BACKGROUND: Helminth extracellular vesicles (EVs) are known to have a three-way communication function among parasitic helminths, their host and the host-associated microbiota. They are considered biological containers that may carry virulence factors, being therefore appealing as therapeutic and prophylactic target candidates. This study aims to describe and characterise EVs secreted by Sparicotyle chrysophrii (Polyopisthocotyla: Microcotylidae), a blood-feeding gill parasite of gilthead seabream (Sparus aurata), causing significant economic losses in Mediterranean aquaculture. METHODS: To identify proteins involved in extracellular vesicle biogenesis, genomic datasets from S. chrysophrii were mined in silico using known protein sequences from Clonorchis spp., Echinococcus spp., Fasciola spp., Fasciolopsis spp., Opisthorchis spp., Paragonimus spp. and Schistosoma spp. The location and ultrastructure of EVs were visualised by transmission electron microscopy after fixing adult S. chrysophrii specimens by high-pressure freezing and freeze substitution. EVs were isolated and purified from adult S. chrysophrii (n = 200) using a newly developed ultracentrifugation-size-exclusion chromatography protocol for Polyopisthocotyla, and EVs were characterised via nanoparticle tracking analysis and tandem mass spectrometry. RESULTS: Fifty-nine proteins involved in EV biogenesis were identified in S. chrysophrii, and EVs compatible with ectosomes were observed in the syncytial layer of the haptoral region lining the clamps. The isolated and purified nanoparticles had a mean size of 251.8 nm and yielded 1.71 × 108 particles · mL-1. The protein composition analysis identified proteins related to peptide hydrolases, GTPases, EF-hand domain proteins, aerobic energy metabolism, anticoagulant/lipid-binding, haem detoxification, iron transport, EV biogenesis-related, vesicle-trafficking and other cytoskeletal-related proteins. Several identified proteins, such as leucyl and alanyl aminopeptidases, calpain, ferritin, dynein light chain, 14-3-3, heat shock protein 70, annexin, tubulin, glutathione S-transferase, superoxide dismutase, enolase and fructose-bisphosphate aldolase, have already been proposed as target candidates for therapeutic or prophylactic purposes. CONCLUSIONS: We have unambiguously demonstrated for the first time to our knowledge the secretion of EVs by an ectoparasitic flatworm, inferring their biogenesis machinery at a genomic and transcriptomic level, and by identifying their location and protein composition. The identification of multiple therapeutic targets among EVs' protein repertoire provides opportunities for target-based drug discovery and vaccine development for the first time in Polyopisthocotyla (sensu Monogenea), and in a fish-ectoparasite model.

• Klíčová slova
• Drug target candidates, Ectosomes, Electron microscopy, Exosomes, Monogenea, Peptidases, Polyopisthocotyla, Prophylactic target candidates,
• MeSH
• extracelulární vezikuly * MeSH
• mořan zlatý * parazitologie   MeSH
• ploštěnci * MeSH
• proteomika MeSH
• Trematoda * MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

BACKGROUND: Monogenea (Platyhelminthes, Neodermata) are the most species-rich class within the Neodermata superclass of primarily fish parasites. Despite their economic and ecological importance, monogenean research tends to focus on their morphological, phylogenetic, and population characteristics, while comprehensive omics analyses aimed at describing functionally important molecules are few and far between. We present a molecular characterisation of monogenean representative Eudiplozoon nipponicum, an obligate haematophagous parasite infecting the gills of the common carp. We report its nuclear and mitochondrial genomes, present a functional annotation of protein molecules relevant to the molecular and biochemical aspect of physiological processes involved in interactions with the fish hosts, and re-examinate the taxonomic position of Eudiplozoon species within the Diplozoidae family. RESULTS: We have generated 50.81 Gbp of raw sequencing data (Illumina and Oxford Nanopore reads), bioinformatically processed, and de novo assembled them into a genome draft 0.94 Gbp long, consisting of 21,044 contigs (N50 = 87 kbp). The final assembly represents 57% of the estimated total genome size (~ 1.64 Gbp), whereby repetitive and low-complexity regions account for ~ 64% of the assembled length. In total, 36,626 predicted genes encode 33,031 proteins and homology-based annotation of protein-coding genes (PCGs) and proteins characterises 14,785 (44.76%) molecules. We have detected significant representation of functional proteins and known molecular functions. The numbers of peptidases and inhibitors (579 proteins), characterised GO terms (16,016 unique assigned GO terms), and identified KEGG Orthology (4,315 proteins) acting in 378 KEGG pathways demonstrate the variety of mechanisms by which the parasite interacts with hosts on a macromolecular level (immunomodulation, feeding, and development). Comparison between the newly assembled E. nipponicum mitochondrial genome (length of 17,038 bp) and other diplozoid monogeneans confirms the existence of two distinct Eudiplozoon species infecting different fish hosts: Cyprinus carpio and Carassius spp. CONCLUSIONS: Although the amount of sequencing data and characterised molecules of monogenean parasites has recently increased, a better insight into their molecular biology is needed. The E. nipponicum nuclear genome presented here, currently the largest described genome of any monogenean parasite, represents a milestone in the study of monogeneans and their molecules but further omics research is needed to understand these parasites' biological nature.

• Klíčová slova
• Annotation, Assembly, Genome, Helminths, Host–parasite interaction, Illumina, Mitochondrial genome, Monogenea, Nanopore, Sequencing,
• MeSH
• fylogeneze MeSH
• genomika MeSH
• kapři * genetika   MeSH
• paraziti * MeSH
• Trematoda * MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

BACKGROUND: Ectoparasites from the family Diplozoidae (Platyhelminthes, Monogenea) belong to obligate haematophagous helminths of cyprinid fish. Current knowledge of these worms is for the most part limited to their morphological, phylogenetic, and population features. Information concerning the biochemical and molecular nature of physiological processes involved in host-parasite interaction, such as evasion of the immune system and its regulation, digestion of macromolecules, suppression of blood coagulation and inflammation, and effect on host tissue and physiology, is lacking. In this study, we report for the first time a comprehensive transcriptomic/secretome description of expressed genes and proteins secreted by the adult stage of Eudiplozoon nipponicum (Goto, 1891) Khotenovsky, 1985, an obligate sanguivorous monogenean which parasitises the gills of the common carp (Cyprinus carpio). RESULTS: RNA-seq raw reads (324,941 Roche 454 and 149,697,864 Illumina) were generated, de novo assembled, and filtered into 37,062 protein-coding transcripts. For 19,644 (53.0%) of them, we determined their sequential homologues. In silico functional analysis of E. nipponicum RNA-seq data revealed numerous transcripts, pathways, and GO terms responsible for immunomodulation (inhibitors of proteolytic enzymes, CD59-like proteins, fatty acid binding proteins), feeding (proteolytic enzymes cathepsins B, D, L1, and L3), and development (fructose 1,6-bisphosphatase, ferritin, and annexin). LC-MS/MS spectrometry analysis identified 721 proteins secreted by E. nipponicum with predominantly immunomodulatory and anti-inflammatory functions (peptidyl-prolyl cis-trans isomerase, homolog to SmKK7, tetraspanin) and ability to digest host macromolecules (cathepsins B, D, L1). CONCLUSIONS: In this study, we integrated two high-throughput sequencing techniques, mass spectrometry analysis, and comprehensive bioinformatics approach in order to arrive at the first comprehensive description of monogenean transcriptome and secretome. Exploration of E. nipponicum transcriptome-related nucleotide sequences and translated and secreted proteins offer a better understanding of molecular biology and biochemistry of these, often neglected, organisms. It enabled us to report the essential physiological pathways and protein molecules involved in their interactions with the fish hosts.

