Potential Involvement of the South American Lungfish Intelectin-2 in Innate-Associated Immune Modulation.

Intelectins belong to a family of lectins with specific and transitory carbohydrate interaction capabilities. These interactions are related to the activity of agglutinating pathogens, as intelectins play a significant role in immunity. Despite the prominent immune defense function of intelectins, limited information about its structural characteristics and carbohydrate interaction properties is available. This study investigated an intelectin transcript identified in RNA-seq data obtained from the South American lungfish (Lepidosiren paradoxa), namely LpITLN2-B. The structural analyses predicted LpITLN2-B to be a homo-trimeric globular protein with the fibrinogen-like functional domain (FReD), exhibiting a molecular mass of 57 kDa. The quaternary structure is subdivided into three monomers, A, B, and C, and each domain comprises 11 ß-sheets: an anti-parallel ß-sheet, a ß-hairpin, and a disordered ß-sheet structure. Molecular docking demonstrates a significant interaction with disaccharides rather than monosaccharides. The preferential interaction with disaccharides highlights the potential interaction with pathogen molecules, such as LPS and Poly(I:C). The hemagglutination assay inhibited lectins activity, especially maltose and sucrose, highlighting lectin activity in L. paradoxa samples. Overall, our results show the potential relevance of LpITLN2-B in L. paradoxa immune defense against pathogens.

Copy number variation and elevated genetic diversity at immune trait loci in Atlantic and Pacific herring.

BACKGROUND: Genome-wide comparisons of populations are widely used to explore the patterns of nucleotide diversity and sequence divergence to provide knowledge on how natural selection and genetic drift affect the genome. In this study we have compared whole-genome sequencing data from Atlantic and Pacific herring, two sister species that diverged about 2 million years ago, to explore the pattern of genetic differentiation between the two species. RESULTS: The genome comparison of the two species revealed high genome-wide differentiation but with islands of remarkably low genetic differentiation, as measured by an FST analysis. However, the low FST observed in these islands is not caused by low interspecies sequence divergence (dxy) but rather by exceptionally high estimated intraspecies nucleotide diversity (π). These regions of low differentiation and elevated nucleotide diversity, termed high-diversity regions in this study, are not enriched for repeats but are highly enriched for immune-related genes. This enrichment includes genes from both the adaptive immune system, such as immunoglobulin, T-cell receptor and major histocompatibility complex genes, as well as a substantial number of genes with a role in the innate immune system, e.g. novel immune-type receptor, tripartite motif and tumor necrosis factor receptor genes. Analysis of long-read based assemblies from two Atlantic herring individuals revealed extensive copy number variation in these genomic regions, indicating that the elevated intraspecies nucleotide diversities were partially due to the cross-mapping of short reads. CONCLUSIONS: This study demonstrates that copy number variation is a characteristic feature of immune trait loci in herring. Another important implication is that these loci are blind spots in classical genome-wide screens for genetic differentiation using short-read data, not only in herring, likely also in other species harboring qualitatively similar variation at immune trait loci. These loci stood out in this study because of the relatively high genome-wide baseline for FST values between Atlantic and Pacific herring.

Role of alternative splicing in fish immunity.

Alternative splicing serves as a pivotal source of complexity in the transcriptome and proteome, selectively connecting various coding elements to generate a diverse array of mRNAs. This process encodes multiple proteins with either similar or distinct functions, contributing significantly to the intricacies of cellular processes. The role of alternative splicing in mammalian immunity has been well studied. Remarkably, the immune system of fish shares substantial similarities with that of humans, and alternative splicing also emerges as a key player in the immune processes of fish. In this review, we offer an overview of alternative splicing and its associated functions in the immune processes of fish, and summarize the research progress on alternative splicing in the fish immunity. Furthermore, we review the impact of alternative splicing on the fish immune system's response to external stimuli. Finally, we present our perspectives on future directions in this field. Our aim is to provide valuable insights for the future investigations into the role of alternative splicing in immunity.

Understanding the host response of farmed fish to blood flukes (Trematoda: Aporocotylidae) for developing new treatment strategies.

Aporocotylids (Trematoda: Digenea), also known as fish blood flukes infect the circulatory system of fish leading to serious health problems and mortality. Aporocotylids are a particular concern for farmed fish as infection intensity can increase within the farming environment and lead to mortalities. In the context of managing these infections, one of the most crucial aspects to consider is the host response of the infected fish against these blood flukes. Understanding the response is essential to improving current treatment strategies that are largely based on the use of anthelmintic praziquantel to manage infections in aquaculture. This review focuses on the current knowledge of farmed fish host responses against the different life stages of aporocotylids. New treatment strategies that are able to provide protection against reinfections should be a long-term goal and is not possible without understanding the fish response to infection and the interactions between host and parasite.

The novel fish miRNA, Soc-miR-118, functions as a negative regulator in NF-κB-mediated inflammation by targeting IL-6 in teleost fish.

Inflammation is initiated as a protective response of the organism to remove invading bacterial and initiate the healing process. Prolonged inflammation and excessive production of inflammatory cytokines lead to inflammatory disorders or autoimmune diseases. Thus, different layers of negative regulators are needed to achieve balances between protective immunity and inflammatory pathology. Accumulating evidences show that miRNAs act as significant and multifunctional regulators involved in regulating networks of host-pathogen interactions. However, the functions and mechanisms of miRNAs in directly targeting and regulating inflammatory cytokines remains largely unknown in lower vertebrates. In this study, we report a novel miRNA, Soc-miR-118, identified from Sciaenops ocellatus, which plays a negative role in antibacterial immunity by regulating Interleukin-6 (IL-6). Specifically, we found that Soc-miR-118 directly targets IL-6 and suppresses the production of inflammatory cytokines through the NF-κB signaling pathway, thereby avoiding excessive inflammatory response. Particularly, the mechanism by which Soc-miR-118 regulates IL-6 expression also exist in other fish, suggesting that the miRNA in fish has evolutionarily conserved regulatory systems. The collective results that Soc-miR-118 acts as a negative regulator involved in host antibacterial immunity through directly regulating inflammatory cytokines, will greatly enrich the intricate networks of host-pathogen interaction in lower vertebrates.

Regulatory roles of miRNA-530 in the post-transcriptional regulation of NF-κB signaling pathway through targeted modulation of IκBα in Sebastesschlegelii.

MicroRNAs (miRNAs) are a crucial type of non-coding RNAs involved in post-transcriptional regulation. The playing essential regulatory roles in the NF-κB signaling pathway and modulate the host immune response to diverse pathogens by targeting IκBα. However, the regulatory mechanism of miRNAs in relation with IκBα in Sebastes schlegelii remains unclear. In our study, we identified two copies of IkBα gene in black rockfish (Sebastes schlegelii), namely IkBα1 and IkBα2. Moreover, we have discovered that miRNA-530 can activate the NF-κB signaling pathway by inhibiting the expression of IκBα, thereby inducing the inflammatory response. This project comprehensively investigated the interactive regulatory roles of miRNA-530 in the NF-κB signaling pathway at both cellular and in vivo levels, while also elucidating the regulatory relationships between miRNA-530 and IκBα. In conclusion, our research confirmed that miRNA-530 can target the 3'UTR region of IκBα, resulting in a decrease in the expression of IκBα at the post-transcriptional level and inhibiting its translation. The findings contribute to the understanding of the regulatory network of non-coding RNA in teleosts and its subsequent regulation of the NF-κB signaling pathway by miRNAs.

Effects of Citrobacter freundii on sturgeon: Insights from skin mucosal immunology and microbiota.

Skin mucus analysis has recently been used as a non-invasive method to evaluate for fish welfare. The present research study was conducted to examine the skin mucosal immunity and skin microbiota profiles of sturgeons infected with Citrobacter freundii. Our histology results showed that the thickness of the epidermal layer of skin remained thinner, and the number of mucous cells was significantly decreased in sturgeons after infection (p < 0.05). Total protein, alanine aminotransferase, aspartate aminotransferase, superoxide dismutase, and creatine kinase levels in the mucus showed biphasic pattern (decrease and then increase). Lactate dehydrogenase, lysozyme, and acid phosphatase activities in the mucus showed an increasing trend after infection. Furthermore, 16S rRNA sequencing also revealed that C. freundii infection also affected the diversity and community structure of the skin mucus microbiota. An increase in microbial diversity (p > 0.05) and a decrease in microbial abundance (p < 0.05) after infection were noted. The predominant bacterial phyla in the skin mucus were Proteobacteria, Fusobacteria, Bacteroidetes, Firmicutes, and Actinobacteria. Specifically, the relative abundance of Fusobacteria increased after infection. The predominant bacterial genera in the skin mucus were Cetobacterium, Pelomonas, Bradyrhizobium, Flavobacterium, and Pseudomonas. The relative abundance of Cetobacterium, Pseudomonas, and Flavobacterium increased after infection. Our current research findings will provide new insights into the theoretical basis for future research studies exploring the mechanism of sturgeon infection with C. freundii.

Establishment and characterization of a golden pompano (Trachinotus blochii) fin cell line for applications in marine fish pathogen immunology.

Pompano fishes have been widely farmed worldwide. As a representative commercial marine species of the Carangidae family, the golden pompano (Trachinotus blochii) has gained significant popularity in China and worldwide. However, because of rapid growth and high-density aquaculture, the golden pompano has become seriously threatened by various diseases. Cell lines are the most cost-effective resource for in vitro studies and are widely used for physiological and pathological research owing to their accessibility and convenience. In this study, we established a novel immortal cell line, GPF (Golden pompano fin cells). GPF has been passaged over 69 generations for 10 months. The morphology, adhesion and extension processes of GPF were evaluated using light and electron microscopy. GPF cells were passaged every 3 days with L-15 containing 20 % fetal bovine serum (FBS) at 1:3. The optimum conditions for GPF growth were 28 °C and a 20 % FBS concentration. DNA sequencing of 18S rRNA and mitochondrial 16S rRNA confirmed that GPF was derived from the golden pompano. Chromosomal analysis revealed that the number pattern of GPF was 48 chromosomes. Transfection experiments demonstrated that GPF could be utilized to express foreign genes. Furthermore, heavy metals (Cd, Cu, and Fe) exhibited dose-dependent cytotoxicity against GPF. After polyinosinic-polycytidylic acid (poly I:C) treatment, transcription of the retinoic acid-inducible gene I-like receptor (RLR) pathway genes, including mda5, mita, tbk1, irf3, and irf7 increased, inducing the expression of interferon (IFN) and anti-viral proteins in GPF cells. In addition, lipopolysaccharide (LPS) stimulation up-regulated the expression of inflammation-related factors, including myd88, irak1, nfκb, il1ß, il6, and cxcl10 expression. To the best of our knowledge, this is the first study on the immune response signaling pathways of the golden pompano using an established fin cell line. In this study, we describe a preliminary investigation of the GPF cell line immune response to poly I:C and LPS, and provide a more rapid and efficient experimental material for research on marine fish immunology.

Functional characterization of NOD1 from golden pompano Trachinotus ovatus.

Fish rely on innate immune system for immunity, and nucleotide-binding oligomerization domain-like receptors (NLRs) are a vital group of receptor for recognition. In the present study, NOD1 gene was cloned and characterized from golden pompano Trachinotus ovatus, a commercially important aquaculture fish species. The ORF of T. ovatus NOD1 was 2820 bp long, encoding 939 amino acid residues with a highly conserved domains containing CARD-NACHT-LRRs. Phylogenetic analysis revealed that the T. ovatus NOD1 clustered with those of fish and separated from those of birds and mammals. T. ovatus NOD1 has wide tissue distribution with the highest expression in gills. Bacterial challenges (Streptococcus agalactiae and Vibrio alginolyticus) significantly up-regulated the expression of NOD1 with different response time. The results of T. ovatus NOD1 ligand recognition and signaling pathway analysis revealed that T. ovatus NOD1 could recognize iE-DAP at the concentration of ≧ 100 ng/mL and able to activate NF-κB signaling pathway. This study confirmed that NOD1 play a crucial role in the innate immunity of T. ovatus. The findings of this study improve our understanding on the immune function of NOD1 in teleost, especially T. ovatus.

Possible transport routes of IgM to the gut of teleost fish.

