Hybrid generations usually face either a heterosis advantage or a breakdown, that can be expressed by the level of parasite infection in hybrid hosts. Hybrids are less infected by parasites than parental species (especially F1 generations) or more infected than parental species (especially post-F1 generations). We performed the experiment with blood-feeding gill parasite Paradiplozoon homoion (Monogenea) infecting leuciscid species, Abramis brama and Rutilus rutilus, their F1 generation and two backcross generations. Backcross generations tended to be more parasitized than parental lines and the F1 generation. The number of differentially expressed genes (DEGs) was lower in F1 hybrids and higher in backcross hybrids when compared to each of the parental lines. The main groups of DEGs were shared among lines; however, A. brama and R. rutilus differed in some of the top gene ontology (GO) terms. DEG analyses revealed the role of heme binding and erythrocyte differentiation after infection by blood-feeding P. homoion. Two backcross generations shared some of the top GO terms, representing mostly downregulated genes associated with P. homoion infection. KEGG analysis revealed the importance of disease-associated pathways; the majority of them were shared by two backcross generations. Our study revealed the most pronounced DEGs associated with blood-feeding monogeneans in backcross hybrids, potentially (but not exclusively) explainable by hybrid breakdown. The lower DEGs reported in F1 hybrids being less parasitized than backcross hybrids is in line with the hybrid advantage.

• Klíčová slova
• Monogenea, Paradiplozoon homoion, RNA seq, differential gene expression, freshwater fish, hybrid breakdown, hybrid heterosis, hybridization,
• Publikační typ
• časopisecké články MeSH

BACKGROUND: Monogeneans, in general, show a range of unique adaptations to a parasitic lifestyle, making this group enormously diverse. Due to their unique biological properties, diplozoid monogeneans represent an attractive model group for various investigations on diverse biological interactions. However, despite numerous studies, there are still gaps in our knowledge of diplozoid biology and morphofunctional adaptations. RESULTS: In this study, we provide a comprehensive microscopic analysis of systems/structures involved in niche searching, sensing and self-protection against the host environment, and excretory/secretory processes in Eudiplozoon nipponicum. Freeze-etching enabled us to detect syncytium organisational features not visible by TEM alone, such as the presence of a membrane subjacent to the apical plasma membrane (separated by a dense protein layer) and a lack of basal plasma membrane. We located several types of secretory/excretory vesicles and bodies, including those attached to the superficial membranes of the tegument. Giant unicellular glands were seen accumulating predominantly in the apical forebody and hindbody haptor region. Muscle layer organisation differed from that generally described, with the outer circular and inner longitudinal muscles being basket-like interwoven by diagonal muscles with additional perpendicular muscles anchored to the tegument. Abundant muscles within the tegumentary ridges were detected, which presumably assist in fixing the parasite between the gill lamellae. Freeze-etching, alongside transmission electron and confocal microscopy with tubulin labelling, enabled visualisation of the protonephridia and nervous system, including the peripheral network and receptor innervation. Three types of receptor were identified: 1) uniciliated sensory endings with a subtle (or missing) tegumentary rim, 2) obviously raised uniciliated receptors with a prominent tegumentary rim (packed with massive innervation and muscles) and 3) non-ciliated papillae (restricted to the hindbody lateral region). CONCLUSIONS: This study points to specific morphofunctional adaptations that have evolved in diplozoid monogeneans to confront their fish host. We clearly demonstrate that the combination of different microscopic techniques is beneficial and can reveal hidden differences, even in much-studied model organisms such as E. nipponicum.

• Klíčová slova
• Excretory system, Freeze-etching, Host-parasite interactions, Immunofluorescence, Musculature, Nervous system, Secretion, Sensory structures, Tegument, Ultrastructure,
• 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

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

BACKGROUND: Cysteine peptidases of clan CA, family C1 account for a major part of proteolytic activity in the haematophagous monogenean Eudiplozoon nipponicum. The full spectrum of cysteine cathepsins is, however, unknown and their particular biochemical properties, tissue localisation, and involvement in parasite-host relationships are yet to be explored. METHODS: Sequences of cathepsins L and B (EnCL and EnCB) were mined from E. nipponicum transcriptome and analysed bioinformatically. Genes encoding two EnCLs and one EnCB were cloned and recombinant proteins produced in vitro. The enzymes were purified by chromatography and their activity towards selected substrates was characterised. Antibodies and specific RNA probes were employed for localisation of the enzymes/transcripts in tissues of E. nipponicum adults. RESULTS: Transcriptomic analysis revealed a set of ten distinct transcripts that encode EnCLs. The enzymes are significantly variable in their active sites, specifically the S2 subsites responsible for interaction with substrates. Some of them display unusual structural features that resemble cathepsins B and S. Two recombinant EnCLs had different pH activity profiles against both synthetic and macromolecular substrates, and were able to hydrolyse blood proteins and collagen I. They were localised in the haematin cells of the worm's digestive tract and in gut lumen. The EnCB showed similarity with cathepsin B2 of Schistosoma mansoni. It displays molecular features typical of cathepsins B, including an occluding loop responsible for its exopeptidase activity. Although the EnCB hydrolysed haemoglobin in vitro, it was localised in the vitelline cells of the parasite and not the digestive tract. CONCLUSIONS: To our knowledge, this study represents the first complex bioinformatic and biochemical characterisation of cysteine peptidases in a monogenean. Eudiplozoon nipponicum adults express a variety of CLs, which are the most abundant peptidases in the worms. The properties and localisation of the two heterologously expressed EnCLs indicate a central role in the (partially extracellular?) digestion of host blood proteins. High variability of substrate-binding sites in the set of EnCLs suggests specific adaptation to a range of biological processes that require proteolysis. Surprisingly, a single cathepsin B is expressed by the parasite and it is not involved in digestion, but probably in vitellogenesis.

• Klíčová slova
• Blood digestion, Cathepsin, Cysteine peptidase, Diplozoidae, Eudiplozoon nipponicum, Fish parasite, Haematophagy, Monogenea, Protease, S2 subsite,
• MeSH
• gastrointestinální trakt parazitologie   MeSH
• hydrolýza MeSH
• interakce hostitele a parazita MeSH
• kapři parazitologie   MeSH
• kathepsin B chemie   genetika   izolace a purifikace   metabolismus   MeSH
• kathepsin L chemie   genetika   izolace a purifikace   metabolismus   MeSH
• proteolýza MeSH
• rekombinantní proteiny analýza   genetika   izolace a purifikace   MeSH
• stanovení celkové genové exprese MeSH
• Trematoda enzymologie   genetika   MeSH
• zavlečené druhy MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• kathepsin B MeSH
• kathepsin L MeSH
• rekombinantní proteiny 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

Parasite inhibitors of cysteine peptidases are known to influence a vast range of processes linked to a degradation of either the parasites' own proteins or proteins native to their hosts. We characterise a novel type I cystatin (stefin) found in a sanguinivorous fish parasite Eudiplozoon nipponicum (Platyhelminthes: Monogenea). We have identified a transcript of its coding gene in the transcriptome of adult worms. Its amino acid sequence is similar to other stefins except for containing a legumain-binding domain, which is in this type of cystatins rather unusual. As expected, the recombinant form of E. nipponicum stefin (rEnStef) produced in Escherichia coli inhibits clan CA peptidases - cathepsins L and B of the worm - via the standard papain-binding domain. It also blocks haemoglobinolysis by cysteine peptidases in the worm's excretory-secretory products and soluble extracts. Furthermore, we had confirmed its ability to inhibit clan CD asparaginyl endopeptidase (legumain). The presence of a native EnStef in the excretory-secretory products of adult worms, detected by mass spectrometry, suggests that this protein has an important biological function at the host-parasite interface. We discuss the inhibitor's possible role in the regulation of blood digestion, modulation of antigen presentation, and in the regeneration of host tissues.

