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

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

Developmental stages of the diplozoid monogenean Eudiplozoon nipponicum, comprising oncomiracidium, diporpa, juvenile, and adult, were investigated using light and scanning electron microscopy in conjunction with confocal scanning laser microscopy in order to examine body organization and identify explicit morphological adaptations to the ectoparasitic life in each stage. The parasite exhibits a complex digestive tract well equipped for hematophagous feeding. It consists of a mouth opening with prominent buccal suckers, eversible pharynx with adjacent glandular structures, and a blind-ending gut with cecal lining. Glandulo-muscular organs, located apically and opened into the mouth corner, are considered to be a part of the digestive tract. Based on our observations of pharynx eversion and in light of the presence of several glandular or gland-like structures, we propose a new hypothesis on the possibility of extracorporeal digestion of this parasite. The hindbody bears an attachment apparatus, comprising haptor, lobular extensions, and tegumental folds, responsible for the parasite's firm attachment to the host gills. The possibility of buccal suckers assisting in the parasite's translocation while searching for an optimal niche or their temporary attachment function during feeding is discussed. The body of each compound adult (i.e., permanent copula) is almost completely filled by two complete reproductive tracts comprising the female as well as male organs. Such a reproductive strategy, in which two independent heterogenic individuals fuse into a single hermaphrodite organism without the need to search for mating partner, represents a high specialization of diplozoids to their parasitic life.

• MeSH
• fyziologická adaptace * MeSH
• hermafroditické organismy fyziologie   MeSH
• infestace ektoparazity parazitologie   patofyziologie   MeSH
• interakce hostitele a parazita fyziologie   MeSH
• kapři parazitologie   MeSH
• mikroskopie elektronová rastrovací MeSH
• parazitární onemocnění kůže parazitologie   patofyziologie   MeSH
• ploštěnci anatomie a histologie   fyziologie   ultrastruktura   MeSH
• rozmnožování fyziologie   MeSH
• stadia vývoje fyziologie   MeSH
• stravovací zvyklosti MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH