Helminthic host defense peptides (HDP) are pleiotropic, multifunctional effector molecules of helminth immunity, efficient against Gram-negative and Gram-positive bacteria. Among them, anisaxin-2S (A-2S), membranolytic cecropin-like HDPs produced by the zoonotic nematodes of the genus Anisakis, shows remarkable efficacy even against multidrug-resistant Gram-negative bacteria, yet its immunomodulatory, antiproliferative and antiviral properties have not been elucidated. Therefore, we tested A-2S immunomodulation in the common carp (Cyprinus carpio) blood cells exposed to two pathogens, the zoonotic bacterium Aeromonas hydrophila and the fish parasite Sphaerospora molnari, and in carp in vivo challenged with the parasite. Furthermore, the A-2S antiproliferative activity was tested in vitro in human bladder and lung cancer cell line, while the antiviral protection was tested in common carp brain cell culture exposed to carp rhabdovirus, alloherpesvirus and paramyxovirus, and in a human immortalized myelogenous leukemia cell line infected with tick-borne encephalitis virus. A-2S exerts an immunostimulatory effect on fish blood cells through upregulation of cytokine expression, with the proinflammatory or anti-inflammatory repertoire conditioned by the presence or absence of co-stimulatory antigen. Surprisingly, in the majority of assays conducted, red blood cells demonstrate equal or even stronger regulation of innate immunity genes compared to white blood cells, along with a more extensive repertoire of differentially expressed markers. In contrast, A-2S has only a limited anticancer activity in human bladder cancer and lung adenocarcinoma cells and limited antiviral activity against the three fish viruses and a human tick-borne encephalitis virus. This study provides the first evidence of red blood cell and platelet immunomodulation by an antimicrobial peptide and highlights the induction of a cytokine repertoire. However, future research should address the study's limitations, including the need for longer in vitro assays (e.g., 3-4 days), testing different white blood cell lineages, to better understand antigen-processing interactions, and evaluating the anticipated adaptive immune response. Powerful antimicrobial activity of A-2S, coupled with immunostimulatory properties, warrant further pursuing of preclinical trials with this anisaxin.

• Keywords
• anisaxin, antimicrobial peptide, immunomodulation, red blood cells, white blood cells,
• MeSH
• Antiviral Agents * pharmacology   MeSH
• Cecropins * pharmacology   MeSH
• Immunologic Factors * pharmacology   MeSH
• Immunomodulation MeSH
• Carps * immunology   parasitology   MeSH
• Humans MeSH
• Cell Line, Tumor MeSH
• Fish Diseases * immunology   MeSH
• Cell Proliferation drug effects   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Antiviral Agents * MeSH
• Cecropins * MeSH
• Immunologic Factors * MeSH

BACKGROUND: Parasitism as a life strategy has independently evolved multiple times within the eukaryotic tree of life. Each lineage has developed mechanisms to invade hosts, exploit resources, and ensure replication, but our knowledge of survival mechanisms in many parasitic taxa remain extremely limited. One such group is the Myxozoa, which are obligate, dixenous cnidarians. Evidence suggests that myxozoans evolved from free-living ancestors to endoparasites around 600 million years ago and are likely one of the first metazoan parasites on Earth. Some myxozoans pose significant threats to farmed and wild fish populations, negatively impacting aquaculture and fish stocks; one such example is Sphaerospora molnari, which forms spores in the gills of common carp (Cyprinus carpio), disrupting gill epithelia and causing somatic and respiratory failure. Sphaerospora molnari undergoes sequential development in different organs of its host, with large numbers of morphologically distinct stages occurring in the blood, liver, and gills of carp. We hypothesize that these parasite life-stages differ in regards to their host exploitation, pathogenicity, and host immune evasion strategies and mechanisms. We performed stage-specific transcriptomic profiling to identify differentially expressed key functional gene groups that relate to these functions and provide a fundamental understanding of the mechanisms S. molnari uses to optimize its parasitic lifestyle. We aimed to identify genes that are likely related to parasite pathogenicity and host cell exploitation mechanisms, and we hypothesize that genes unique to S. molnari might be indicative of evolutionary innovations and specific adaptations to host environments. RESULTS: We used parasite isolation protocols and comparative transcriptomics to study early proliferative and spore-forming stages of S. molnari, unveiling variation in gene expression between each stage. We discovered several apparent innovations in the S. molnari transcriptome, including proteins that are likely to function in the uptake of previously unknown key nutrients, immune evasion factors that may contribute to long-term survival in hosts, and proteins that likely improve adhesion to host cells that may have arisen from horizontal gene transfer. Notably, we identified genes that are similar to known virulence factors in other parasitic organisms, particularly blood and intestinal parasites like Plasmodium, Trypanosoma, and Giardia. Many of these genes are absent in published cnidarian and myxozoan datasets and appear to be specific to S. molnari; they may therefore represent potential innovations enabling Sphaerospora to exploit the host's blood system. CONCLUSIONS: In order to address the threat posed by myxozoans to both cultured fish species and wild stocks, it is imperative to deepen our understanding of their genetics. Sphaerospora molnari offers an appealing model for stage-specific transcriptomic profiling and for identifying differentially expressed key functional gene groups related to parasite development. We identified genes that are thus far unique to S. molnari, which reveal their evolutionary novelty and likely role as adaptations to specific host niches. In addition, we describe the pathogenicity-associated genetic toolbox of S. molnari and discuss the implications of our discoveries for disease control by shedding light on specific targets for potential intervention strategies.

