Eudiplozoon nipponicum (Monogenea, Diplozoidae) and its adaptation to haematophagy as revealed by transcriptome and secretome profiling.

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.

Annulotrema (Monogenea: Dactylogyridae) from the gills of African tetras (Characiformes: Alestidae) in Lake Turkana, Kenya, with descriptions of four new species and a redescription of A. elongata Paperna and Thurston, 1969.

Four new and four previously described species of Annulotrema were collected from the gills of four species (three genera, i.e. Alestes, Hydrocynus and Brycinus) of African tetras from Lake Turkana, Kenya: Annulotrema alestesnursi Paperna, 1973 from Brycinus nurse; Annulotrema ansatum n. sp., Annulotrema besalis Rehulková, Musilová and Gelnar, 2014, Annulotrema bipatens n. sp., Annulotrema cucullatum n. sp., Annulotrema nili Paperna, 1973, and Annulotrema pontile n. sp. from Hydrocynus forskahlii; and Annulotrema elongata Paperna and Thurston, 1969 from Alestes baremoze and Alestes dentex. A. elongata is re-described on the basis of new material from A. baremoze. The sclerotized structures of the haptor and male copulatory organ of A. alestesnursi and A. elongata are illustrated from their type material. H. forskahlii is a new host record for A. besalis. The findings of A. besalis and A. elongata in Kenya represent a new locality records for these helminths. Three Annulotrema spp., namely A. besalis, A. elongata and A. pontile n. sp., share the same type of male copulatory organ, which may indicate a close relationship among these species.

Annulotrema (Monogenea: Dactylogyridae) from Hydrocynus brevis (Characiformes: Alestidae) in Senegal, with descriptions of two new species and remarks on Annulotrema pikei.

Two new species of Annulotrema Paperna & Thurston, 1969 were collected from the gills of the African tiger fish, Hydrocynus brevis, from the Gambia River basin in the Niokolo-Koba National Park, Senegal. Annulotrema besalis n. sp. is characterized by having a male copulatory organ (MCO) composed of an arcuate copulatory tube articulated to an eight-shaped accessory piece with terminal claw. The new species resembles Annulotrema pikei (Price, Peebles & Bamford, 1969) in having morphologically similar types of haptoral sclerites and MCO. As a result of the differential diagnosis made for A. besalis n. sp., new information on taxonomically important features of A. pikei is provided based on illustrations of the sclerotized parts of the holotype from Hydrocynus vittatus. The report of A. pikei on the gills of Hydrocynus forskahlii by Paperna in 1979 is shown to be erroneous. Annulotrema uncata n. sp. is similar to Annulotrema alestesimberi Paperna, 1973 in its possession of a coiled copulatory tube with about two and a half rings. Features distinguishing the new species include the sharply curved shaft of the ventral anchor, the base of the copulatory tube extending to a sock-like structure and a leech-shaped vagina. The necessity of emending the generic diagnosis of Annulotrema is briefly discussed.

Eudiplozoon nipponicum in focus: monogenean exhibiting a highly specialized adaptation for ectoparasitic lifestyle.

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.

Eudiplozoon nipponicum: morphofunctional adaptations of diplozoid monogeneans for confronting their host.

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.

Redescription of Paradiplozoon opsariichthydis (Jiang, Wu et Wang 1984) Jiang, Wu et Wang, 1989 (Monogenea, Diplozoidae).

Paradiplozoon opsariichthydis (Jiang, Wu et Wang, 1984) Jiang, Wu et Wang, 1989 (Platyhelminthes, Monogenea, Diplozoidae) is blood-feeding parasite from the gills of Asian cyprinid fish Opsariichthys bidens Günther, 1873. In this study, we present a morphological redescription of P. opsariichthydis neotype main morphological features e.g. size of body and clamps due to the fact that the type material is missing. We decided to supplement morphological descriptions by the relevant molecular data (internal transcribed spacer - ITS2) related to P. opsariichthydis adult worm isolates and other representatives of genus Paradiplozoon to cross verify our findings. In addition to that, this study also brings an attention to the host identification. Thus, parasite data were complemented by the determinant cytochrome oxidase b (cytb) sequences of its hosts. All novel sequences are deposited in GenBank. This combination of the morphological and molecular data related to both the parasite and its host seems to be the optimal approach to the general process of (re)description of highly host-specific parasitic organisms, which can then lead to a meaningful phylogenetic analysis.