Fish rely on mucosal surfaces as their first defence barrier against pathogens. Maintaining mucosal homeostasis is therefore crucial for their overall well-being, and it is likely that secreted immunoglobulins (sIg) play a pivotal role in sustaining this balance. In mammals, the poly-Ig receptor (pIgR) is an essential component responsible for transporting polymeric Igs across mucosal epithelia. In teleost fish, a counterpart of pIgR has been identified and characterized, exhibiting structural differences and broader mRNA expression patterns compared to mammals. Despite supporting evidence for the binding of Igs to recombinant pIgR proteins, the absence of a joining chain (J-chain) in teleosts challenges the conventional understanding of Ig transport mechanisms. The transport of IgM to the intestine via the hepatobiliary route is observed in vertebrates and has been proposed in a few teleosts. Investigations on the stomachless fish, ballan wrasse, revealed a significant role of the hepatobiliary route and interesting possibilities for alternative IgM transport routes that might include pancreatic tissue. These findings highlight the importance of gaining a thorough understanding of the mechanisms behind Ig transport to the gut in various teleosts. This review aims to gather existing information on pIgR-mediated transport across epithelial cells and immunoglobulin transport pathways to the gut lumen in teleost fish. It provides comparative insights into the hepatobiliary transport of Igs to the gut, emphasizing the current understanding in teleost fish while exploring potential alternative pathways for Ig transport to the gut lumen. Despite significant progress in understanding various aspects, there is still much to uncover, especially concerning the diversity of mechanisms across different teleost species.

Climate change can impair bacterial pathogen defences in sablefish via hypoxia-mediated effects on adaptive immunity.

Low-oxygen levels (hypoxia) in aquatic habitats are becoming more common because of global warming and eutrophication. However, the effects on the health/disease status of fishes, the world's largest group of vertebrates, are unclear. Therefore, we assessed how long-term hypoxia affected the immune function of sablefish, an ecologically and economically important North Pacific species, including the response to a formalin-killed Aeromonas salmonicida bacterin. Sablefish were held at normoxia or hypoxia (100% or 40% air saturated seawater, respectively) for 6-16 weeks, while we measured a diverse array of immunological traits. Given that the sablefish is a non-model organism, this involved the development of a species-specific methodological toolbox comprised of qPCR primers for 16 key immune genes, assays for blood antibacterial defences, the assessment of blood immunoglobulin (IgM) levels with ELISA, and flow cytometry and confocal microscopy techniques. We show that innate immune parameters were typically elevated in response to the bacterial antigens, but were not substantially affected by hypoxia. In contrast, hypoxia completely prevented the ∼1.5-fold increase in blood IgM level that was observed under normoxic conditions following bacterin exposure, implying a serious impairment of adaptive immunity. Since the sablefish is naturally hypoxia tolerant, our results demonstrate that climate change-related deoxygenation may be a serious threat to the immune competency of fishes.

Transcriptome profiling of lumpfish (Cyclopterus lumpus) head kidney to Renibacterium salmoninarum at early and chronic infection stages.

Renibacterium salmoninarum causes Bacterial Kidney Disease (BKD) in several fish species. Atlantic lumpfish, a cleaner fish, is susceptible to R. salmoninarum. To profile the transcriptome response of lumpfish to R. salmoninarum at early and chronic infection stages, fish were intraperitoneally injected with either a high dose of R. salmoninarum (1 × 109 cells dose-1) or PBS (control). Head kidney tissue samples were collected at 28- and 98-days post-infection (dpi) for RNA sequencing. Transcriptomic profiling identified 1971 and 139 differentially expressed genes (DEGs) in infected compared with control samples at 28 and 98 dpi, respectively. At 28 dpi, R. salmoninarum-induced genes (n = 434) mainly involved in innate and adaptive immune response-related pathways, whereas R. salmoninarum-suppressed genes (n = 1537) were largely connected to amino acid metabolism and cellular processes. Cell-mediated immunity-related genes showed dysregulation at 98 dpi. Several immune-signalling pathways were dysregulated in response to R. salmoninarum, including apoptosis, alternative complement, JAK-STAT signalling, and MHC-I dependent pathways. In summary, R. salmoninarum causes immune suppression at early infection, whereas lumpfish induce a cell-mediated immune response at chronic infection. This study provides a complete depiction of diverse immune mechanisms dysregulated by R. salmoninarum in lumpfish and opens new avenues to develop immune prophylactic tools to prevent BKD.

The structure of the teleost Immunoglobulin M core provides insights on polymeric antibody evolution, assembly, and function.

Polymeric (p) immunoglobulins (Igs) serve broad functions during vertebrate immune responses. Typically, pIgs contain between two and six Ig monomers, each with two antigen binding fragments and one fragment crystallization (Fc). In addition, many pIgs assemble with a joining-chain (JC); however, the number of monomers and potential to include JC vary with species and heavy chain class. Here, we report the cryo-electron microscopy structure of IgM from a teleost (t) species, which does not encode JC. The structure reveals four tIgM Fcs linked through eight C-terminal tailpieces (Tps), which adopt a single ß-sandwich-like domain (Tp assembly) located between two Fcs. Specifically, two of eight heavy chains fold uniquely, resulting in a structure distinct from mammalian IgM, which typically contains five IgM monomers, one JC and a centrally-located Tp assembly. Together with mutational analysis, structural data indicate that pIgs have evolved a range of assembly mechanisms and structures, each likely to support unique antibody effector functions.

A novel protein encoded by circVPS13D attenuates antiviral innate immunity by targeting MAVS in teleost fish.

IMPORTANCE: The expression of circVPS13D was upregulated with SCRV invasion, which proved that circVPS13D was involved in the regulation of the antiviral immune response. Our study revealed that the existence of circVPS13D promoted the replication of SCRV. Functionally, circVPS13D negatively regulates the antiviral responses of fish. Mechanistically, we confirmed that circVPS13D inhibited RLRs antiviral signaling pathway via the encoded protein VPS13D-170aa by targeting MAVS. Our study provided novel insights into the roles of protein-coding circRNAs and supported VPS13D-170aa as a negative regulator in the antiviral immune responses of teleost fish.

circCBL and its host gene CBL collaboratively enhance the antiviral immunity and antibacterial immunity by targeting MITA in fish.

IMPORTANCE: Increasing evidence indicates that circular RNAs exert crucial functions in regulating gene expression in mammals. However, the function of circRNAs in lower vertebrates still needs further exploration. Our research results demonstrated that circRNA, namely circCBL, is involved in modulating antiviral and antibacterial immune responses in lower vertebrates. In addition, our study also found that circCBL can serve as a competing endogenous RNA to facilitate MITA expression, thereby modulating MITA-mediated innate immunity. Further research has proved that the host gene CBL also promotes the expression of MITA, enhancing antiviral and antibacterial immune responses. Our study not only elucidated the underlying biological mechanism of the circRNA-miRNA-mRNA axis in the innate immune response of lower vertebrates but also unveiled the synergistic antibacterial and antiviral mechanisms between circRNA and its host gene in lower vertebrates.

Cytokine Receptor-Like Factor 3 Negatively Regulates Antiviral Immunity by Promoting the Degradation of TBK1 in Teleost Fish.

The cytokine receptor-like factor 3 (Crlf3) belongs to the orphan class I cytokine receptors and is identified as a neuroprotective erythropoietin receptor. In previous studies of Crlf3, few focused on its role in innate immunity. Therefore, this study explored the regulatory role of Crlf3 in innate immunity. TANK-binding kinase 1 (TBK1) is a vital adaptor protein for the activation of the RLRs-MVAS-IRF3 antiviral signaling axis; thus, its expression and activity must be tightly regulated to maintain immune homeostasis and avoid undesirable effects. Here, we report that Crlf3 is a negative regulator of type I interferon production. The expression of Crlf3 is induced by poly(I·C) or Siniperca chuatsi rhabdovirus (SCRV) treatment. Silencing of Crlf3 enhanced poly(I·C)- and SCRV-induced type I interferon production, whereas overexpression of Crlf3 suppressed type I interferon production. Mechanistically, Crlf3 interacted with TBK1 via its N domain and then inhibited type I interferon production by promoting TBK1 proteasomal degradation through K48-linked polyubiquitination. Our study shows that Crlf3 is a key factor for viral escape from innate antiviral immunity in fish and provides a new perspective on mammalian resistance to viral invasion. IMPORTANCE The expression of Crlf3 was upregulated with SCRV invasion, which proved that Crlf3 was involved in the regulation of the antiviral immune response. In this study, we found that the existence of Crlf3 promoted the replication of SCRV. Therefore, it is reasonable to believe that SCRV evades innate immune attack with the assistance of Crlf3. In addition, we report that Crlf3 negatively regulates interferon (IFN) induction by promoting the degradation of TBK1 in fish. We showed that Crlf3 is evenly distributed in the cytoplasm and interacts with TBK1. Further studies showed that Crlf3 specifically mediates K48-linked ubiquitination of TBK1 and promotes TBK1 degradation, resulting in a marked inhibition of retinoic acid-inducible gene I (RIG-I) downstream signaling.

Molecular Identification of Nocardia seriolae and Comparative Analysis of Spleen Transcriptomes of Hybrid Snakehead (Channa maculata Female × Channa argus Male) With Nocardiosis Disease.

Hybrid snakehead (Channa maculata female × Channa argus male) is a new freshwater aquaculture fish species in southern China. During intensive aquaculture, hybrid snakeheads are often infected by Nocardia seriolae. In this study, hybrid snakehead infected suspiciously by N. seriolae in an artificial breeding pond were examined. Diseased hybrid snakeheads swam slowly without food intake, and the clinical symptoms included skin wound, anal swelling and ascites, and white granulomatous in liver, spleen, and kidney of fish. Through bacterial isolation, 16S rDNA sequencing, fluorescence in situ hybridization (FISH) and artificial infection experiment, the pathogen was identified as N. seriolae. Furthermore, the spleen samples from diseased and healthy male hybrid snakeheads in the same pond were used for RNA-Seq analysis. A total of 3,512 unique transcripts (unigenes) were identified as differentially expressed genes (DEGs), and 1,886 of them were up-regulated in diseased fish. The expression patterns of 20 DEGs were verified by quantitative polymerase chain reaction (qPCR). Several immune-related pathways and many immune-related genes were identified. qPCR results showed that the expression patterns of immune-related genes in the liver and kidney of diseased fish were comparable to that in the spleen. This study provides deep-sequencing data of hybrid snakehead spleen and will help understand the immune response of hybrid snakehead to N. seriolae. It is also helpful for the biomarker screening of fish-borne Nocardia spp. and the breeding of nocardiosis-resistant fish species.

RNA-Seq of Single Fish Cells - Seeking Out the Leukocytes Mediating Immunity in Teleost Fishes.

The immune system is a complex and sophisticated biological system, spanning multiple levels of complexity, from the molecular level to that of tissue. Our current understanding of its function and complexity, of the heterogeneity of leukocytes, is a result of decades of concentrated efforts to delineate cellular markers using conventional methods of antibody screening and antigen identification. In mammalian models, this led to in-depth understanding of individual leukocyte subsets, their phenotypes, and their roles in health and disease. The field was further propelled forward by the development of single-cell (sc) RNA-seq technologies, offering an even broader and more integrated view of how cells work together to generate a particular response. Consequently, the adoption of scRNA-seq revealed the unexpected plasticity and heterogeneity of leukocyte populations and shifted several long-standing paradigms of immunology. This review article highlights the unprecedented opportunities offered by scRNA-seq technology to unveil the individual contributions of leukocyte subsets and their crosstalk in generating the overall immune responses in bony fishes. Single-cell transcriptomics allow identifying unseen relationships, and formulating novel hypotheses tailored for teleost species, without the need to rely on the limited number of fish-specific antibodies and pre-selected markers. Several recent studies on single-cell transcriptomes of fish have already identified previously unnoticed expression signatures and provided astonishing insights into the diversity of teleost leukocytes and the evolution of vertebrate immunity. Without a doubt, scRNA-seq in tandem with bioinformatics tools and state-of-the-art methods, will facilitate studying the teleost immune system by not only defining key markers, but also teaching us about lymphoid tissue organization, development/differentiation, cell-cell interactions, antigen receptor repertoires, states of health and disease, all across time and space in fishes. These advances will invite more researchers to develop the tools necessary to explore the immunology of fishes, which remain non-conventional animal models from which we have much to learn.

Rodlet cells, fish immune cells and a sentinel of parasitic harm in teleost organs.

Rodlet cells (RCs) are the enigmatic and distinctive pear-shaped cells had found in many tissues of marine and freshwater teleosts. They have a distinctive fibrous capsule or the cell cortex that envelopes conspicuous inclusions called rodlets, basally situated nucleus, and poorly developed mitochondria. The contraction of the cell cortex results in the expulsion of the cell contents through an apical opening. One hundred and thirty years since rodlet cells were first reported, many questions remain about their origin and a function. This review will present new evidence regarding the relationship between RCs and metazoan parasites, and a protozoan infecting organs of different fish species, and update the state of knowledge about the origin, structure and the function of these intriguing fish cells.