• Keywords
• Sphaerospora molnari, Differential expression, Myxozoans, Pathogenicity related, Species specific genes,
• MeSH
• Adaptation, Physiological * genetics   MeSH
• Host-Parasite Interactions genetics   MeSH
• Carps parasitology   MeSH
• Myxozoa * genetics   physiology   growth & development   MeSH
• Gene Expression Profiling * MeSH
• Transcriptome * MeSH
• Gills parasitology   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Comparative Study MeSH

From ancient cold-blooded fishes to mammals, all vertebrates are protected by adaptive immunity, and retain immunological memory. Although immunologists can demonstrate these phenomena in all fish, the responding cells remain elusive, without the tools to study them nor markers to define them. Fundamentally, we posited that it is longevity that defines a memory cell, like how it is antibody production that defines a plasma cell. We infected the common carp with Sphaerospora molnari, a cnidarian parasite which causes seasonal outbreaks to which no vaccine is available. B cells proliferated and expressed gene signatures of differentiation. Despite a half-year gap between EdU labeling and sampling, IgM+ B cells retained the thymidine analogue, suggesting that these are at least six-month-old resting memory cells stemming from proliferating precursors. Additionally, we identified a lymphoid organ-resident population of plasma cells by the exceptional levels of IgM they express. Thus, we demonstrate that a teleost fish produces the lymphocytes key to vaccination success and long-term disease protection, supporting the idea that immunological memory is observable and universal across vertebrates.

• Keywords
• antibody, antibody-secreting cell (ASC), humoral memory, immunoglobulin, myxozoa,
• MeSH
• B-Lymphocytes immunology   MeSH
• Cell Differentiation * immunology   MeSH
• Immunoglobulin M * immunology   MeSH
• Immunologic Memory * MeSH
• Carps * immunology   parasitology   MeSH
• Fish Diseases immunology   parasitology   MeSH
• Memory B Cells immunology   MeSH
• Plasma Cells * immunology   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Immunoglobulin M * MeSH

Myxozoans are microscopical parasites widely distributed in fish, with over 2,600 described species, but their actual diversity is still underestimated. Among salmonids, more than 70 myxozoan species have been identified. This study focuses on species of Chloromyxum Mingazzini, 1890 that infect salmonid kidneys, particularly C. majori Yasutake et Wood, 1957 and C. schurovi Shulman et Ieshko, 2003. Despite their similar spore morphology, they exhibit distinct host preferences, tissue affinities and geographical distributions. Chloromyxum schurovi predominantly infects the renal tubules of Salmo salar Linnaues and S. trutta Linnaeus in Europe, while C. majori targets the glomeruli of Oncorhynchus mykiss (Walbaum) and O. tshawytscha (Walbaum) in North America. The sequence data for C. majori and C. schurovi have been either missing or questionable. In our study, we examined the kidneys of two salmonid species for chloromyxid infections, using both morphological and molecular data to characterise Chloromyxum species in salmonids. The sequence of C. schurovi obtained in our study did not match the previously published parasite data. Instead, it clustered as an independent lineage sister to the Paramyxidium Freeman et Kristmundsson, 2018 clade gathering the species from various fish organs, including the urinary tract. Our findings clarified the taxonomic origin of the previous C. schurovi sequence as Myxidium giardi Cépède, 1906, highlighting the risks associated with the presence of myxozoan blood stages in the bloodstream of their fish host and the challenges of non-specific PCR amplification. We redescribe C. schurovi, thus contributing to a better understanding of the diversity and phylogeny of kidney-infecting species of Chloromyxum.

• Keywords
• Myxozoan diversity, PCR screening, Salmo trutta, phylogeny,
• MeSH
• Phylogeny * MeSH
• Kidney parasitology   MeSH
• Myxozoa * classification   genetics   anatomy & histology   isolation & purification   MeSH
• Fish Diseases * parasitology   MeSH
• Parasitic Diseases, Animal * parasitology   epidemiology   MeSH
• Trout * parasitology   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH

The myxozoan parasite Tetracapsuloides bryosalmonae is the causative agent of proliferative kidney disease (PKD)-a disease of salmonid fishes, notably of the commercially farmed rainbow trout Oncorhynchus mykiss. Both wild and farmed salmonids are threatened by this virulent/deadly disease, a chronic immunopathology characterized by massive lymphocyte proliferation and hyperplasia, which manifests as swollen kidneys in susceptible hosts. Studying the immune response towards the parasite helps us understand the causes and consequences of PKD. While examining the B cell population during a seasonal outbreak of PKD, we unexpectedly detected the B cell marker immunoglobulin M (IgM) on red blood cells (RBCs) of infected farmed rainbow trout. Here, we studied the nature of this IgM and this IgM+ cell population. We verified the presence of surface IgM via parallel approaches: flow cytometry, microscopy, and mass spectrometry. The levels of surface IgM (allowing complete resolution of IgM- RBCs from IgM+ RBCs) and frequency of IgM+ RBCs (with up to 99% of RBCs being positive) have not been described before in healthy fishes nor those suffering from disease. To assess the influence of the disease on these cells, we profiled the transcriptomes of teleost RBCs in health and disease. Compared to RBCs originating from healthy fish, PKD fundamentally altered RBCs in their metabolism, adhesion, and innate immune response to inflammation. In summary, RBCs play a larger role in host immunity than previously appreciated. Specifically, our findings indicate that the nucleated RBCs of rainbow trout interact with host IgM and contribute to the immune response in PKD.

• Keywords
• anemia, antibody, bony fish, erythrocytes, innate immunity, proliferative kidney disease (PKD), renal disease,
• MeSH
• B-Lymphocytes MeSH
• Erythrocytes MeSH
• Immunoglobulin M MeSH
• Kidney Diseases * MeSH
• Oncorhynchus mykiss * MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Immunoglobulin M MeSH

Poly lactic-co-glycolic acid (PLGA) particles safely and effectively deliver pharmaceutical ingredients, with many applications approved for clinical use in humans. In fishes, PLGA particles are being considered as carriers of therapeutic drugs and vaccine antigens. However, existing studies focus mainly on vaccine antigens, the endpoint immune responses to these (e.g., improved antibody titres), without deeper understanding of whether fishes react to the carrier. To test whether or not PLGA are recognized by or interact at all with the immune system of a teleost fish, we prepared, characterized and injected PLGA microparticles intraperitoneally into common carp. The influx, phenotype of inflammatory leukocytes, and their capacity to produce reactive oxygen species and phagocytose PLGA microparticles were tested by flow cytometry, qPCR, and microscopy. PLGA microparticles were indeed recognized. However, they induced only transient recruitment of inflammatory leukocytes that was resolved 4 days later whereas only the smallest µm-sized particles were phagocytosed. The overall response resembled that described in mammals against foreign materials. Given the similarities between our findings and those described in mammals, PLGA particles can be adapted to play a dual role as both antigen and drug carriers in fishes, depending on the administered dose and their design.

• Keywords
• PLGA, antigen, aquaculture, carrier, common carp, foreign body, inflammation, microparticle, teleost fish, vaccine,
• MeSH
• Antigens MeSH
• Glycols MeSH
• Immunity MeSH
• Carps * MeSH
• Polylactic Acid-Polyglycolic Acid Copolymer MeSH
• Lactic Acid MeSH
• Polyglycolic Acid MeSH
• Mammals MeSH
• Vaccines * pharmacology   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Antigens MeSH
• Glycols MeSH
• Polylactic Acid-Polyglycolic Acid Copolymer MeSH
• Lactic Acid MeSH
• Polyglycolic Acid MeSH
• Vaccines * MeSH

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.

• Keywords
• RNA-seq, antibody, flow cytometry, leukocytes, lymphocytes, phenotype, single-cell transcriptome, teleost bony fish,
• MeSH
• Single-Cell Analysis * methods   MeSH
• Immunity MeSH
• Leukocytes immunology   metabolism   MeSH
• Fishes genetics   immunology   MeSH
• RNA-Seq * MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH

Myxozoans are a diverse group of microscopic cnidarian parasites and some representatives are associated with important diseases in fish, in both marine and freshwater aquaculture systems. Research on myxozoans has been largely hampered by the inability to isolate myxozoan parasites from their host tissues. In this study, we developed and optimized a method to isolate the myxozoan proliferative stages of different size and cellularity from fish blood, using DEAE-cellulose ion exchange chromatography. We optimized several parameters and obtained 99-100% parasite purity, as well as high survival and infectivity. Using polyclonal pan-carp blood cell-specific antibodies, we further developed a rapid cytometric assay for quantification of the proliferative stages, not only in highly concentrated DEAE-C isolates but also in dilute conditions in full blood. Early developmental stages of myxozoans are key to parasite proliferation, establishment, and pathology in their hosts. The isolation of these stages not only opens new possibilities for in vivo and in vitro studies, but also for obtaining purified DNA and protein extracts for downstream analyses. Hence, we provide a long-desired tool that will advance the functional research into the mechanisms of host exploitation and immune stimulation/evasion in this group, which could contribute greatly to the development of therapeutic strategies against myxozoans.