• Klíčová slova
• Annotation, Assembly, Eudiplozoon nipponicum, Mass spectrometry, Monogenea, NGS, Secretome, Transcriptome,
• MeSH
• anotace sekvence MeSH
• chromatografie kapalinová MeSH
• fylogeneze MeSH
• kapři * genetika   MeSH
• stanovení celkové genové exprese MeSH
• tandemová hmotnostní spektrometrie MeSH
• transkriptom MeSH
• Trematoda * genetika   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články 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.

• Klíčová slova
• cysteine protease inhibitor, diversification, parasite, phylogenetic analysis, protein structure, signal peptide, stefin,
• Publikační typ
• časopisecké články MeSH

Eudiplozoon nipponicum (Goto, 1891) is a hematophagous monogenean ectoparasite which inhabits the gills of the common carp (Cyprinus carpio). Heavy infestation can lead to anemia and in conjunction with secondary bacterial infections cause poor health and eventual death of the host. This study is based on an innovative approach to protein localization which has never been used in parasitology before. Using laser capture microdissection, we dissected particular areas of the parasite body without contaminating the samples by surrounding tissue and in combination with analysis by mass spectrometry obtained tissue-specific proteomes of tegument, intestine, and parenchyma of our model organism, E. nipponicum. We successfully verified the presence of certain functional proteins (e.g. cathepsin L) in tissues where their presence was expected (intestine) and confirmed that there were no traces of these proteins in other tissues (tegument and parenchyma). Additionally, we identified a total of 2,059 proteins, including 72 peptidases and 33 peptidase inhibitors. As expected, the greatest variety was found in the intestine and the lowest variety in the parenchyma. Our results are significant on two levels. Firstly, we demonstrated that one can localize all proteins in one analysis and without using laboratory animals (antibodies for immunolocalization of single proteins). Secondly, this study offers the first complex proteomic data on not only the E. nipponicum but within the whole class of Monogenea, which was from this point of view until recently neglected.

• MeSH
• kapři parazitologie   MeSH
• kathepsiny analýza   metabolismus   MeSH
• laserová záchytná mikrodisekce MeSH
• parenchymatická tkáň metabolismus   MeSH
• ploštěnci metabolismus   MeSH
• proteasy analýza   metabolismus   MeSH
• proteom analýza   MeSH
• proteomika metody   MeSH
• střevní sliznice metabolismus   MeSH
• tandemová hmotnostní spektrometrie MeSH
• vysokoúčinná kapalinová chromatografie MeSH
• žábry parazitologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• kathepsiny MeSH
• proteasy MeSH
• proteom MeSH

BACKGROUND: Serpins are a superfamily of serine peptidase inhibitors that participate in the regulation of many physiological and cell peptidase-mediated processes in all organisms (e.g. in blood clotting, complement activation, fibrinolysis, inflammation, and programmed cell death). It was postulated that in the blood-feeding members of the monogenean family Diplozoidae, serpins could play an important role in the prevention of thrombus formation, activation of complement, inflammation in the host, and/or in the endogenous regulation of protein degradation. RESULTS: In silico analysis showed that the DNA and primary protein structures of serpin from Eudiplozoon nipponicum (EnSerp1) are similar to other members of the serpin superfamily. The inhibitory potential of EnSerp1 on four physiologically-relevant serine peptidases (trypsin, factor Xa, kallikrein, and plasmin) was demonstrated and its presence in the worm's excretory-secretory products (ESPs) was confirmed. CONCLUSION: EnSerp1 influences the activity of peptidases that play a role in blood coagulation, fibrinolysis, and complement activation. This inhibitory potential, together with the serpin's presence in ESPs, suggests that it is likely involved in host-parasite interactions and could be one of the molecules involved in the control of feeding and prevention of inflammatory responses.