• MeSH
• cystatiny metabolismus   MeSH
• cysteinové endopeptidasy metabolismus   MeSH
• Escherichia coli MeSH
• fylogeneze MeSH
• kapři parazitologie   MeSH
• klonování DNA MeSH
• konformace proteinů MeSH
• ploštěnci metabolismus   MeSH
• počítačová simulace MeSH
• proteinové domény MeSH
• proteiny červů genetika   metabolismus   MeSH
• rekombinantní proteiny genetika   metabolismus   MeSH
• sekvenční analýza proteinů MeSH
• sekvenční seřazení MeSH
• vazba proteinů MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• asparaginylendopeptidase MeSH Prohlížeč
• cystatiny MeSH
• cysteinové endopeptidasy MeSH
• proteiny červů MeSH
• rekombinantní proteiny MeSH

BACKGROUND: Species of Diplozoon Palombi, 1949 (Monogenea: Diplozoidae) are blood-feeding ectoparasites mainly parasitising the gills of cyprinid fishes. Although these parasites have been the subject of numerous taxonomic, phylogenetic and ecological studies, the ultrastructure of the surface and haptor attachment structures remains almost unknown. In this study, we used transmission electron microscopy to examine the ultrastructure of attachment clamps and neodermal surface of Paradiplozoon homoion (Bychowsky & Nagibina, 1959), family Diplozoidae Palombi, 1949, thereby broadening our knowledge of platyhelminth biology. RESULTS: The hindbody surface of P. homoion is distinctly ridged, each ridge being supported by several muscle fibers and equipped with scales on the surface plasma membrane. Such structures have not been recorded previously in species of the family Diplozoidae. Comparisons of the surface structure of different body parts revealed slight differences in the thickness and number of organelles. Each of the clamps has a flattened bowl-like structure composed of sclerites, movable skeletal-like structures that are anchored by robust, radially oriented muscle bundles. The base of the posterior median plate sclerites is equipped with glandular cells possessing secretory vesicles. CONCLUSION: This study brings detailed ultrastructural data for the surface and haptoral attachment clamps of P. homoion and provides new insights into the ultrastructure of Diplozoidae. Glandular cells at the base of the attachment clamps responsible for sclerite development in diplozoid species were observed for the first time. Our findings support the hypothesis that the structure of particular neodermal compartments is similar within the Platyhelminthes. On the other hand, the diplozoid glandular system and the mechanism of sclerite development clearly merits further attention.

• Klíčová slova
• Attachment clamps, Neodermis, Paradiplozoon homoion, Tegument, Ultrastructure,
• MeSH
• Cyprinidae parazitologie   MeSH
• fylogeneze MeSH
• infekce červy třídy Trematoda parazitologie   veterinární   MeSH
• nemoci ryb parazitologie   MeSH
• transmisní elektronová mikroskopie MeSH
• Trematoda izolace a purifikace   fyziologie   ultrastruktura   MeSH
• žábry parazitologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Diplozoons are representatives of blood-feeding ectoparasites from the family Diplozoidae (Polyopisthocotylea, Monogenea). Although these worms have been the subject of numerous taxonomical, phylogenetic, and ecological studies, the detailed study of their excretory system has remained relatively neglected. Our observations focused on the morphological and ultrastructural features of the excretory apparatus of four diplozoid species: Diplozoon paradoxum, Eudiplozoon nipponicum, Paradiplozoon bliccae, and Paradiplozoon homoion. Observations were obtained using two microscope methods: light microscopy, equipped with differential interference contrast (Nomarski DIC) and transmission electron microscopy (TEM). The ultrastructure of two basic compartments which forms the excretory apparatus, flame cells with filtration apparatus, and canal cells forming the protonephridial ducts is revealed in this study. A unique consecutive sequence of longitudinal semi-thin sections of the excretory pore of E. nipponicum is visualized there for the first time.

• Klíčová slova
• Diplozoidae, Excretory system, Flame cell, Monogenea, Protonephridia, Ultrastructure,
• MeSH
• druhová specificita MeSH
• Trematoda ultrastruktura   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH