopoiesis in addition to urinary function in H. labeo. [ABSTRACT FROM AUTHOR]

The physiology of ectothermic animals, including fish, is strictly regulated by season-related external factors such as temperature or photoperiod. The immune response and the production of hormones, such as estrogens, are therefore also subject to seasonal changes. This study in common carp aimed to determine how the season affects the estrogen system and the immune response, including the antibacterial response during Aeromonas salmonicida infection. We compared the immune reaction in spring and autumn in the head kidney and liver and found that carp have higher levels of blood 17β-estradiol in autumn, while in the liver of these fish there is a higher constitutive expression of genes encoding vitellogenin, estrogen receptors and Cyp19 aromatase than in spring. Fish sampled in autumn also exhibited higher expression of immune-related genes in the liver. In contrast, in the head kidney from fish sampled in the autumn, the expression of genes encoding estrogen receptors and aromatase was lower than in spring, and a similar profile of expression was also measured in the head kidney for inos, arginases and il-10. In turn, during bacterial infection, we observed higher upregulation of the expression of inos, il-12p35, ifnγ-2, arginase 2 and il-10 in the liver of carp sampled in spring. In the liver of carp infected in spring a higher upregulation of the expression of the genes encoding CRPs was observed compared to fish infected during autumn. The opposite trend occurred in the head kidney, where the upregulation of the expression of the genes involved in the immune response was higher in fish infected in autumn than in those infected in spring. During the infection, also season-dependent changes occurred in the estrogen system. In conclusion, we demonstrated that season differentially affects the estrogenic and immune activity of the head kidney and liver. These results reinforce our previous findings that the endocrine and immune systems cooperate in maintaining homeostasis and fighting infection. [ABSTRACT FROM AUTHOR]

Inflammatory responses are modulated by sex steroids, and hence, not surprisingly, in seasonally breeding organisms, the immune responses are reported to vary according to the reproductive phases. NOD-like receptors (NLRs) act as intracellular sentinels and play a key role in inflammation. Interestingly, to date, there are no reports on their reproductive season-dependent expression and their regulation by sex steroids in teleosts. In the current study, the expression of NOD-like receptors (NLRs) in primary and secondary lymphoid tissues in female Channa punctata during different reproductive phases has been investigated. It was observed that NLRs show a dramatic increase in expression during the spawning phase of Channa punctata. Since estradiol is the primary sex steroid in females that shows the highest plasma level during the reproductively active phase, we investigated whether estradiol was responsible for the increased transcription of NLRs. The effect of estradiol on NLR expression in female Channa punctata was diverse, depending upon the lymphoidic tissue, dose, duration, and mode of estradiol treatment. However, the increased expression of NLRs in the spawning phase could not be attributed to estradiol alone as evident from the study. Other factors acting alone or collaborating with estrogen might be involved in the increased NLR expression during spawning phase in Channa punctata. [ABSTRACT FROM AUTHOR]

Sex steroids are known to modulate immune responses and as a result many of the immune parameters in seasonally breeding organisms show reproductive‐phase dependent variation. Androgens, the male sex steroids, are largely reported to be immunosuppressive. Together with other pattern recognition receptors, the nucleotide‐binding and oligomerization domain‐like receptors (NLRs) serve as intracellular sentinels and are essential to defense mechanisms. Interestingly, to date the transcriptional modulation of NLRs by androgens has not been explored. In the present study, we investigated the reproductive‐phase dependent expression of NLRs in the male spotted snakehead Channa punctata. Furthermore, the effect of dihydrotestosterone (DHT) on NLR expression was studied. The expression of NLRs was observed to be most pronounced during the spawning phase of the fish, which is marked by the highest testosterone level. In vivo as well as in vitro studies showed the diverse effect of DHT on NLR expression depending on the duration and mode of treatment, as well as the immune tissue studied. [ABSTRACT FROM AUTHOR]

Simple Summary: Bisphenol A (BPA) is an organic compound used in the manufacture of plastic products and is considered a common pollutant in the aquatic environment. Its impact on aquatic organisms is manifested in several reactions, particularly in changes in peripheral blood and disorders of hematopoiesis. In the present study, the effects of high concentrations of BPA on several indices of zebrafish blood were investigated. Differences in the effects of high and low concentrations on blood composition and disorders in hematopoietic organs were identified. The genotoxic effect of BPA at concentrations of 6 and 8 mg/L was confirmed. The test parameters most affected by the toxicant, such as the number of young erythrocytes in peripheral blood, nuclear anomalies of erythrocytes, and disturbances in hematopoiesis processes in the head kidney, expressed as changes in the ratio of immature/undifferentiated cell elements, were determined. In this study, the results of evaluating the acute toxicity of Bisphenol A on Danio rerio are presented, encompassing peripheral blood parameters, the composition of hematopoietic cells of erythroid and myeloid lines in the head kidney, and data from histological studies. The LC50 values of Bisphenol A for adult zebrafish individuals for 12, 24, and 48–96 h were determined, which were 18.04, 7.55, and 6.22 mg/L, respectively. The study includes data on the morphology and quantitative frequency of specific cells in the hematopoietic tissue of the head kidney, along with the consideration of adaptive mechanisms in hematopoiesis under BPA exposure. The application of polynomial regression analysis to reveal the concentration–effect relationship for some hematological and histological parameters was demonstrated. Significant increases in the frequency of erythrocyte nuclear abnormalities were observed at BPA concentrations of 6 and 8 mg/L, which indicates a genotoxic effect. BPA's impact on fish peripheral blood parameters manifested as an increase in the number of erythrocytes (RBC) and immature erythrocytes, as well as a decrease in the number of lymphocytes. The most notable pathological changes in the head kidney's hematopoietic tissue included circulatory disturbances and the formation of inflammation/degradation foci, as confirmed by histopathologic indices. At BPA concentrations of 2 and 4 mg/L, the observed changes were compensated for by hematopoietic adaptation mechanisms; however, at concentrations of 6 and 8 mg/L, acute systemic toxicity was evident. [ABSTRACT FROM AUTHOR]

This study investigated the effects of acute ammonia nitrogen (NH3-N) exposure on kidney antioxidant ability and phosphatases and related gene expression in juvenile yellowfin tuna (Thunnus albacares). The 180 juvenile yellowfin tuna (260.39 ± 55.99 g, 22.33 ± 2.28 cm) were exposed to ammonia for 6, 24, and 36 h using natural seawater (0 mg/L) as a control and NH3-N at 5 and 10 mg/L. The lipid peroxidation byproduct malondialdehyde (MDA) and the levels of antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-PX), alkaline phosphatase (AKP), and acid phosphatase (ACP), were measured using the colorimetric method in the trunk kidney to determine changes in antioxidant ability and phosphatase activity of juvenile yellowfin tuna exposed to NH3-N. Results indicated that, at 36 h, MDA, SOD, CAT, and GSH-PX levels rose in the 5 mg/L group versus the control. In the 10 mg/L group, MDA and SOD, CAT, and GSH-PX activities significantly increased after 24 and 36 h exposure compared to the control. Phosphatases play a pivotal role in the immune system. AKP activity significantly increased at 6 h, and ACP activity markedly rose at 36 h in the 5 mg/L group versus the control. Real-time fluorescence quantitative PCR was applied to detect alterations in the antioxidant genes SOD2, CAT, and glutathione peroxidase 1b (GPX1b) and immune cytokines-related genes Interleukin 10 (IL-10) and Interleukin 6 receptor (IL-6r) expression in the head kidney in juvenile tuna. Relative to the control, antioxidant gene expression in the 5 mg/L group significantly rose at 6 and 36 h, and in the 10 mg/L group, SOD2 and GPX1b were significantly elevated at 36 h. Compared to the control group, IL-10 expression in the 5 mg/L group significantly increased at 6 h, whereas IL-6r expression decreased. In the 10 mg/L group, both IL-10 and IL-6r levels were observed to be lower. Low ammonia nitrogen concentrations boost antioxidant defenses, phosphatase activities, and gene expression levels, whereas higher levels may induce suppressive effects. In yellowfin tuna juvenile farming, NH3-N concentration significantly affects the health of the juveniles. When the NH3-N concentration is between 5–10 mg/L, the stress duration should be limited to 24 h; if the concentration is below 5 mg/L, the stress duration can be extended to 36 h.

Current salmonid hatchery practices typically include high rearing densities without structure within the water column. In the present study, we assessed the effects of low (conservation hatchery) and high (production hatchery) rearing densities on the plasma cortisol response during simulated transport stress and whether density might interact with in-tank structure to further mitigate the stress response. After 10 months of rearing in their respective treatments, we subjected fish to a transportation simulation stressor that encompassed fish being caught, bucketed, and crowded at a lowered water depth in a smaller tank for 3 h, then caught and bucketed to a novel tank of similar size and flow. We sampled stressed and unstressed fish for plasma cortisol determination over a 23 h time period. Additionally, we determined resting cortisol secretion from head kidneys collected from unstressed fish at time 0 h from each rearing treatment to determine if resting cortisol secretion pattern was affected by the rearing conditions. There was no significant difference in size of the fish at the time of sampling; overall, they averaged 24.7 g in weight and 136.2 mm in fork length. Fish reared with in-tank structure at low density had the lowest plasma cortisol concentration during the stressor, while fish reared without in-tank structure at high density had the highest plasma cortisol concentration in response to the stressor. In-tank structure, high-density reared fish had less plasma cortisol than no-structure high-density fish, but was elevated over low-density in-tank structure fish. Rearing condition had no meaningful effect on resting cortisol secretion by the interrenals. We speculate that low density with in-tank structure rearing would ultimately lead to the highest survival rates when hatchery Chinook salmon are released into the wild due to their reduced plasma cortisol concentration in response to a stressor.

Nephrons are generally not distributed in the head kidney of teleost. Nonetheless, in this study, the nephron structure was identified in the head kidney of three species of Sebastes (Sebastes inermis, Sebastes schlegelii and Sebastes thompsoni). The kidney is Y shaped, with the development in the head kidney. The nephron structure was confirmed in the head kidney and body kidney. In addition, the nephron consisted of renal corpuscles and tubules, and the renal corpuscle consisted of the Bowman's capsule and glomerulus. Histologically, previous studies reported that the nephron structure is similar to that of other marine teleost. The renal tubule is a simple columnar epithelial layer with microvilli and cilia on the free surface, which is observed as a brush border. The Rrk (relative area ratio of kidney to body surface) was 5.14%, 7.58% and 5.17% in S. inermis, S. schlegelii and S. thompsoni, respectively. The Gar (glomerular area ratio of the head kidney) was higher in the central area than in the peripheral area, and species, which showed significant difference (P < 0.05), were in the following order: S. thompsoni (1.60%) > S. schlegelii (0.90%) > S. inermis (0.66%). [ABSTRACT FROM AUTHOR]

Simple Summary: Hematological analysis is one of the essential tools to assess the influence of organic and inorganic substances on fish. Thus, it is widely used in ecotoxicological and pharmacotoxicological studies. In this review, we demonstrate and discuss the use, strengths, and limitations of hematological as well as hematopoietic analysis in the context of fish toxicology. Since hematopoietic analysis can help to explain and understand the mechanisms of studied hematological changes, we highly recommend this method as a good complement to standard hematological tests. Hematological analysis is commonly used to assess the physiological state of fish. It includes red blood cell parameters, white blood cell parameters, and the number of thrombocytes per blood volume unit. Hematological analysis is one of the basic tools (often accompanied by biochemical and histopathological analyses) to assess the influence of organic and inorganic substances on fish. It is, therefore, applicable in both ecotoxicology and pharmacotoxicology. The advantages of this research method are the lack of need for specialized laboratory equipment and low costs, and the limitations are the need for extensive experience among the personnel performing the tests. One of the recommended methods of supplementing routinely determined hematological parameters is assessing the cellular composition and activity of hematopoietic tissue. As there is very little scientific data available on the issue of the effects of xenobiotics on the cellular structure of fish head kidney hematopoietic tissue, filling this gap should be considered an urgent need. Therefore, we recommend conducting research with the simultaneous use of hematological and hematopoietic analysis as reliable and complementary methods of assessing the impact of toxic substances on fish. [ABSTRACT FROM AUTHOR]

Changes in an erythrocyte count, as well as erythropoiesis in hematopoietic tissue, were studied in the golden grey mullet (Chelon auratus Risso, 1810) over an annual cycle. Fish capture and delivery to the laboratory were provided at a monthly basis. The content of immature erythroid forms (erythroblasts, basophilic and polychromatophilic normoblasts) was counted in the head kidney (pronephros) and circulating blood. It was found that erythropoiesis in C. auratus proceeds irregularly. The active production of erythroid mass is mainly confined to the post-spawning period, as evidenced by an increase in the content of immature erythroid forms in the head kidney and blood. This coincides with an overall increase in the number of red blood cells (RBCs) in the circulatory system and indicates a shift in the erythrocyte balance toward RBC production. Outside the pre-spawning period, the processes are opposite. It is assumed that the above findings are due to peculiar organization of the RBC system in Ch. auratus, which excludes the regular production of erythropoietin in the kidneys. [ABSTRACT FROM AUTHOR]

The cellular composition of leukocytes in the peripheral blood, head kidney and spleen was studied in individuals of three species of chars of the genus Salvelinus, white char S. albus, long–headed char S. kronocius and long-nosed char S. schmidti, as well as two forms, small-mouth and large-mouth chars living in Lake Kronotskoe. In the studied individuals, the proportion of lymphocytes of leukocytes in the peripheral blood was significant. The forms (species) of chars differed in the proportion of cell types, the size of leukocytes, as well as the intensity of leukopoiesis. The most significant differences in the proportion of different forms of leukocytes were found in the large-mouth char compared to other species and small–mouth char: high content of lymphocytes in blood smears, low content of monocytes in all tissues and organs, blast forms in the blood and neutrophils in the pronephros. [ABSTRACT FROM AUTHOR]

Streptococcus iniae is a Gram-positive bacterium and is considered a harmful aquaculture pathogen worldwide. In this study, S. iniae strains were isolated from East Asian fourfinger threadfin fish (Eleutheronema tetradactylum) reared on a farm in Taiwan. A transcriptome analysis of the head kidney and spleen was performed in the fourfinger threadfin fish 1 day after infection using the Illumina HiSeq™ 4000 platform for RNA-seq to demonstrate the host immune mechanism against S. iniae. A total of 7333 genes based on the KEGG database were obtained after the de novo assembly of transcripts and functional annotations. Differentially expressed genes (DEGs) (2-fold difference) were calculated by comparing the S. iniae infection and phosphate-buffered saline control group gene expression levels in each tissue sample. We identified 1584 and 1981 differentially expressed genes in the head kidney and spleen, respectively. Based on Venn diagrams, 769 DEGs were commonly identified in both the head kidney and spleen, and 815 and 1212 DEGs were specific to the head kidney and spleen, respectively. The head-kidney-specific DEGs were enriched in ribosome biogenesis. The spleen-specific and common DEGs were found to be significantly enriched in immune-related pathways such as phagosome, Th1, and Th2 cell differentiation; complement and coagulation cascades; hematopoietic cell lineage; antigen processing and presentation; and cytokine–cytokine receptor interactions, based on the KEGG database. These pathways contribute to immune responses against S. iniae infection. Inflammatory cytokines (IL-1β, IL-6, IL-11, IL-12, IL-35, and TNF) and chemokines (CXCL8 and CXCL13) were upregulated in the head kidney and spleen. Neutrophil-related genes, including phagosomes, were upregulated post-infection in the spleen. Our results could offer a strategy for the treatment and prevention of S. iniae infection in fourfinger threadfin fish. [ABSTRACT FROM AUTHOR]

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A teleost's kidney was divided into head kidney and trunk kidney. The head kidney is an important lymphatic organ, while the trunk kidney mainly performs osmotic pressure regulation and excretion functions. Previous studies have shown that the teleost's head kidney exerts a strong immune response against pathogen invasion, while the mechanism of immune response in the trunk kidney is still rarely reported. Therefore, in this study, we established an Infectious hematopoietic necrosis virus (IHNV) immersion infection model to compare the similarities and differences of immune response mechanisms between the head kidney and trunk kidney against viral infection. The results showed that IHNV infection causes severe tissue damage and inflammatory reaction in the head and trunk kidney, triggers a series of interferon cascade reactions, and produces strong immune response. In addition, the transcriptome data showed that the head kidney and trunk kidney had similar immune response mechanisms, which showed that the NOD-like receptor signaling pathway and Toll-like receptor signaling pathway were activated. In conclusion, despite functional differentiation, the teleost's trunk kidney still has a strong immune response, especially the interferon-stimulated genes, which have stronger immune response in the trunk kidney than in the head kidney when responding to IHNV infection. This study contributes to a more comprehensive understanding of the teleost immune system and enriches the theory of kidney immunity in teleosts. [ABSTRACT FROM AUTHOR]

In this study, the results of evaluating the acute toxicity of Bisphenol A on Danio rerio are presented, encompassing peripheral blood parameters, the composition of hematopoietic cells of erythroid and myeloid lines in the head kidney, and data from histological studies. The LC50 values of Bisphenol A for adult zebrafish individuals for 12, 24, and 48–96 h were determined, which were 18.04, 7.55, and 6.22 mg/L, respectively. The study includes data on the morphology and quantitative frequency of specific cells in the hematopoietic tissue of the head kidney, along with the consideration of adaptive mechanisms in hematopoiesis under BPA exposure. The application of polynomial regression analysis to reveal the concentration–effect relationship for some hematological and histological parameters was demonstrated. Significant increases in the frequency of erythrocyte nuclear abnormalities were observed at BPA concentrations of 6 and 8 mg/L, which indicates a genotoxic effect. BPA’s impact on fish peripheral blood parameters manifested as an increase in the number of erythrocytes (RBC) and immature erythrocytes, as well as a decrease in the number of lymphocytes. The most notable pathological changes in the head kidney’s hematopoietic tissue included circulatory disturbances and the formation of inflammation/degradation foci, as confirmed by histopathologic indices. At BPA concentrations of 2 and 4 mg/L, the observed changes were compensated for by hematopoietic adaptation mechanisms; however, at concentrations of 6 and 8 mg/L, acute systemic toxicity was evident.

Teleosts have a unique immune system because their head kidney (HK) and trunk kidney (TK) are sites for hematopoiesis. However, the immune functions of the HK and TKs require further elucidation in yellow catfish (Pelteobagrus fulvidraco). In the present study, imprints of the HK and TK were examined using the Wright's-Giemsa staining method. Morphological characteristics of the blood cell lineages revealed that the HK and TK were hematopoietic organs. To explore its immune function, transcriptome sequencing was performed after infection with Aeromonas hydrophila. A total of 1139 genes showed significant alterations in their expression in the kidney; these genes included 737 upregulated and 402 downregulated genes. Furthermore, 1117 differentially expressed genes were observed in the HK, which included 784 upregulated and 333 downregulated genes. Both organs showed 357 upregulated genes and 85 downregulated genes. Some immune-related genes were only expressed in the TK, such as ATP-dependent RNA helicase DDX58, the gene encoding the immunoglobulin heavy chain and light chain. The immune responses in the HK and TK were differential and the TK played a critical role in the mechanism underlying the immune response. The purpose of the present study was to facilitate the elucidation of the immune defense mechanism of yellow catfish and other teleosts. • The HK and TK are sites for hematopoiesis. • The immune responses in HK and TKD are differential. • Some immune-related genes are only expressed in TK. [ABSTRACT FROM AUTHOR]

Overcoming a stress situation, such as hypoxia episodes, which involve an allostatic load, will depend on the ability of fish to modulate physiological and biochemical systems to maintain homeostasis. The aim of the study was to determine the integrated stress response after acute hypoxia of the rainbow trout considering the different elements and areas of the stress response: systemic and mucosal, local and global, and from the systemic hypothalamic-pituitary-interrenal axis to skin mucosa. For this purpose, trout were subjected to acute hypoxia (dissolved O2 down to 2 mg/L) for 1 h and then recovered and sampled at 1, 6, and 24 h after reoxygenation. Physiological responses were significantly affected by hypoxic stress and their interaction with time after the challenge, being significant for plasma lactate and cortisol levels, in both plasma and skin mucus. At the central brain level, only trh expression was modulated 1 h after hypoxia which indicates that brain function is not heavily affected by this particular stress. Unlike the brain, the head kidney and skin were more affected by hypoxia and reoxygenation. In the head kidney, an upregulation in the expression of most of the genes studied (gr, il1β, il6, tgfβ1, lysozyme, caspase 3, enolase, hif-1, myoglobin, sod2, gpx, gst, and gsr) took place 6 h after recovery, whereas only hsp70 and il10 were upregulated after 1 h. On the contrary, in the skin, most of the analyzed genes showed a higher upregulation during 1 h after stress suggesting that, in the skin, a local response took place as soon as the stressor was detected, thus indicating the importance of the skin in the building of a stress response, whereas the interrenal tissue participated in a later time point to help prevent further alteration at the central level. The present results also show that, even though the stressor is a physical/environmental stressor, all components of the biological systems participate in the regulation of the response process and the recovery process, including neuroendocrine, metabolism, and immunity. [ABSTRACT FROM AUTHOR]

Aflatoxin B1 (AFB1) is kind of a common mycotoxin in food and feedstuff. Aquafeeds are susceptible to contamination of AFB1. In teleost fish, the spleen and head kidney are key immune organ. Moreover, the fish skin is a critical mucosal barrier system. However, there was little study on the effects of dietary AFB1 on the immune response of these immune organs in fish. This study aimed to explore the impacts of oral AFB1 on the immune competence and its mechanisms in the skin, spleen, and head kidney of grass carp. Our work indicated that dietary AFB1 reduced antibacterial compounds and immunoglobulins contents, and decreased the transcription levels of antimicrobial peptides in grass carp immune organs. In addition, dietary AFB1 increased the transcription levels of pro-inflammatory cytokines and reduced the transcription levels of anti-inflammatory cytokines in the grass carp immune organs, which might be regulated by NF-κB and TOR signaling, respectively. Meanwhile, we evaluated the content of AFB1 in the grass carp diet should not exceed 29.48 mg/kg diet according to the levels of acid phosphatase and lysozyme. In summary, dietary AFB1 impaired immune response in grass carp skin, spleen, and head kidney. [ABSTRACT FROM AUTHOR]

Prolactin (Prl) and growth hormone (Gh) as well as insulin-like growth factor 1 (Igf1) are involved in the physiological adaptation of fish to varying salinities. The Igfs have been also ascribed other physiological roles during development, growth, reproduction and immune regulation. However, the main emphasis in the investigation of osmoregulatory responses has been the endocrine, liverderived Igf1 route and local regulation within the liver and osmoregulatory organs. Few studies have focused on the impact of salinity alterations on the Gh/Igf-system within the neuroendocrine and immune systems and particularly in a salinity-tolerant species, such as the blackchin tilapia Sarotherodon melanotheron. This species is tolerant to hypersalinity and saline variations, but it is confronted by severe climate changes in the Saloum inverse estuary. Here we investigated bidirectional effects of increased salinity followed by its decrease on the gene regulation of prl, gh, igf1, igf2, Gh receptor and the tumor-necrosis factor a. A mixed population of sexually mature 14-month old blackchin tilapia adapted to freshwater were first exposed to seawater for one week and then to fresh water for another week. Brain, pituitary, head kidney and spleen were excised at 4 h, 1, 2, 3 and 7 days after both exposures and revealed differential expression patterns. This investigation should give us a better understanding of the role of the Gh/Igf system within the neuroendocrine and immune organs and the impact of bidirectional saline challenges on fish osmoregulation in non-osmoregulatory organs, notably the complex orchestration of growth factors and cytokines. [ABSTRACT FROM AUTHOR]

Moritella viscosa is a bacterial pathogen causing winter-ulcer disease in Atlantic salmon. The lesions on affected fish lead to increased mortality, decreased fish welfare, and inferior meat quality in farmed salmon. MicroRNAs (miRNAs) are small non-coding RNAs involved in post-transcriptional regulation by guiding the miRNA-induced silencing complex to specific mRNA transcripts (target genes). The goal of this study was to identify miRNAs responding to Moritella viscosa in salmon by investigating miRNA expression in the head-kidney and the muscle/skin from lesion sites caused by the pathogen. Protein coding gene expression was investigated by microarray analysis in the same materials. Seventeen differentially expressed guide-miRNAs (gDE-miRNAs) were identified in the head-kidney, and thirty-nine in lesion sites, while the microarray analysis reproduced the differential expression signature of several thousand genes known as infection-responsive. In silico target prediction and enrichment analysis suggested that the gDE-miRNAs were predicted to target genes involved in immune responses, hemostasis, angiogenesis, stress responses, metabolism, cell growth, and apoptosis. The majority of the conserved gDE-miRNAs (e.g., miR-125, miR-132, miR-146, miR-152, miR-155, miR-223 and miR-2188) are known as infection-responsive in other vertebrates. Collectively, the findings indicate that gDE-miRNAs are important post-transcriptional gene regulators of the host response to bacterial infection. [ABSTRACT FROM AUTHOR]

Myxozoa (Cnidaria) is a large group of microscopic obligate endoparasites that can cause emerging diseases, affecting wild fish populations and fisheries. Recently, the myxozoan Myxobolus bejeranoi was found to infect the gills of hybrid tilapia (Nile tilapia (Oreochromis niloticus) × Jordan/blue tilapia (O. aureus)), causing high morbidity and mortality. Here, we used comparative transcriptomics to elucidate the molecular processes occurring in the fish host following infection by M. bejeranoi. Fish were exposed to pond water containing actinospores for 24 h and the effects of minor, intermediate, and severe infections on the sporulation site, the gills, and on the hematopoietic organs, head kidney and spleen, were compared. Enrichment analysis for GO and KEGG pathways indicated immune system activation in gills at severe infection, whereas in the head kidney a broad immune suppression included deactivation of cytokines and GATA3 transcription factor responsible for T helper cell differentiation. In the spleen, the cytotoxic effector proteins perforin and granzyme B were downregulated and insulin, which may function as an immunomodulatory hormone inducing systemic immune suppression, was upregulated. These findings suggest that M. bejeranoi is a highly efficient parasite that disables the defense mechanisms of its fish host hybrid tilapia. [ABSTRACT FROM AUTHOR]

In the present work, we studied the effect of short-term acute hypoxia on the cellular composition of the blood and the head kidney of the black scorpionfish. Dissolved oxygen concentration was decreased from 8.5–8.7 mg O2 l−1 (normoxia) to 3–5 mg O2 l−1 (relative normoxia), 1–3 mg O2 l−1 (moderate hypoxia), and 0–1 mg O2 l−1 (acute hypoxia) within 1.5–2 h by bubbling of water with N2. Exposure period was 4 h, water temperature was adjusted to 14–16 °C, and photoperiod was 12 h (light). Short-time acute hypoxia induced a rapid release of blast and immature cells from the head kidney into the circulating blood of the black scorpionfish, which was associated with reduction in erythropoietic reserves in 2.5 times. The number of immature erythroid cells (pronormoblasts, basophilic and polychromatophilic normoblasts) significantly increased in blood, and the simultaneously relative decrease of the number of abnormal red blood cell (RBC) and the increase of the number of RBC ghosts (lysed RBCs) in circulating blood were observed. The significant correlation between methemoglobin concentration and the number of RBC ghosts was shown (R2 = 0.640 or r = 0.800). Hypoxia induced RBC swelling on 5–6% compared to control. The number of RBC ghosts in the blood is likely involved in the stimulation of erythropoietin production under hypoxia. [ABSTRACT FROM AUTHOR]

The teleost kidneys are anatomically divided into head kidney and trunk kidney, each performing distinct physiological functions. Although previous research has elucidated the role of the head kidney in immune responses, there is a paucity of literature on the comparative studies of the head and trunk kidney response to bacterial infection. Therefore, an Edwardsiella ictaluri infection model of yellow catfish was constructed to investigate and compare the immune responses between the two kidney types. The findings indicated that E. ictaluri infection induced significant pathological changes in both the head and trunk kidney. Despite variances in structure, both the head and trunk kidney of yellow catfish exhibit robust immune responses following E. ictaluri infection. Unexpectedly, the up-regulation level of IgM was found to be higher in the trunk kidney compared to the head kidney. Additionally, both the IgM + and IgD +  B cells were increased after bacterial infection. This research elucidates the parallels and distinctions in immune functions between both the head and trunk kidney in fish, enriching the immune theory of the fish kidney, and also providing a theoretical basis for the immune response of teleost kidney against bacterial infections., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Overcoming a stress situation, such as hypoxia episodes, which involve an allostatic load, will depend on the ability of fish to modulate physiological and biochemical systems to maintain homeostasis. The aim of the study was to determine the integrated stress response after acute hypoxia of the rainbow trout considering the different elements and areas of the stress response: systemic and mucosal, local and global, and from the systemic hypothalamic–pituitary–interrenal axis to skin mucosa. For this purpose, trout were subjected to acute hypoxia (dissolved O2 down to 2 mg/L) for 1 h and then recovered and sampled at 1, 6, and 24 h after reoxygenation. Physiological responses were significantly affected by hypoxic stress and their interaction with time after the challenge, being significant for plasma lactate and cortisol levels, in both plasma and skin mucus. At the central brain level, only trh expression was modulated 1 h after hypoxia which indicates that brain function is not heavily affected by this particular stress. Unlike the brain, the head kidney and skin were more affected by hypoxia and reoxygenation. In the head kidney, an upregulation in the expression of most of the genes studied (gr, il1β, il6, tgfβ1, lysozyme, caspase 3, enolase, hif-1, myoglobin, sod2, gpx, gst, and gsr) took place 6 h after recovery, whereas only hsp70 and il10 were upregulated after 1 h. On the contrary, in the skin, most of the analyzed genes showed a higher upregulation during 1 h after stress suggesting that, in the skin, a local response took place as soon as the stressor was detected, thus indicating the importance of the skin in the building of a stress response, whereas the interrenal tissue participated in a later time point to help prevent further alteration at the central level. The present results also show that, even though the stressor is a physical/environmental stressor, all components of the biological systems participate in the regulation of the response process and the recovery process, including neuroendocrine, metabolism, and immunity.

Hematological analysis is commonly used to assess the physiological state of fish. It includes red blood cell parameters, white blood cell parameters, and the number of thrombocytes per blood volume unit. Hematological analysis is one of the basic tools (often accompanied by biochemical and histopathological analyses) to assess the influence of organic and inorganic substances on fish. It is, therefore, applicable in both ecotoxicology and pharmacotoxicology. The advantages of this research method are the lack of need for specialized laboratory equipment and low costs, and the limitations are the need for extensive experience among the personnel performing the tests. One of the recommended methods of supplementing routinely determined hematological parameters is assessing the cellular composition and activity of hematopoietic tissue. As there is very little scientific data available on the issue of the effects of xenobiotics on the cellular structure of fish head kidney hematopoietic tissue, filling this gap should be considered an urgent need. Therefore, we recommend conducting research with the simultaneous use of hematological and hematopoietic analysis as reliable and complementary methods of assessing the impact of toxic substances on fish.

Atlantic salmon is one of the most famous and economically important fish species globally. However, bacterial diseases constantly constrain salmon aquaculture. Thereinto, Aeromonas salmonicida subsp. masoucida (ASM), classified as atypical A. salmonicida , caused huge losses to salmonid industry in China. In this regard, we conducted transcriptome analysis in Atlantic salmon head kidney following the administration of ASM vaccination to reveal genes, their expression patterns, and pathways involved in immune responses. A total of 448.71 million clean reads were obtained, and 397.69 million reads were mapped onto the Atlantic salmon reference genome. In addition, 117, 1891, 741, 207, and 377 genes were significantly up-regulated, and 183, 1920, 695, 83, and 539 genes were significantly down-regulated post ASM vaccination at 12 h, 24 h, 1 m, 2 m, and 3 m, respectively. Furthermore, KEGG pathway analysis revealed that many differentially expressed genes (DEGs) following ASM vaccination were involved in cell adhesion molecules (H2-Aa-l and CD28-l),cytokine-cytokine receptor interaction (IL10 , CXCL9 , CXCL11 , CXCR3 , and CCL19), herpes simplex infection (IL1B , SOCS3-l , and C3-l), HTLV-I infection (Il1r2 and BCL2L1), influenza A (CXCL8 and Il12b) , and PI3K-Akt signaling pathway (PIK3R3-l and Ddit4-l). Finally, the results of qRT-PCR showed a significant correlation with RNA-Seq results, suggesting the reliability of RNA-Seq for gene expression analysis. This study sets the foundation for further study on the vaccine protective mechanism in Atlantic salmon as well as other teleost species. • RNA-seq was conducted to reveal the immune responses in Atlantic salmon head kidney following the administration of vaccine. • A total of 300, 3811, 1436, 290, and 916 genes were differentially expressed following vaccination at 12 h, 24 h, 1 m, 2 m, and 3 m, respectively. • Many DEGs were involved in immune-related pathways such as Cytokine-cytokine receptor interaction and PI3K-Akt signaling pathway. • The results of RT-qPCR showed a significant correlation with RNA-Seq results, suggesting the reliability of RNA-Seq. [ABSTRACT FROM AUTHOR]

This dissertation was primarily focused on the immune response, inflammatory response and molecular mechanisms in the skin, head kidney and spleen of grown-up grass carp (Ctenopharyngodon idella). Six iso-nitrogen diets differing in lysine concentrations (5.6, 8.5, 11.6, 14.4, 17.5 and 20.7 g/kg) were fed to 540 grass carp (164.85 ± 0.79 g) for 60 d. After that, grass carp were challenged by Aeromonas hydrophila for 6 d. This study revealed that lysine deficiency (1) suppressed the growth performance of the fish and decreased their ability to resist skin lesion morbidity, (2) impaired the immune organ's immune response by decreasing the gene expressions of mucin, liver-expressed antimicrobial peptide (LEAP)-2B, β-defensin-1 and LEAP-2A and the production of antibacterial compounds of grown-up grass carp, and (3) aggravated the inflammatory response of immune organs in the fish by increasing the gene expressions of pro-inflammatory cytokines (interferon γ2 [IFN-γ2], tumor necrosis factor α [TNF-α], interleukin [IL]-15, IL-17D, IL-12p40, IL-6 and IL-8) and down-regulating anti-inflammatory cytokines (IL-11, transforming growth factor β1 [TGF-β1], IL-10 and IL-4/13A), which were tightly correlated with signal transducer and activator of transcription (STAT)1 and STAT3 signaling pathway, respectively. The different phenomenon in the skin, spleen and head kidney of fish may be correlated with the difference in gene subtype. In addition, using quadratic regression analysis of percent weight gain (PWG), skin lesion morbidity, and the lysozyme activities in the spleen and head kidney, the dietary lysine requirements for grown-up grass carp were estimated to be 13.58, 13.51, 14.56 and 14.18 g/kg, respectively.

The yellowfin seabream (Acanthopagrus latus) is a crucial marine resource owing to its economic significance. Acanthopagrus latus aquaculture faces numerous challenges from viral diseases, but a robust in-vitro research model to understand and address these threats is lacking. Therefore, we developed a novel A. latus cell line from head kidney cells called ALHK1. This study details the development, characterisation, and viral susceptibility properties of ALHK cells. This cell line primarily comprises fibroblast-like cells and has robust proliferative capacity when cultured at 28 °C in Leibovitz's L-15 medium supplemented with 10–20% foetal bovine serum. It exhibited remarkable stability after more than 60 consecutive passages and validation through cryopreservation techniques. The specificity of the ALHK cell line's origin from A. latus was confirmed via polymerase chain reaction (PCR) amplification of the cytochrome B gene, and a chromosomal karyotype analysis revealed a diploid count of 48 (2n = 48). Furthermore, the lipofection-mediated transfection efficiency using the pEGFP-N3 plasmid was high, at nearly 40%, suggesting that ALHK cells could be used for studies involving exogenous gene manipulation. In addition, ALHK cells displayed heightened sensitivity to the large mouth bass virus (LMBV), substantiated through observations of cytopathic effects, quantitative real-time PCR, and viral titration assays. Finally, the response of ALHK cells to LMBV infection resulted in differentially expressed antiviral genes associated with innate immunity. In conclusion, the ALHK cell line is a dependable in-vitro platform for elucidating the mechanisms of viral diseases in yellowfin seabream. Moreover, this cell line could be valuable for immunology, vaccine development, and host-pathogen interaction studies. • Established a new cell line (ALHK) from the head kidney of yellowfin seabream (Acanthopagrus latus). • Verified the cell line's origin using PCR amplification of the cytochrome B gene. • Karyotype analysis showed that the standard chromosome numbers were 48. • A nearly 40% transfection efficiency was achieved in ALHK cells, showing potential for genetic research. • ALHK cells are sensitive to Large Mouth Bass Virus (LMBV). [ABSTRACT FROM AUTHOR]

We previously observed that exposure to a complex mixture of high molecular weight polycyclic aromatic hydrocarbons (PAHs) increased sensitivity of rainbow trout (Oncorhynchus mykiss) to subsequent challenge with Aeromonas salmonicida, the causative agent of furunculosis. In this study, we evaluate potential mechanisms associated with disease susceptibility from combined environmental factors of dietary PAH exposure and pathogen challenge. Rainbow trout were fed a mixture of ten high molecular weight PAHs at an environmentally relevant concentration (7.82μg PAH mixture/g fish/day) or control diet for 50 days. After 50 days of PAH exposure, fish were challenged with either Aeromonas salmonicida at a lethal concentration 30 (LC30) or growth media without the pathogen (mock challenge). Head kidneys were collected 2, 4, 10 and 20 days after challenge and gene expression (q

Antarctic icefish is the only known vertebrate species that lacks oxygen-carrying hemoglobin and functional erythrocytes. To reveal the unique hematopoietic process of icefish, we used an integrated approach including tandem mass tag (TMT) labeling and liquid chromatography–tandem mass spectrometry (LC–MS/MS) to quantify the dynamic changes in the head kidney whole proteome of a white-blooded icefish, Chionodraco hamatus, compared to those in two other red-blooded Antarctic fish, Trematomus bernacchii and Notothenia coriiceps. Of the 4672 identified proteins, in the Antarctic icefish head kidney, 123 proteins were significantly upregulated and 95 proteins were downregulated. The functional grouping of differentially expressed proteins (DEPs) based on KEGG pathway analysis shows that white blood fish and red blood fish have significant differences in erythropoiesis, heme biogenesis, leucocyte and platelet cell development. The proteins involved in the hematopoietic process in icefish showed a clear trend of downregulation of erythroid lineage marker proteins (β-Spectrin, hemoglobin, CTCF and tfr1a) and upregulation of lymphoid and megakaryocytic lineage marker proteins, including CD9, ITGB2, and mTOR, which suggests a shift in hematopoiesis in the icefish head kidney due to the loss of erythrocytes. Moreover, western blotting analysis showed that a significantly down expression level of CTCF protein was detected in the icefish head kidney, which was in line with the proteomics data. The results of the present study not only provide basic datasets for the head kidney proteins of Antarctic fishes, but also provide important references for studies on immunity and hematopoiesis in various species. [ABSTRACT FROM AUTHOR]

Aquaculture management involves regular handling procedures, but these can evoke stress responses in farmed fish. We compiled an extensive list of published parameters that indicate the most likely handling-induced physiological deviations from the norm. However, since these parameters are based almost exclusively on studies of rainbow trout and Atlantic salmon, we conducted a handling-challenge experiment with maraena whitefish (Coregonus maraena). This salmonid fish was sampled at either 3 or 24 h after a single 1-min handling or after 10 days of daily repeated 1-min handling. The cortisol levels were strongly elevated in some individuals at 3 h after the single handling challenge, but these elevations were not significantly different between the challenged and control cohorts. The phagocytic capacity of myeloid head-kidney cells stimulated with fluorophore-labeled, inactivated Aeromonas salmonicida was significantly decreased in maraena whitefish at 3 h after the handling challenge compared to control fish. Microarray analysis of head-kidney samples from the challenged and control fish revealed 12 differentially expressed genes at 3 h and 70 at 24 h after the single handling episode, but only 5 differentially expressed genes after 10 days of repeated daily handling. The identified genes were assigned to numerous stress- and immune-relevant functional pathways, including “glucocorticoid receptor signaling” (3 h post-challenge), “HIF1A signaling” (24 h post-challenge), or “complement system” (10 days of repeated challenge). Our data reveal the tight interconnection of immune and stress pathways in the head kidney of maraena whitefish and corroborate several parameters previously found regulated in other tissues of handling-stressed rainbow trout. These findings indicate that handling may compromise the health and welfare of maraena whitefish in aquaculture.

As molecular mediators, long noncoding RNAs (lncRNAs) are essential for multiple biological processes, such as activating immune response genes and regulating the inflammatory process. The draft genome has been sequenced in yellow catfish (Pelteobagrus fulvidraco), but the expression profile and role of lncRNAs in this fish are still unclear. To identify the immunity-associated lncRNAs in the head kidney of yellow catfish, lncRNA sequences were acquired after Aeromonas hydrophila infection. After high-throughput sequencing, 202 differentially expressed lncRNAs were obtained, including 131 upregulated and 71 downregulated lncRNAs. The functional annotation of cis- and trans-regulatory target genes revealed that many DE-lncRNAs were involved in multiple immunity-associated GO terms and KEGG pathways, such as C-C motif chemokine 3 and interferon regulatory factor 1. Moreover, some lncRNAs might give rise to miRNA. The present study not only revealed the immunomodulatory lncRNAs in head kidney, but also provided a new perspective on the immune response mechanism. The results will facilitate subsequent studies in this species.

The study of soft anatomy has always been a useful tool in the understanding of an organism’s structure and function as well as in establishing its taxonomic status and interrelationships. The present study deals with the light and electronmicroscopic study of the adrenal gland in a catfish, Heteropneustes fossilis (Bloch, 1794). The findings reveal some significant features of the adrenal gland in this fish viz. (i) paired bilobed head kidney bearing the adrenal tissues, which is separate from the main trunk kidney (opisthonephros) (ii) the adrenal tissues being primarily haemopoetic in nature and possessing diffusely distributed interrenal and chromaffin cells (iii) chromaffin cells displaying both adrenaline and noradrenaline granules (iv) steroidogenic interrenal cells having numerous tubulo-lamellar mitochondria and abundant electron lucent lipid-like spaces (v) a characteristic absence of nervous tissues in the vicinity of these cells. Several of these features as observed in this catfish are characteristically absent in advanced teleosteans. The presence of an adrenal tissue in H. fossilis that has several resemblances to the lower bony fishes is suggested to have phylogenetic considerations. [ABSTRACT FROM AUTHOR]

Streptococcus iniae is a Gram-positive bacterium and is considered a harmful aquaculture pathogen worldwide. In this study, S. iniae strains were isolated from East Asian fourfinger threadfin fish (Eleutheronema tetradactylum) reared on a farm in Taiwan. A transcriptome analysis of the head kidney and spleen was performed in the fourfinger threadfin fish 1 day after infection using the Illumina HiSeq™ 4000 platform for RNA-seq to demonstrate the host immune mechanism against S. iniae. A total of 7333 genes based on the KEGG database were obtained after the de novo assembly of transcripts and functional annotations. Differentially expressed genes (DEGs) (2-fold difference) were calculated by comparing the S. iniae infection and phosphate-buffered saline control group gene expression levels in each tissue sample. We identified 1584 and 1981 differentially expressed genes in the head kidney and spleen, respectively. Based on Venn diagrams, 769 DEGs were commonly identified in both the head kidney and spleen, and 815 and 1212 DEGs were specific to the head kidney and spleen, respectively. The head-kidney-specific DEGs were enriched in ribosome biogenesis. The spleen-specific and common DEGs were found to be significantly enriched in immune-related pathways such as phagosome, Th1, and Th2 cell differentiation; complement and coagulation cascades; hematopoietic cell lineage; antigen processing and presentation; and cytokine–cytokine receptor interactions, based on the KEGG database. These pathways contribute to immune responses against S. iniae infection. Inflammatory cytokines (IL-1β, IL-6, IL-11, IL-12, IL-35, and TNF) and chemokines (CXCL8 and CXCL13) were upregulated in the head kidney and spleen. Neutrophil-related genes, including phagosomes, were upregulated post-infection in the spleen. Our results could offer a strategy for the treatment and prevention of S. iniae infection in fourfinger threadfin fish.

The effects of two potential antibacterial agents of plant origin: trans -cinnamaldehyde (TC) and thyme oil (TO) on the peripheral blood parameters and cellular composition of hematopoietic tissue of Cyprinus carpio were studied. Both phytochemicals were used in the doses based on the bactericidal concentrations against Aeromonas spp. developed earlier in in vitro study. The fish were fed for 2 weeks on a commercial feed supplemented with 10 μl/kg of TC or 20 μl/kg of TO. Groups TC1 and TO1 were fed diets containing phytochemicals daily, while groups TC2 and TO2 every 2 days. Control group and groups TC2 and TO2 on the remaining days were fed plain feed. Peripheral blood and head kidney hematopoietic tissue were sampled from all the fish at the end of the experiment. In all the groups, hematological values were within the reference ranges for the healthy common carp juveniles. However, blood hemoglobin (Hb) concentration and mean corpuscular hemoglobin concentration (MCHC) were significantly lower in all the groups exposed to TC and TO, while MCH in TC1, TO1, and TO2 compared to the control. TC and TO did not affect leukocyte count [white blood cell (WBC)], differential leukocyte count, the oxidative activity of phagocytes [nitroblue tetrazolium (NBT)], or thrombocyte count (Thro). No significant alterations were observed in the hematopoietic tissue. The results showed that TC and TO exhibited no considerable hematotoxic effects and trials of their use in the treatment of fish infected with Aeromonas spp. may be undertaken. [ABSTRACT FROM AUTHOR]

Tumor necrosis factor (TNF)-like weak inducer of apoptosis or TWEAK is a member of the TNF superfamily involved in the regulation of many biological processes. In mammals, TWEAK has been shown to play a role in some autoimmune or inflammatory conditions, but its immune role is not yet clearly defined. In teleost fish, although a few studies have identified homologues to mammalian TWEAK, their biological effects have never been investigated. In the current study, we have studied the transcriptional regulation of two TWEAK homologues (TWEAK 1 and 2) identified in rainbow trout (Oncorhynchus mykiss) throughout different tissues, in response to parasitic or viral infections, or in head kidney (HK) leukocytes stimulated with different stimuli. Although the transcription of both homologues was modulated when HK leukocytes were exposed to several immune stimuli, only TWEAK 1 was significantly modulated upon pathogenic exposure. Thus, we performed a characterization of the functions exerted by this cytokine in HK leukocytes. Recombinant TWEAK 1 strongly up-regulated the transcription of pro-inflammatory genes and antimicrobial peptides in HK leukocytes, with differential transcriptional effects in IgM+ B cells, IgM- lymphocytes and myeloid cells. TWEAK 1 also increased the survival and promoted the differentiation of B cells in HK leukocyte cultures. Our results demonstrate that in teleost fish, TWEAK 1 is involved in the response to different types of pathogens, through the modulation of antimicrobial and pro-inflammatory genes in different leukocytes subsets. Furthermore, a role for TWEAK as a B cell differentiation factor has also been established in rainbow trout. [ABSTRACT FROM AUTHOR]

The effects of two potential antibacterial agents of plant origin: trans-cinnamaldehyde (TC) and thyme oil (TO) on the peripheral blood parameters and cellular composition of hematopoietic tissue of Cyprinus carpio were studied. Both phytochemicals were used in the doses based on the bactericidal concentrations against Aeromonas spp. developed earlier in in vitro study. The fish were fed for 2 weeks on a commercial feed supplemented with 10 μl/kg of TC or 20 μl/kg of TO. Groups TC1 and TO1 were fed diets containing phytochemicals daily, while groups TC2 and TO2 every 2 days. Control group and groups TC2 and TO2 on the remaining days were fed plain feed. Peripheral blood and head kidney hematopoietic tissue were sampled from all the fish at the end of the experiment. In all the groups, hematological values were within the reference ranges for the healthy common carp juveniles. However, blood hemoglobin (Hb) concentration and mean corpuscular hemoglobin concentration (MCHC) were significantly lower in all the groups exposed to TC and TO, while MCH in TC1, TO1, and TO2 compared to the control. TC and TO did not affect leukocyte count [white blood cell (WBC)], differential leukocyte count, the oxidative activity of phagocytes [nitroblue tetrazolium (NBT)], or thrombocyte count (Thro). No significant alterations were observed in the hematopoietic tissue. The results showed that TC and TO exhibited no considerable hematotoxic effects and trials of their use in the treatment of fish infected with Aeromonas spp. may be undertaken.