Contexte : Les serpines sont une super-famille d’inhibiteurs de sérine peptidases qui participent, dans tous les organismes, à la régulation de nombreux processus physiologiques et à médiation par les peptidases cellulaires (par exemple la coagulation sanguine, l’activation du complément, la fibrinolyse, l’inflammation et la mort cellulaire programmée). Il a été postulé que chez les Monogènes de la famille Diplozoidae, qui sont hématophages, les serpines pourraient jouer un rôle important dans la prévention de la formation de thrombus, l’activation du complément, l’inflammation chez l’hôte et/ou la régulation endogène de la dégradation des protéines. Résultats : Une analyse in silico a montré que l’ADN et les structures primaires protéiques de la serpine d’Eudiplozoon nipponicum (EnSerp1) sont similaires aux autres membres de la superfamille des serpines. Le potentiel inhibiteur d’EnSerp1 sur quatre sérine peptidases physiologiquement pertinentes (la trypsine, le facteur Xa, la kallikréine et la plasmine) a été démontré et sa présence dans les produits excréteurs de sécrétion du ver (ESP) a été confirmée. Conclusion : EnSerp1 influence l’activité des peptidases qui jouent un rôle dans la coagulation sanguine, la fibrinolyse et l’activation du complément. Ce potentiel inhibiteur, ainsi que la présence de la serpine dans les ESP, suggèrent qu’elle est probablement impliquée dans les interactions hôte-parasite et pourrait être l’une des molécules impliquées dans le contrôle de l’alimentation et la prévention des réponses inflammatoires.

• MeSH
• DNA helmintů chemie   MeSH
• fylogeneze MeSH
• infekce červy třídy Trematoda parazitologie   veterinární   MeSH
• inhibitory serinových proteinas chemie   genetika   izolace a purifikace   metabolismus   MeSH
• kapři parazitologie   MeSH
• nemoci ryb parazitologie   MeSH
• počítačová simulace MeSH
• polymerázová řetězová reakce MeSH
• rekombinantní proteiny genetika   izolace a purifikace   metabolismus   MeSH
• sekvence aminokyselin MeSH
• sekvence nukleotidů MeSH
• sekvenční seřazení MeSH
• serpiny chemie   genetika   izolace a purifikace   metabolismus   MeSH
• Trematoda chemie   klasifikace   enzymologie   genetika   MeSH
• žábry parazitologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• DNA helmintů MeSH
• inhibitory serinových proteinas MeSH
• rekombinantní proteiny MeSH
• serpiny MeSH

Diplozoidae (Monogenea) are blood-feeding freshwater fish gill ectoparasites with extraordinary body architecture and a unique sexual behaviour in which two larval worms fuse and transform into one functioning individual. In this study, we describe the body organisation of Paradiplozoon homoion adult stage using a combined approach of confocal laser scanning and electron microscopy, with emphasis on the forebody and hindbody. Special attention is given to structures involved in functional adaptation to ectoparasitism, i.e. host searching, attachment and feeding/metabolism. Our observations indicate clear adaptations for blood sucking, with a well-innervated mouth opening surrounded by sensory structures, prominent muscular buccal suckers and a pharynx. The buccal cavity surface is covered with numerous tegumentary digitations that increase the area in contact with host tissue and, subsequently, with its blood. The buccal suckers and the well-innervated haptor (with sclerotised clamps controlled by noticeable musculature) cooperate in attaching to and moving over the host. Putative gland cells accumulate in the region of apical circular structures, pharynx area and in the haptor middle region. Paired club-shaped sacs lying laterally to the pharynx might serve as secretory reservoirs. Furthermore, we were able to visualise the body wall musculature, including peripheral innervation, the distribution of uniciliated sensory structures essential for reception of external environmental information, and flame cells involved in excretion. Our results confirm in detail that P. homoion displays a range of sophisticated adaptations to an ectoparasitic life style, characteristic for diplozoid monogeneans.

• MeSH
• infestace ektoparazity parazitologie   MeSH
• interakce hostitele a parazita MeSH
• konfokální mikroskopie MeSH
• mikroskopie elektronová rastrovací MeSH
• ploštěnci anatomie a histologie   patogenita   MeSH
• ryby parazitologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH