Authors: Victoria A Sleight, J Andrew Gillis

Paired fins are a defining feature of the jawed vertebrate body plan, but their evolutionary origin remains unresolved. Gegenbaur proposed that paired fins evolved as gill arch serial homologues, but this hypothesis is now widely discounted, owing largely to the presumed distinct embryonic origins of these structures from mesoderm and neural crest, respectively. Here, we use cell lineage tracing to test the embryonic origin of the pharyngeal and paired fin skeleton in the skate (). We find that while the jaw and hyoid arch skeleton derive from neural crest, and the pectoral fin skeleton from mesoderm, the gill arches are of dual origin, receiving contributions from both germ layers. We propose that gill arches and paired fins are serially homologous as derivatives of a continuous, dual-origin mesenchyme with common skeletogenic competence, and that this serial homology accounts for their parallel anatomical organization and shared responses to axial patterning signals.

Topics: Animal Fins; Animals; Embryo, Nonmammalian; Embryonic Development; Gills; Neural Crest; RNA, Messenger; Skates, Fish; Skeleton

PubMed: 33198887
DOI: 10.7554/eLife.60635

Authors: Sonja Fiedler, Hannah Wünnemann, Isabel Hofmann...

Rainbow trout (Oncorhynchus mykiss) are frequently used as experimental animals in ecotoxicological studies, in which they are experimentally exposed to defined concentrations of test substances, such as heavy metals, pesticides, or pharmaceuticals. Following exposure to a broad variety of aquatic pollutants, early morphologically detectable toxic effects often manifest in alterations of the gills. Suitable methods for an accurate and unbiased quantitative characterization of the type and the extent of morphological gill alterations are therefore essential prerequisites for recognition, objective evaluation and comparison of the severity of gill lesions. The aim of the present guidelines is to provide practicable, standardized and detailed protocols for the application of unbiased quantitative stereological analyses of relevant morphological parameters of the gills of rainbow trout. These gill parameters inter alia include the total volume of the primary and secondary gill lamellae, the surface area of the secondary gill lamellae epithelium (i.e., the respiratory surface) and the thickness of the diffusion barrier. The featured protocols are adapted to fish of frequently used body size classes (300-2000 g). They include well-established, conventional sampling methods, probes and test systems for unbiased quantitative stereological analyses of light- and electron microscopic 2-D gill sections, as well as the application of modern 3-D light sheet fluorescence microscopy (LSFM) of optically cleared gill samples as an innovative, fast and efficient quantitative morphological analysis approach. The methods shown here provide a basis for standardized and representative state-of-the-art quantitative morphological analyses of trout gills, ensuring the unbiasedness and reproducibility, as well as the intra- and inter-study comparability of analyses results. Their broad implementation will therefore significantly contribute to the reliable identification of no observed effect concentration (NOEC) limits in ecotoxicological studies and, moreover, to limit the number of experimental animals by reduction of unnecessary repetition of experiments.

Topics: Animals; Female; Gills; Male; Microscopy, Fluorescence; Oncorhynchus mykiss; Reproducibility of Results; Water Pollutants, Chemical

PubMed: 33296424
DOI: 10.1371/journal.pone.0243462

Authors: Mar Marcos-López, Josep A Calduch-Giner, Luca Mirimin...

Amoebic gill disease (AGD) is one of the main diseases affecting Atlantic salmon (Salmo salar L.) mariculture. Hallmarks of AGD are hyperplasia of the lamellar epithelium and increased production of gill mucus. This study investigated the expression of genes involved in mucus secretion, cell cycle regulation, immunity and oxidative stress in gills using a targeted 21-gene PCR array. Gill samples were obtained from experimental and natural Neoparamoeba perurans infections, and sampling points included progressive infection stages and post-freshwater treatment. Up-regulation of genes related to mucin secretion and cell proliferation, and down-regulation of pro-inflammatory and pro-apoptotic genes were associated with AGD severity, while partial restoration of the gill homeostasis was detected post-treatment. Mucins and Th2 cytokines accoun ted for most of the variability observed between groups highlighting their key role in AGD. Two mucins (muc5, muc18) showed differential regulation upon disease. Substantial up-regulation of the secreted muc5 was detected in clinical AGD, and the membrane bound muc18 showed an opposite pattern. Th2 cytokines, il4/13a and il4/13b2, were significantly up-regulated from 2 days post-infection onwards, and changes were lesion-specific. Despite the differences between experimental and natural infections, both yielded comparable results that underline the importance of the studied genes in the respiratory organs of fish, and during AGD progression.

Topics: Amoeba; Animals; Down-Regulation; Fish Diseases; Gene Expression; Gills; Inflammation; Interleukin-13; Interleukin-4; Mucins; Salmo salar; Up-Regulation

PubMed: 30209326
DOI: 10.1038/s41598-018-32019-8

Authors: Mohamed Emam, Albert Caballero-Solares, Xi Xue...

