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

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