Contrasting Host-Parasite Population Structure: Morphology and Mitogenomics of a Parasitic Flatworm on Pelagic Deepwater Cichlid Fishes from Lake Tanganyika.

Little phylogeographic structure is presumed for highly mobile species in pelagic zones. Lake Tanganyika is a unique ecosystem with a speciose and largely endemic fauna famous for its remarkable evolutionary history. In bathybatine cichlid fishes, the pattern of lake-wide population differentiation differs among species. We assessed the congruence between the phylogeographic structure of bathybatine cichlids and their parasitic flatworm Cichlidogyrus casuarinus to test the magnifying glass hypothesis. Additionally, we evaluated the use of a PoolSeq approach to study intraspecific variation in dactylogyrid monogeneans. The lake-wide population structure of C. casuarinus ex Hemibates stenosoma was assessed based on a portion of the cox1 gene combined with morphological characterisation. Additionally, intraspecific mitogenomic variation among 80 parasite samples from one spatially constrained metapopulation was assessed using shotgun NGS. While no clear geographic genetic structure was detected in parasites, both geographic and host-related phenotypic variation was apparent. The incongruence with the genetic north-south gradient observed in H. stenosoma may be explained by the broad host range of this flatworm including eupelagic bathybatine host species that form panmictic populations across the lake. In addition, we present the first parasite mitogenome from Lake Tanganyika and propose a methodological framework for studying the intraspecific mitogenomic variation of dactylogyrid monogeneans.

Parasitic flatworms infecting thorny skate, Amblyraja radiata: Infection by the monogeneans Acanthocotyle verrilli and Rajonchocotyle emarginata in Svalbard.

Parasite diversity above the Arctic circle remains understudied even for commercially valuable host taxa. Thorny skate, Amblyraja radiata, is a common bycatch species with a growing commercial value. Its natural range covers both sides of the North Atlantic including the Arctic zone. Svalbard is a Norwegian archipelago located on the northwest corner of the Barents Shelf which sustains a spectacular species diversity. So far, several monogenean species have been reported infecting thorny skate across the Atlantic Ocean. In the present study, we intend to fill in the knowledge gap on monogenean parasites infecting thorny skate in the northern part of its range and thus indirectly assess the connectivity between the thorny skate populations off the Svalbard coast and from previously studied locations. 46 monogenean individuals were recovered from 11 specimens of thorny skate. Following morphological and molecular assessment, two species of monogeneans, Acanthocotyle verrilli and Rajonchocotyle emarginata, were identified. The results serve as the northernmost record for both parasite genera and the first record of monogenean species off Svalbard. Detailed morphometric evaluation revealed a relatively high level of morphological variation in A. verrilli compared to its congeners. Phylogenetic reconstruction placed A. verrilli in a well-supported clade with A. imo. Our study also suggests high diagnostic significance of sclerotised structures in the identification of Rajonchocotyle. Even though the occurrence of two directly transmitted parasite species supports the previously suggested long-distance migration of A. radiata, future studies employing highly variable genetic markers are needed to assess the ongoing and historical migration patterns.

Five new monogenean species from the gills of Mulloidichthys vanicolensis (Perciformes: Mullidae) off New Caledonia, with the proposal of Volsellituba n. g. and Pennulituba n. g. (Monogenea: Dactylogyridae).

Three species of Volsellituba n. g. and two species of Pennulituba n. g. are described from the gills of the yellowfin goatfish Mulloidichthys vanicolensis (Valenciennes) off New Caledonia (South Pacific). Volsellituba and Pennulituba resemble dactylogyrid genera whose species lack eye-spots and possess tandem or slightly overlapping gonads, a single prostatic reservoir, a male copulatory organ without an accessory piece, a dextroventral non-sclerotised vagina, ventral and dorsal anchor/bar complexes, and hooks with shanks comprising a single slender unit. Volsellituba n. g. is characterised by its species having ventral tegumental folds at the level of the vaginal duct, a male copulatory organ with a subterminal basal opening and a two-piece dorsal bar. The following new species of Volsellituba are described: V. orchidea n. sp. (type-species), V. nabla n. sp. and V. elephantina n. sp. Pennulituba n. g. is distinguished by its species possessing a male copulatory organ with a wing-shaped membrane and a dorsal bar with a poorly to non-sclerotised medial part. This genus includes P. piratifalx n. sp. (type-species) and P. cymansis n. sp. A tabular summary of the species of Haliotrema Johnston & Tiegs, 1922 reported from mullids is provided.

Laser capture microdissection in combination with mass spectrometry: Approach to characterization of tissue-specific proteomes of Eudiplozoon nipponicum (Monogenea, Polyopisthocotylea).

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.

Uncharted digenean diversity in Lake Tanganyika: cryptogonimids (Digenea: Cryptogonimidae) infecting endemic lates perches (Actinopterygii: Latidae).

BACKGROUND: Lake Tanganyika is considered a biodiversity hotspot with exceptional species richness and level of endemism. Given the global importance of the lake in the field of evolutionary biology, the understudied status of its parasite fauna is surprising with a single digenean species reported to date. Although the most famous group within the lake's fish fauna are cichlids, the pelagic zone is occupied mainly by endemic species of clupeids (Actinopterygii: Clupeidae) and lates perches (Actinopterygii: Latidae, Lates Cuvier), which are an important commercial source for local fisheries. In this study, we focused on the lake's four lates perches and targeted their thus far unexplored endoparasitic digenean fauna. METHODS: A total of 85 lates perches from four localities in Lake Tanganyika were examined. Cryptogonimid digeneans were studied by means of morphological and molecular characterisation. Partial sequences of the nuclear 28S rRNA gene and the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene were sequenced for a representative subset of the specimens recovered. Phylogenetic analyses were conducted at the family level under Bayesian inference. RESULTS: Our integrative approach revealed the presence of six species within the family Cryptogonimidae Ward, 1917. Three out of the four species of Lates were found to be infected with at least one cryptogonimid species. Two out of the three reported genera are new to science. Low interspecific but high intraspecific phenotypic and genetic diversity was found among Neocladocystis spp. Phylogenetic inference based on partial 28S rDNA sequences revealed a sister group relationship for two of the newly erected genera and their close relatedness to the widely distributed genus Acanthostomum Looss, 1899. CONCLUSIONS: The present study provides the first comprehensive characterisation of the digenean diversity in a fish family from Lake Tanganyika which will serve as a baseline for future explorations of the lake's digenean fauna. Our study highlights the importance of employing an integrative approach for revealing the diversity in this unique host-parasite system.

Weak population structure and recent demographic expansion of the monogenean parasite Kapentagyrus spp. infecting clupeid fishes of Lake Tanganyika, East Africa.

Lake Tanganyika, East Africa, is the oldest and deepest African Great Lake and harbours one of the most diverse fish assemblages on earth. Two clupeid fishes, Limnothrissa miodon and Stolothrissa tanganicae, constitute a major part of the total fish catch, making them indispensable for local food security. Parasites have been proposed as indicators of stock structure in highly mobile pelagic hosts. We examined the monogeneans Kapentagyrus limnotrissae and Kapentagyrus tanganicanus (Dactylogyridae) infecting these clupeids to explore the parasites' lake-wide population structure and patterns of demographic history. Samples were collected at seven sites distributed across three sub-basins of the lake. Intraspecific morphological variation of the monogeneans (n = 380) was analysed using morphometrics and geomorphometrics of sclerotised structures. Genetic population structure of both parasite species (n = 246) was assessed based on a 415 bp fragment of the mitochondrial cytochrome c oxidase subunit I (COI) gene. Overall, we observed a lack of clear geographical morphological differentiation in both parasites along a north-south axis. This lack of geographical population structure was also reflected by a large proportion of shared haplotypes, and a pattern of seemingly unrestricted gene flow between populations. Significant morphological and genetic differentiation between some populations might reflect temporal differentiation rather than geographical isolation. Overall, the shallow population structure of both species of Kapentagyrus reflects the near-panmictic population structure of both host species as previously reported. Morphological differences related to host species identity of K. tanganicanus were consistent with incipient speciation at the genetic level. Both parasite species experienced a recent demographic expansion, which might be linked to paleohydrological events. Finally, interspecific hybridisation was found in Kapentagyrus, representing the first case in dactylogyrid monogeneans.

The interaction between an introduced fish host and local parasite fauna: Neogobius kessleri in the middle Danube River.

Parasite communities of introduced fish Neogobius kessleri Günther (Gobiidae) were studied at five localities in the Slovak section of the Danube River during 2002-2005. Thirty-three metazoan parasite species were identified. All fish were infected with at least two parasite species; most of the parasite species were generalists. At all sampling sites, high susceptibility to local parasites was observed. The parasite community was dominated by three parasite species: glochidia of Anodonta anatina, larval or subadult acanthocephalan Pomphorhynchus laevis, and larval nematode Raphidascaris acus. The infection of both A. anatina and P. laevis was affected by season and habitat type, with higher abundance in spring and more frequent occurrence of A. anatina in side channels and P. laevis in main river channels. At both the component and infracommunity levels, a more diverse parasite community was found in side channels. This habitat was dominated by actively transmitted parasites, whilst endoparasites were more abundant in fish from the main river channel. Larval stages of parasites dominated the endoparasite community at all sampling sites. The introduced N. kessleri was used as intermediate host for most of the recorded parasites, in some cases also as a paratenic host. Finally, the importance of gobies as suitable hosts for local non-native parasite species (Anguillicoloides crassus, Anodonta woodina, Hydrozetes lacustris) is discussed.

Gyrodactylus species (Monogenea: Gyrodactylidae) on the cichlid fishes of Senegal, with the description of Gyrodactylus ergensi n. sp. from Mango tilapia, Sarotherodon galilaeus L. (Teleostei: Cichilidae).

The first record of Gyrodactylus species Nordmann, 1832 on the cichlid fish from Senegal is reported. Gyrodactylus parasites were found on four host species studied, Hemichromis fasciatus, Hemichromis letournaeuxi, Oreochromis niloticus, and Sarotherodon galilaeus. Gyrodactylus cichlidarum Paperna (Bamidgeh 13:14-29, 1968) were collected from H. fasciatus, and its identity was confirmed based on the morphometrical comparison with the holotype specimens of G. cichlidarum. Only one specimen of Gyrodactylus sp. was noted on H. letournaeuxi. The gyrodactylid specimens on O. niloticus and S. galilaeus were described as Gyrodactylus ergensi n. sp. based on the detailed morphometrical analysis supported by sequencing. The haptoral sclerites of G. ergensi n. sp., which closely resemble those of Gyrodactylus nyanzae Paperna, 1973, are compared with the type material and discussed. A partial sequence of the internal transcribed spacer recombinant DNA (ITS rDNA) was obtained from two specimens of G. ergensi, and a close relationship between G. ergensi n. sp. and G. cichlidarum is suggested based on 92% similarity in ITS rDNA region.

Species of Characidotrema Paperna & Thurston, 1968 (Monogenea: Dactylogyridae) from fishes of the Alestidae (Characiformes) in Africa: new species, host-parasite associations and first insights into the phylogeny of the genus.

BACKGROUND: African tetras (Alestidae) belonging to Brycinus Valenciennes are known to be parasitized with monogeneans attributed to two genera, Annulotrema Paperna & Thurston, 1969 and Characidotrema Paperna & Thurston, 1968 (Dactylogyridae). During a survey of monogeneans parasitizing alestids, species of Characidotrema were collected in Cameroon, D. R. Congo, Senegal, South Africa, Sudan and Zimbabwe. This paper provides new morphological data and the first molecular analysis broadening our knowledge on the diversity of these parasites. RESULTS: Seven species (four known and three new) of Characidotrema are reported from two species of Brycinus: C. auritum n. sp. and C. vespertilio n. sp. from B. imberi (Peters); and C. brevipenis Paperna, 1969, C. nursei Ergens, 1973, C. pollex n. sp., C. spinivaginus (Paperna, 1973) and C. zelotes Kritsky, Kulo & Boeger, 1987 from B. nurse (Rüppell). Species identification was based on morphological analysis of the sclerotized structures supported by nuclear ribosomal DNA (partial 18S rDNA, ITS1, and 28S rDNA) sequence data. Morphological analysis confirmed that the most apparent character distinguishing species in the genus is the morphology of the male copulatory organ and vagina. Observations on the haptoral sclerotized elements of these parasites by means of phase contrast microscopy revealed the presence of a sheath-like structure relating to the ventral anchor, a feature that supplements the generic diagnosis of Characidotrema. Maximum Likelihood and Bayesian analyses of the large subunit (28S) rDNA sequences recovered Characidotrema species isolated from the two Brycinus hosts as monophyletic, and indicated a closer relationship of this group to monogeneans parasitizing African cyprinids (Dactylogyrus spp.) and cichlids (species of Cichlidogyrus Paperna, 1960, Scutogyrus Pariselle & Euzet, 1995, and Onchobdella Paperna, 1968) than to those from catfishes (species of Quadriacanthus Paperna, 1961, Schilbetrema Paperna & Thurston, 1968 and Synodontella Dossou & Euzet, 1993). The overall agreement between the morphological diversification of the MCOs and the molecular tree observed in this study indicates that significant phylogenetic signals for clarifying relationships among species of Characidotrema are present in the characteristics of the MCO. CONCLUSIONS: It seems that intra-host speciation is an important force shaping the present distribution and diversity of Characidotrema but further studies are necessary to confirm this hypothesis and assess questions related to the phylogeny of these parasites. To identify potential co-speciation events, co-phylogenetic analyses of these monogeneans and their alestid hosts are required.

Cryptosporidia: epicellular parasites embraced by the host cell membrane.

The ultrastructure of two gastric cryptosporidia, Cryptosporidium muris from experimentally infected rodents (Mastomys natalensis) and Cryptosporidium sp. 'toad' from naturally infected toads (Duttaphrynus melanostictus), was studied using electron microscopy. Observations presented herein allowed us to map ultrastructural aspects of the cryptosporidian invasion process and the origin of a parasitophorous sac. Invading parasites attach to the host cell, followed by gradual envelopment, with the host's cell membrane folds, eventually forming the parasitophorous sac. Cryptosporidian developmental stages remain epicellular during the entire life cycle. The parasite development is illustrated in detail using high resolution field emission scanning electron microscopy. This provides a new insight into the ultrastructural detail of host-parasite interactions and species-specific differences manifested in frequency of detachment of the parasitophorous sac, radial folds of the parasitophorous sac and stem-formation of the parasitised host cell.

Architecture of Paradiplozoon homoion: A diplozoid monogenean exhibiting highly-developed equipment for ectoparasitism.

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.

Redescription of Paradiplozoon hemiculteri (Monogenea, Diplozoidae) from the type host Hemiculter leucisculus, with neotype designation.

Paradiplozoon hemiculteri (Ling, 1973), a member of the Diplozoidae, parasitizes the gills of Asian fish. Not only is the type material unavailable for this species, the original description was poor and somewhat conflicting, and adequate molecular data were not available. What is more, the available morphological and molecular data are inconsistent and fluctuate significantly. Here, we present a redescription of P. hemiculteri based on morphological and molecular data from new isolates collected from the type host, the sharpbelly Hemiculter leucisculus (Basilewsky, 1855), captured at the neotype locality (Shaoguan, Guangdong Province, southern China); a neotype for P. hemiculteri was designated from this collection. The length and width of the body, buccal suckers, pharynx, attachment clamps, sickle and the central hook handle were all measured and the shape of the anterior and posterior part of the median plate and anterior and posterior joining sclerites accurately documented. Phylogenetic analyses based on the sequences of the second rDNA internal transcribed spacer (ITS2) indicated that all new samples clustered together and differed clearly from sequences attributed to P. hemiculteri, which are deposited in GenBank. Our results confirm that P. hemiculteri is the only diplozoid that has demonstrably been found on the gills of H. leucisculus to date.

Redescription of the genus Afrodiplozoon Khotenovski, 1981 and its only known species Afrodiplozoon polycotyleus (Paperna, 1973) (Monogenea: Diplozoidae) using a combined multidisciplinary approach.

The present study redescribes the monotypic genus Afrodiplozoon Khotenovski, 1981, a diplozooid parasite endemic to Africa, based on material collected during surveys carried out in the Limpopo Province, South Africa, from two cyprinid hosts Labeobarbus marequensis and Enteromius paludinosus. Morphometrical analysis of the composition of internal organs and attachment clamps, using light microscopy and scanning electron microscopy, provided details for the genus and species redescriptions. Posterior attachment apparatus can bear up the 15 pairs of the clamps with the first pair of clamps distinctly smaller and not fully developed. Molecular characterization of the second internal transcribed spacer (ITS2) rDNA and phylogenetic analysis revealed the position of Afrodiplozoon polycotyleus as a sister taxon to Paradiplozoon krugerense and Paradiplozoon bingolenisis, distant from other African representatives of the genus Paradiplozoon, Paradiplozoon vaaleense and Paradiplozoon ichtyoxanthon.

Identification and partial characterization of a novel serpin from Eudiplozoon nipponicum (Monogenea, Polyopisthocotylea).

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.