Seabass (Lates calcarifer) are high value fish occupying a market niche, and is susceptible to infection by pathogenic bacteria such as Vibriosis. In this study the effect of intraperitoneal administration of recombinant outer membrane protein (OMP) of inactivated cells vaccine; r-OmpK on hematological parameters for monitoring fish health and histopathological examination of seabass were investigated. The results revealed that r-OmpK affect some hematological parameters positively in juvenile bass. The r-OmpK vaccinated seabass showed mild histological alterations in the vital organs (spleen, kidney and intestine) when compared to PBS control groups. Further we sought to understand the mechanisms of molecular immunity of L. calcarifer against V. harveyi infection. We carried out an extensive analysis of the transcriptome of L. calcarifer head kidney upon vaccination trial with r-OmpK recombinant cell vaccine and challenged with V. harveyi pathogenic strain VH1. Average reads of 92.74 million and 116.17 million were obtained from head kidney of juvenile L. calcarifer immunized with phosphate buffered saline (PBS) and r-OmpK (10 9 cfu/mL) recombinant cell vaccine, respectively. Also, the analysis revealed that a higher number of genes and pathways were regulated in response to infection with V. harveyi at 48 h post infection compared to the PBS controls. Based on the differential expressed genes (DEGs) around 0.31 % of total unigenes were differentially expressed between the two vaccinated groups. Protein-protein interaction analysis predicts glycolysis pathway, alanine, aspartate and glutamate metabolism pathway, pyruvate metabolism pathway, MAPK signaling pathway, metabolic pathways, insulin signaling pathway and focal adhesion pathway in head kidney of infected fish, which might be useful in understanding biological processes and further studies in the direction of pathways. We reported the novel immune-related signalling pathways, namely complement and coagulation cascades, chemokine signalling pathway, and toll-like receptor signalling pathway having great importance in immunity and anti-inflammation.

Red sea bream (Pagrus major) is described as one of the marine fishes with high economic value, which is widely cultivated in East Asia. With large-scale and intensive aquaculture, bacterial diseases outbreak more frequently than before. In order to effectively prevent and control Vibrio anguillarum disease in red sea bream, we systematically analyzed the immune response mechanism of the head kidney and spleen of P. major infected with Vibrio anguillarum by RNAseq technology. In this study, we obtained 225,106,036 clean reads. By using Nr, Nt, Pfam, KOG/COG, Swiss-prot, KEGG, GO databases, we annotated 85,595 genes. In the head kidney and spleen, we obtained 3963 and 4981 DEGs (different expressed genes) respectively. To further study the function of DEGs, we performed GO and KEGG enrichment analysis on them. In pathway enrichment, the pathways which significantly enriched in both head kidney and spleen included Antigen processing and presentation, Phagocytosis and Cell adhesion molecules. In the head kidney, Ubiquitin-mediated proteolysis was specially enriched; while Intestinal immune network for IgM production, Complement and coagulation cascade were specially enriched in the spleen. This study provide effective data support for the comprehensive analysis of mechanism in P. major immune response following V. anguillarum infection, which would lay the foundation for further disease prevention and treatment.

Grouper is an important marine fish mainly cultured in China and Southeast Asia. Here, a continuous cell line, named as ELHK, was established from head kidney of giant grouper (Epinephelus lanceolatus). The origin of cell line was confirmed by sequencing of 18s rRNA genes. ELHK cells consisted predominantly of fibroblast-like cells, and multiplied well in Leibovitz’s L-15 medium supplemented with 10% fetal bovine serum at 28 °C. Karyotyping analysis indicated that the modal chromosome number of ELHK cell was 60 at 50 passages. The cells were successfully transfected with GFP reporter gene suggesting that ELHK cell could be used for exogenous gene expression in vitro. Moreover, ELHK cells was susceptible to Singapore grouper iridovirus (SGIV) and red-spotted grouper nervous necrosis virus (RGNNV), demonstrated by the occurrence of cytopathic effects (CPEs) and increased transcription of the viral genes during virus infection. Furthermore, numerous viral particles were observed in SGIV- or RGNNV-infected ELHK cells using electron microscopy, which further confirmed that ELHK cells could be used for fish virus propagation in vitro. In addition, quantitative PCR analysis indicated that SGIV and RGNNV infection in ELHK cells significantly increased the transcripts of several interferon- or pro-inflammation-related cytokines, suggested that both of them up-regulated host interferon and inflammatory immune response in vitro. Taken together, our established ELHK cell line could be used as an in vitro tool for gene expression, as well as virus propagation and virus-host interaction studies.

Tumor necrosis factor (TNF)-like weak inducer of apoptosis or TWEAK is a member of the TNF superfamily involved in the regulation of many biological processes. In mammals, TWEAK has been shown to play a role in some autoimmune or inflammatory conditions, but its immune role is not yet clearly defined. In teleost fish, although a few studies have identified homologues to mammalian TWEAK, their biological effects have never been investigated. In the current study, we have studied the transcriptional regulation of two TWEAK homologues (TWEAK 1 and 2) identified in rainbow trout (Oncorhynchus mykiss) throughout different tissues, in response to parasitic or viral infections, or in head kidney (HK) leukocytes stimulated with different stimuli. Although the transcription of both homologues was modulated when HK leukocytes were exposed to several immune stimuli, only TWEAK 1 was significantly modulated upon pathogenic exposure. Thus, we performed a characterization of the functions exerted by this cytokine in HK leukocytes. Recombinant TWEAK 1 strongly up-regulated the transcription of pro-inflammatory genes and antimicrobial peptides in HK leukocytes, with differential transcriptional effects in IgM+ B cells, IgM- lymphocytes and myeloid cells. TWEAK 1 also increased the survival and promoted the differentiation of B cells in HK leukocyte cultures. Our results demonstrate that in teleost fish, TWEAK 1 is involved in the response to different types of pathogens, through the modulation of antimicrobial and pro-inflammatory genes in different leukocytes subsets. Furthermore, a role for TWEAK as a B cell differentiation factor has also been established in rainbow trout.

A teleost’s kidney was divided into head kidney and trunk kidney. The head kidney is an important lymphatic organ, while the trunk kidney mainly performs osmotic pressure regulation and excretion functions. Previous studies have shown that the teleost’s head kidney exerts a strong immune response against pathogen invasion, while the mechanism of immune response in the trunk kidney is still rarely reported. Therefore, in this study, we established an Infectious hematopoietic necrosis virus (IHNV) immersion infection model to compare the similarities and differences of immune response mechanisms between the head kidney and trunk kidney against viral infection. The results showed that IHNV infection causes severe tissue damage and inflammatory reaction in the head and trunk kidney, triggers a series of interferon cascade reactions, and produces strong immune response. In addition, the transcriptome data showed that the head kidney and trunk kidney had similar immune response mechanisms, which showed that the NOD-like receptor signaling pathway and Toll-like receptor signaling pathway were activated. In conclusion, despite functional differentiation, the teleost’s trunk kidney still has a strong immune response, especially the interferon-stimulated genes, which have stronger immune response in the trunk kidney than in the head kidney when responding to IHNV infection. This study contributes to a more comprehensive understanding of the teleost immune system and enriches the theory of kidney immunity in teleosts.