Gill damage represents a significant challenge in the teleost fish aquaculture industry globally, due to the gill's involvement in several vital functions and direct contact with the surrounding environment. To examine the local and systemic effects accompanying gill damage (which is likely to negatively affect gill function) of Atlantic salmon, we performed a field sampling to collect gill and liver tissue after several environmental insults (e.g., harmful algal blooms). Before sampling, gills were visually inspected and gill damage was scored; gill scores were assigned from pristine [gill score 0 (GS0)] to severely damaged gills (GS3). Using a 44K salmonid microarray platform, we aimed to compare the transcriptomes of pristine and moderately damaged (i.e., GS2) gill tissue. Rank Products analysis (5% percentage of false-positives) identified 254 and 34 upregulated and downregulated probes, respectively, in GS2 compared with GS0. Differentially expressed probes represented genes associated with functions including gill remodeling, wound healing, and stress and immune responses. We performed gill and liver qPCR for all four gill damage scores using microarray-identified and other damage-associated biomarker genes. Transcripts related to wound healing (e.g., and ) were significantly upregulated in GS2 compared with GS0 in the gills. Also, transcripts associated with immune and stress-relevant pathways were dysregulated (e.g., downregulation of and upregulation of ) in GS2 compared with GS0 gills. The livers of salmon with moderate gill damage (i.e., GS2) showed significant upregulation of transcripts related to wound healing (i.e., ), apoptosis (e.g., ), blood coagulation (e.g., and ), transcription regulation (i.e., ), and stress-responses (e.g., ) compared with livers of GS0 fish. We performed principal component analysis (PCA) using transcript levels for gill and liver separately. The gill PCA showed that PC1 significantly separated GS2 from all other gill scores. The genes contributing most to this separation were , , , , and The liver PCA showed that PC1 significantly separated GS2 from GS0; levels of , , , , and were the highest contributors to this separation. Also, hepatic acute phase biomarkers (e.g., and ) were positively correlated to each other and to gill damage. Gill damage-responsive biomarker genes and associated qPCR assays arising from this study will be valuable in future research aimed at developing therapeutic diets to improve farmed salmon welfare.

Topics: Animals; Biomarkers; Gills; Liver; PPAR gamma; Salmo salar

PubMed: 35418993
DOI: 10.3389/fimmu.2022.806484

Authors: Marit M Amundsen, Haitham Tartor, Kathrine Andersen...

Salmon Gill Poxvirus Disease (SGPVD) has emerged as a cause of acute mortality in Atlantic salmon () presmolts in Norwegian aquaculture. The clinical phase of the disease is associated with apoptotic cell death in the gill epithelium causing acute respiratory distress, followed by proliferative changes in the regenerating gill in the period after the disease outbreak. In an experimental SGPV challenge trial published in 2020, acute disease was only seen in fish injected with hydrocortisone 24 h prior to infection. SGPV-mediated mortality in the hydrocortisone-injected group was associated with more extensive gill pathology and higher SGPV levels compared to the group infected with SGPV only. In this study based on the same trial, SGPV gene expression and the innate and adaptive antiviral immune response was monitored in gills and spleen in the presence and absence of hydrocortisone. Whereas most SGPV genes were induced from day 3 along with the interferon-regulated innate immune response in gills, the putative SGPV virulence genes of the B22R family were expressed already one day after SGPV exposure, indicating a potential role as early markers of SGPV infection. In gills of the hydrocortisone-injected fish infected with SGPV, expression was delayed until day 10, and then expression skyrocketed along with the viral peak, gill pathology and mortality occurring from day 14. A similar expression pattern was observed for Interferon gamma () and granzyme A () in the gills, indicating a role of acute cytotoxic cell activity in SGPVD. Duplex hybridization demonstrated effects of hydrocortisone on the number and localization of -containing cells, and colocalization with SGPV infected cells in the gill. SGPV was generally not detected in spleen, and gill infection did not induce any corresponding systemic immune activity in the absence of stress hormone injection. However, in fish injected with hydrocortisone, and gene expression was induced in spleen in the days prior to acute mortality. These data indicate that suppressed mucosal immune response in the gills and the late triggered systemic immune response in the spleen following hormonal stress induction may be the key to the onset of clinical SGPVD.

Topics: Animals; Anti-Inflammatory Agents; CD4 Antigens; CD8 Antigens; Fish Diseases; Fish Proteins; Gills; Granzymes; Host-Pathogen Interactions; Hydrocortisone; Immunity, Mucosal; Interferon-gamma; Mucous Membrane; Poxviridae; Poxviridae Infections; Salmo salar

PubMed: 34177946
DOI: 10.3389/fimmu.2021.689302

Authors: Huiling Jiang, Jun He, Hui Wang...