• Keywords
• Sphaerospora, anti-carp antibody, blood stages, cell separation, common carp, cytometry, diethylaminoethyl (DEAE) cellulose, parasite, teleost,
• MeSH
• Genomics MeSH
• Carps * MeSH
• Myxozoa * genetics   MeSH
• Fish Diseases * MeSH
• Antibodies MeSH
• Aquaculture MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Antibodies MeSH

Myxozoans are microscopic, metazoan, obligate parasites, belonging to the phylum Cnidaria. In contrast to the free-living lifestyle of most members of this taxon, myxozoans have complex life cycles alternating between vertebrate and invertebrate hosts. Vertebrate hosts are primarily fish, although they are also reported from amphibians, reptiles, trematodes, mollusks, birds and mammals. Invertebrate hosts include annelids and bryozoans. Most myxozoans are not overtly pathogenic to fish hosts, but some are responsible for severe economic losses in fisheries and aquaculture. In both scenarios, the interaction between the parasite and the host immune system is key to explain such different outcomes of this relationship. Innate immune responses contribute to the resistance of certain fish strains and species, and the absence or low levels of some innate and regulatory factors explain the high pathogenicity of some infections. In many cases, immune evasion explains the absence of a host response and allows the parasite to proliferate covertly during the first stages of the infection. In some infections, the lack of an appropriate regulatory response results in an excessive inflammatory response, causing immunopathological consequences that are worse than inflicted by the parasite itself. This review will update the available information about the immune responses against Myxozoa, with special focus on T and B lymphocyte and immunoglobulin responses, how these immune effectors are modulated by different biotic and abiotic factors, and on the mechanisms of immune evasion targeting specific immune effectors. The current and future design of control strategies for myxozoan diseases is based on understanding this myxozoan-fish interaction, and immune-based strategies such as improvement of innate and specific factors through diets and additives, host genetic selection, passive immunization and vaccination, are starting to be considered.

• Keywords
• B lymphocytes, RNAseq, T lymphocytes, adaptive immunity, immune evasion, immunoglobulin, parasite, teleost,
• MeSH
• Adaptive Immunity * MeSH
• Antiparasitic Agents pharmacology   MeSH
• B-Lymphocytes immunology   metabolism   parasitology   MeSH
• Immune Evasion MeSH
• Immunoglobulins immunology   metabolism   MeSH
• Host-Parasite Interactions MeSH
• Myxozoa drug effects   immunology   pathogenicity   MeSH
• Fish Diseases immunology   metabolism   parasitology   prevention & control   MeSH
• Parasitic Diseases, Animal immunology   metabolism   parasitology   prevention & control   MeSH
• Immunity, Innate * MeSH
• Fishes immunology   metabolism   parasitology   MeSH
• T-Lymphocytes immunology   metabolism   parasitology   MeSH
• Vaccines pharmacology   MeSH
• Aquaculture MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Names of Substances
• Antiparasitic Agents MeSH
• Immunoglobulins MeSH
• Vaccines MeSH

The evolutionary aspects of cystatins are greatly underexplored in early-emerging metazoans. Thus, we surveyed the gene organization, protein architecture, and phylogeny of cystatin homologues mined from 110 genomes and the transcriptomes of 58 basal metazoan species, encompassing free-living and parasite taxa of Porifera, Placozoa, Cnidaria (including Myxozoa), and Ctenophora. We found that the cystatin gene repertoire significantly differs among phyla, with stefins present in most of the investigated lineages but with type 2 cystatins missing in several basal metazoan groups. Similar to liver and intestinal flukes, myxozoan parasites possess atypical stefins with chimeric structure that combine motifs of classical stefins and type 2 cystatins. Other early metazoan taxa regardless of lifestyle have only the classical representation of cystatins and lack multi-domain ones. Our comprehensive phylogenetic analyses revealed that stefins and type 2 cystatins clustered into taxonomically defined clades with multiple independent paralogous groups, which probably arose due to gene duplications. The stefin clade split between the subclades of classical stefins and the atypical stefins of myxozoans and flukes. Atypical stefins represent key evolutionary innovations of the two parasite groups for which their origin might have been linked with ancestral gene chimerization, obligate parasitism, life cycle complexity, genome reduction, and host immunity.

• Keywords
• cysteine protease inhibitor, diversification, parasite, phylogenetic analysis, protein structure, signal peptide, stefin,
• Publication type
• Journal Article MeSH