Moritella viscosa is a bacterial pathogen causing winter-ulcer disease in Atlantic salmon. The lesions on affected fish lead to increased mortality, decreased fish welfare, and inferior meat quality in farmed salmon. MicroRNAs (miRNAs) are small non-coding RNAs involved in post-transcriptional regulation by guiding the miRNA-induced silencing complex to specific mRNA transcripts (target genes). The goal of this study was to identify miRNAs responding to Moritella viscosa in salmon by investigating miRNA expression in the head-kidney and the muscle/skin from lesion sites caused by the pathogen. Protein coding gene expression was investigated by microarray analysis in the same materials. Seventeen differentially expressed guide-miRNAs (gDE-miRNAs) were identified in the head-kidney, and thirty-nine in lesion sites, while the microarray analysis reproduced the differential expression signature of several thousand genes known as infection-responsive. In silico target prediction and enrichment analysis suggested that the gDE-miRNAs were predicted to target genes involved in immune responses, hemostasis, angiogenesis, stress responses, metabolism, cell growth, and apoptosis. The majority of the conserved gDE-miRNAs (e.g., miR-125, miR-132, miR-146, miR-152, miR-155, miR-223 and miR-2188) are known as infection-responsive in other vertebrates. Collectively, the findings indicate that gDE-miRNAs are important post-transcriptional gene regulators of the host response to bacterial infection.

Adjuvants are the helper substances that increase vaccine efficacy by enhancing the potency and longevity of specific immune responses to antigens. Most existing fish vaccines are presented in the form of oil-based emulsions delivered by intraperitoneal injection. The characterization of their mode of action is a valuable aid to future vaccine development, particularly for the potential identification and stimulation of specific immunological pathways related to the desired protective response. This study characterized the expression of selected immune-related genes in the peritoneal cavity, head kidney and spleen following the administration of two adjuvanted-bacterial vaccines thought to induce humoral (Montanide™ ISA 763A VG) or humoral and cell mediated (Montanide™ ISA 761 VG) immune responses, to determine if differences in responsiveness are readily apparent. The most informative site was the spleen, where Montanide™ ISA 763A VG + bacterin gave rise to upregulation of genes driving T-cell/lymphoid responses, namely IL-2, IL-15 and IL-21. This combined with upregulation of IFNγ1 and IFNγ2, IL-4/13B2, p35A1 and p40 (B1 and C) indicated that the induction of Th1 and possibly Th2 immunity was occurring in fish vaccinated with this adjuvant. Perhaps the most intriguing finding was the lack of a detectable Th1 response in fish given Montanide™ ISA 761 VG + bacterin, suggesting some other arm of the immune system is activated to give protection. Whatever the reason for the different responses detected, it is clear from the present study that the adjuvant used has a major impact on the responses elicited. Since these differences are readily detectable it allows, in principle, their use to help select the most appropriate adjuvants for inclusion into fish vaccines, where the type of response elicited may need to be tailored to a particular pathogen to confer protection. • Montanide™ ISA 763A VG and Montanide™ ISA 761 VG were used as adjuvants in trout. • Trout were vaccinated with an Aeromonas salmonicida bacterin emulsified with each adjuvant. • Expression of immune genes in peritoneal, head kidney and spleen cells was analysed. • The adjuvant used has a major impact on which immune pathways stimulated. [ABSTRACT FROM AUTHOR]

Red drum (Sciaenops ocellatus), as an important economical marine fish, has been affected by various bacterial diseases in recent years. Vibrio harveyi cause fatal vibriosis in S. ocellatus , leading to massive mortality and causing significant setbacks in aquaculture. However, the regulatory mechanisms of S. ocellatus response to V. harveyi infection are poorly understood. In this regard, we performed transcriptomic analysis with head kidney tissues of S. ocellatus after V. harveyi infection from 12 h to 48 h to reveal genes, gene expression profiles, and pathways involved in immune and inflammation responses. Specifically, a total of 9,599, 5,728, and 7144 differentially expressed genes (DEGs) were identified after V. harveyi infection at 12 h, 24 h, and 48 h, respectively, and 1,848 shared DEGs have been identified from the above three comparison groups. Subsequent pathway analysis revealed that the shared DEGs following V. harveyi were involved in complement and coagulation cascades (C1R, C1QC, C3, C4, C5, C7, C8A, C8B, C8G, C9, CFB, CFH, and CFI), MAPK signaling pathway, chemokine signaling pathway (CCL19, CXCL8, CXCL12, CXCL14, CCR4, CCR7, and CXCR2), PPAR signaling pathway (PPAR-α, PPAR-γ and PPAR-β), and TNF signaling pathway. Finally, the expression patterns of DEGs in head kidney tissues and S. ocellatus macrophages were validated by qRT-PCR, suggesting the reliability of RNA sequencing for gene expression analysis. This dynamic transcriptome analyses provided insights into gene expression regulation and immune related pathways involved in S. ocellatus after V. harveyi infection, and provides useful information for further study on the immune defense mechanisms in S. ocellatus as well as other teleost species. • First comparative transcriptome analysis of S. ocellatus infected with V. harveyi. • DEGs were identified from 12 h, 24 h, and 48 h after V. harveyi infection. • A total of 1848 DEGs was identified from three comparative transcriptome analysis. • Immune-related DEGs and pathways were identified and classified. [ABSTRACT FROM AUTHOR]

Single-cell transcriptomics is the current gold standard for global gene expression profiling, not only in mammals and model species, but also in non-model fish species. This is a rapidly expanding field, creating a deeper understanding of tissue heterogeneity and the distinct functions of individual cells, making it possible to explore the complexities of immunology and gene expression on a highly resolved level. In this study, we compared two single cell transcriptomic approaches to investigate cellular heterogeneity within the head kidney of healthy farmed Atlantic salmon (Salmo salar). We compared 14,149 cell transcriptomes assayed by single cell RNA-seq (scRNA-seq) with 18,067 nuclei transcriptomes captured by single nucleus RNA-Seq (snRNA-seq). Both approaches detected eight major cell populations in common: granulocytes, heamatopoietic stem cells, erythrocytes, mononuclear phagocytes, thrombocytes, B cells, NK-like cells, and T cells. Four additional cell types, endothelial, epithelial, interrenal, and mesenchymal cells, were detected in the snRNA-seq dataset, but appeared to be lost during preparation of the single cell suspension submitted for scRNA-seq library generation. We identified additional heterogeneity and subpopulations within the B cells, T cells, and endothelial cells, and revealed developmental trajectories of heamatopoietic stem cells into differentiated granulocyte and mononuclear phagocyte populations. Gene expression profiles of B cell subtypes revealed distinct IgM and IgT-skewed resting B cell lineages and provided insights into the regulation of B cell lymphopoiesis. The analysis revealed eleven T cell sub-populations, displaying a level of T cell heterogeneity in salmon head kidney comparable to that observed in mammals, including distinct subsets of cd4 / cd8 -negative T cells, such as tcrγ positive, progenitor-like, and cytotoxic cells. Although snRNA-seq and scRNA-seq were both useful to resolve cell type-specific expression in the Atlantic salmon head kidney, the snRNA-seq pipeline was overall more robust in identifying several cell types and subpopulations. While scRNA-seq displayed higher levels of ribosomal and mitochondrial genes, snRNA-seq captured more transcription factor genes. However, only scRNA-seq-generated data was useful for cell trajectory inference within the myeloid lineage. In conclusion, this study systematically outlines the relative merits of scRNA-seq and snRNA-seq in Atlantic salmon, enhances understanding of teleost immune cell lineages, and provides a comprehensive list of markers for identifying major cell populations in the head kidney with significant immune relevance. [Display omitted] • List of robust markers genes for cell type annotation. • Identification and characterization of 12 major cell populations in Atlantic salmon head kidney. • Insights into B cell and T cell heterogeneity and description of biologically relevant subpopulations. • Comparative analysis of scRNA-seq and snRNA-seq reveals important considerations for experimental design in single cell genomics. [ABSTRACT FROM AUTHOR]