Microcystin-LR (MC-LR) is widely present in waters around the world, but its potential toxic effects and mechanisms on amphibian gills remain unknown. In the present study, tadpoles () were exposed to environmentally realistic concentrations of 0.5, 2 μg/L MC-LR, and 0 μg/L MC-LR (Control) for 30 days with the objective to unveil the impairment of gill health. The lysozyme was downregulated, while pattern recognition receptors and complement and adaptive immune processes were upregulated and the ability of gill supernatant to inhibit pathogenic bacteria decreased in the 0.5 and 2 μg/L MC-LR groups. The transcriptions of epithelial barrier components (e.g., ) were significantly decreased in MC-LR-exposed gills, while the gill content of lipopolysaccharide (LPS) endotoxins and the transcriptions of downstream responsive genes (e.g., and ) were concurrently increased. In addition, the number of eosinophils and the expression of pro-inflammatory cytokines (e.g., IL-1β and ) were increased. These results imply that exposure of tadpoles to low environmentally concentrations of MC-LR leads to inflammation, increased permeability, and a reduced ability to inhibit pathogenic bacteria. The epithelial cells of inner gill filaments increased and transcriptions of hypoxic stress genes (e.g., , , and ) were upregulated within the exposed group. As a consequence, exposure to MC-LR may lead to hypoxic stress. MC-LR exposure also drove gill microbiota to a dysbiosis. The relative abundance of was positively correlated with content of LPS and transcriptions of and . Overall, this study presents the first evidence about the pronounced impacts of MC-LR exposure on gills of amphibians, highlighting the susceptibility of early developing tadpoles to the environmental risks of MC-LR.

Topics: Animals; Arginine; Gills; Larva; Leucine; Lipopolysaccharides; Microcystins; NF-kappa B; Rana catesbeiana; Tumor Necrosis Factor-alpha

PubMed: 35878217
DOI: 10.3390/toxins14070479

Authors: Christine Hirschberger, J Andrew Gillis

The pseudobranch is a gill-like epithelial elaboration that sits behind the jaw of most fishes. This structure was classically regarded as a vestige of the ancestral gill arch-like condition of the gnathostome jaw. However, more recently, hypotheses of jaw evolution by transformation of a gill arch have been challenged, and the pseudobranch has alternatively been considered a specialised derivative of the second (hyoid) pharyngeal arch. Here, we demonstrate in the skate (Leucoraja erinacea) that the pseudobranch does, in fact, derive from the mandibular arch, and that it shares gene expression features and cell types with gills. We also show that the skate mandibular arch pseudobranch is supported by a spiracular cartilage that is patterned by a shh-expressing epithelial signalling centre. This closely parallels the condition seen in the gill arches, where cartilaginous appendages called branchial rays, which support the respiratory lamellae of the gills, are patterned by a shh-expressing gill arch epithelial ridge. Together with similar discoveries in zebrafish, our findings support serial homology of the pseudobranch and gills, and an ancestral origin of gill arch-like anatomical features from the gnathostome mandibular arch.

Topics: Animals; Branchial Region; Gills; Jaw; Skates, Fish; Zebrafish

PubMed: 35762641
DOI: 10.1242/dev.200184

The Atlantic Salmon Gill Transcriptome Response in a Natural Outbreak of Salmon Gill Pox Virus Infection Reveals New Biomarkers of Gill Pathology and Suppression of Mucosal Defense.

Authors: Mona C Gjessing, Aleksei Krasnov, Gerrit Timmerhaus...

The salmon gill poxvirus (SGPV) is a large DNA virus that infects gill epithelial cells in Atlantic salmon and is associated with acute high mortality disease outbreaks in aquaculture. The pathological effects of SGPV infection include gill epithelial apoptosis in the acute phase of the disease and hyperplasia of gill epithelial cells in surviving fish, causing damage to the gill respiratory surface. In this study, we sampled gills from Atlantic salmon presmolts during a natural outbreak of SGPV disease (SGPVD). Samples covered the early phase of infection, the acute mortality phase, the resolving phase of the disease and control fish from the same group and facility. Mortality, the presence and level of SGPV and gill epithelial apoptosis were clearly associated. The gene expression pattern in the acute phase of SGPVD was in tune with the pathological findings and revealed novel transcript-based disease biomarkers, including pro-apoptotic and proliferative genes, along with changes in expression of ion channels and mucins. The innate antiviral response was strongly upregulated in infected gills and chemokine expression was altered. The regenerating phase did not reveal adaptive immune activity within the study period, but several immune effector genes involved in mucosal protection were downregulated into the late phase, indicating that SGPV infection could compromise mucosal defense. These data provide novel insight into the infection mechanisms and host interaction of SGPV.

Topics: Animals; Apoptosis; Biomarkers; Cell Proliferation; Disease Outbreaks; Fish Diseases; Fish Proteins; Gills; Immunity, Mucosal; Immunosuppression Therapy; Ion Channels; Mucins; Norway; Poxviridae; Poxviridae Infections; Salmo salar; Transcriptome

PubMed: 33013908
DOI: 10.3389/fimmu.2020.02154

Authors: Wei-Dan Jiang, Li Zhang, Lin Feng...

In this study, we have investigated the influence of vitamin A on gill barrier function of grass carp () infected with The fish were fed different concentrations of vitamin A diets for 10 weeks and then infected with by immersion. We observed that optimal vitamin A significantly prevented gill rot morbidity in fish infected with . Further investigations revealed that vitamin A boosted the gill immunity by increasing the contents of complements (C3 and C4), activities of acid phosphatase (ACP) and lysozyme, mRNAs of and and , , and anti-inflammatory cytokines like and . It also enhanced the levels of various related signaling molecules including and but downregulated the expression of pro-inflammatory cytokines including , and and related signaling molecules including (rather than (rather than and mRNA levels in fish gills. In addition, dietary vitamin A markedly lowered the concentrations of reactive oxygen species (ROS), malondialdehyde (MDA), and protein carbonyl (PC), increased both the activities and mRNAs of copper/zinc superoxide dismutase (Cu/ZnSOD), MnSOD, glutathione transferases (GSTs), glutathione peroxidase (GPx), and glutathione reductase (GR) associated with upregulation of mRNAs and downregulation of and mRNAs. Moreover, vitamin A decreased the mRNAs of different apoptotic mediators [ (rather than 7)] associated with downregulation of signaling molecule mRNAs in fish gills. Besides, vitamin A promoted tight junction (TJ) complex mRNAs [including , and ] that have been linked to the downregulation of myosin light chain kinase (MLCK) signaling. Taken together, the current study demonstrated for the first time that vitamin A markedly enhanced gill health associated with immune modulation and physical barrier protection. Based on protecting fish against gill rot morbidity, ACP activity, and against lipid peroxidation, optimum vitamin A concentrations in on-growing grass carp (262-997 g) were found to be 1,991, 2,188, and 2,934 IU/kg diet, respectively.

Topics: Animal Feed; Animals; Carps; Cytokines; Dietary Supplements; Fish Proteins; Flavobacterium; Gills; Vitamin A

PubMed: 35401542
DOI: 10.3389/fimmu.2022.833455

Authors: Alf S Dalum, Aurora Kraus, Shanawaz Khan...

The zebrafish is extensively used as an animal model for human and fish diseases. However, our understanding of the structural organization of its immune system remains incomplete, especially the mucosa-associated lymphoid tissues (MALTs). Teleost MALTs are commonly perceived as diffuse and scattered populations of immune cells throughout the mucosa. Yet, structured MALTs have been recently discovered in Atlantic salmon (), including the interbranchial lymphoid tissue (ILT) in the gills. The existence of the ILT was only recently identified in zebrafish and other fish species, highlighting the need for in-depth characterizations of the gill-associated lymphoid tissue (GIALT) in teleosts. Here, using 3-D high-resolution microscopy, we analyze the GIALT of adult zebrafish with an immuno-histology approach that reveals the organization of lymphoid tissues the labeling of T/NK cells with an antibody directed to a highly conserved epitope on the kinase ZAP70. We show that the GIALT in zebrafish is distributed over at least five distinct sub-regions, an organization found in all pairs of gill arches. The GIALT is diffuse in the pharyngeal part of the gill arch, the interbranchial septum and the filaments/lamellae, and structured in two sub-regions: the ILT, and a newly discovered lymphoid structure located along each side of the gill arch, which we named the Amphibranchial Lymphoid Tissue (ALT). Based on RAG2 expression, neither the ILT nor the ALT constitute additional thymi. The ALT shares several features with the ILT such as presence of abundant lymphoid cells and myeloid cells embedded in a network of reticulated epithelial cells. Further, the ILT and the ALT are also a site for T/NK cell proliferation. Both ILT and ALT show structural changes after infection with Spring Viraemia of Carp Virus (SVCV). Together, these data suggest that ALT and ILT play an active role in immune responses. Comparative studies show that whereas the ILT seems absent in most neoteleosts ("Percomorphs"), the ALT is widely present in cyprinids, salmonids and neoteleosts, suggesting that it constitutes a conserved tissue involved in the protection of teleosts the gills.

Topics: Animals; Fish Diseases; Gills; Imaging, Three-Dimensional; Lymphoid Tissue; Viremia; Zebrafish

PubMed: 34880866
DOI: 10.3389/fimmu.2021.769901