Ultrastructural Observations on Fertilization in Dionchus remorae (Platyhelminthes,Monogenea, Dionchidae)

Ultrastructural observations are presented for some of the stages occurring during fertilization in Dionchus remorae (a gill parasite of Echeneis naucrates) and are believed to be the first published concerning a monogenean. Fertilized female germ cells were found in the ovary. Several loops of the spermatozoon were present within the oocyte cytoplasm; the sperm nucleus became electron lucent and the parallel peripheral doublets of the axonemes became increasingly divergent. The cortical granules in the oocyte were not released immediately after penetration by the spermatozoon. The homogeneity apparently found in the oocyte ultrastructure and process of fertilization in the monogeneans and digeneans contrasts with the variety that exists in their sperm ultrastructure.     

Phylogenetic relationships of the Temnocephaloidea (Platyhelminthes)

Temnocephala novaezealandiae (family Temnocephalidae) and Troglocaridicola mrazeki and Scutariella georgica (family Scutariellidae) were studied by electron microscopy in an attempt to reveal characters that would indicate their phylogenetic relationship to other members of the Platyhelminthes. Ultrastructural features of the epidermis in these temnocephaloideans are like those of the neoophoran turbellarians. The epidermis is syncytial, is honeycombed by a multitude of gland necks whose secretions produce an epidermal surface film, and is underlaid by a thick basement membrane. Some cells in the parenchyma are compartmentalized by intrusive cell processes from neighboring parenchymal cells in a fashion similar to parenchymal structure in the Monogenea and Digenea. The spermatozoa have a pair of free 9+1 flagella and contain aligned dense bodies. The Temnocephaloidea is evidently derived from an early rhabdocoel-turbellarian-like ancestor.     

Phylogenetic analyses of the family Tetraonchidae (Platyhelminthes: Monogenea)

A phylogenetic reconstruction of the monogenean family Tetraonchidae was carried out by methods of parsimony-based cladistics. The analysis included 20 species of tetraonchids and two out-groups (Sundanonchus tomanorum and Dactylogyrus amphibothrium) and was based on 34 morphofunctional characters. Software PAUP 4.0 and Winclada were used for the phylogenetic reconstructions. Obtained results allow proposing a preliminary phylogenetic hypothesis of the family Tetraonchidae along with the discussion of host-parasite association. According to the current taxonomic view, the family Tetraonchidae included two genera. Cladistic analysis showed a monophyly of the family and the genus Tetraonchus Diesing, 1858. Two representative of the former genus, Tetraoncus monenteron and T. borealis, parasitize the pikes (Esocoformes: Esocidae) and the grayling (Salmonidae: Thymallinae) respectively. The genus Salmonchus Spassky et Roytman, 1958 has a complicated structure and its intrageneric relationships were not completely resolved; in general, the analysis allows to recognise several species groups: Salmonchus oncorhynchi--the parasite of the Oncorhynchus masou smolt living during the first year of life in fresh water; four species (S. variabilis, S. gussevi, S. grumosus, S. alaskensis) inhabiting specifically the whitefishes (Salmonidae: Coregoninae); all reminder of Salmonchus species occurring on the salmons (Salmonidae: Salmoninae). The bootstrap test gives a support only for the following clades: family Tetraonchidae (75%), genus Tetraonchus (88%); a group of Salmonchus species associated with the whitefishes (93%) and grouping of four species (S. huhonis, S. pseudolenoki, S. skrjabini and S. lenoki) from the lenoks (Brachymystax) and taimens (Hucho) (61%).     

False Vertical Rootlets of Epidermal Cilia in the Oncomiracidia of Neoheterocotyle rhinobatidis and Monocotyle spiremae (Platyhelminthes,Monogenea, Monopisthocotylea)

Epidermal cilia of the oncomiracidium of Neoheterocotyle rhinobatidis (Monogenea, Monopisthocotylea, Monocotylidae) have long cross-striated vertical rootlets that are not extensions of the basal bodies as are the vertical rootlets in all catenulid and rhabditophoran turbellarians examined to date. Instead, they originate in the basal part of the horizontal rootlet a short distance from the basal bodies. In Monocotyle spiremae (Monocotylidae), the vertical rootlets are less distinct, with no apparent cross-striation, but they also originate from the basal part of the horizontal rootlets. Epidermal cilia of the oncomiracidium of Zeuxapta seriolae (Monogenea, Polyopisthocotylea, Axinidae) lack vertical rootlets like all other neodermatans examined, but bundles of fibres extend from the basal bodies a short distance into the cytoplasm of the epidermal cells. Monopisthocotylean Monogenea would be intermediate between rhabditophorans and the other neodermatans (in having weakly developed vertical rootles), if these structures were homologous in the two groups. However, in view of the different origin of vertical rootlets in turbellarians and monopisthocolylean oncomiracidia, it is suggested that they are not homologous, and vertical rootlets in the Monopisthocotylea are therefore named “false vertical rootlets”.     

Phylogenetic relationships of the genus Dugesia (Platyhelminthes, Tricladida, Paludicola)

A phylogenetic analysis was performed on the genera and subgenera within the freshwater triclad family Dugesiidae, based on 19 terminal taxa and 17 morphological characters. The phylogenetic tree proposed has length of 27 steps and consistency index of 0.66. This phylogenetic hypothesis implies that the current genus Dugesia is paraphyletic and that its subgenera Girardia, Schmidiea and Dugesia S.S. should be elevated to the rank of genus. The genera Cura, Spathula and Neppia are presumed monophyletic by default because the database was unable to provide autapomorphies for any of these genera. The genera Dugesia S.S. and Neppia share sistergroup relationship. Several characters are discussed which were previously considered to be of phylogenetic importance but were not included in the present analysis. It is emphasized that sensory organs form potentially useful set of phylogenetic characters for the Dugesiidae.     

Ultrastructure of spermiogenesis, spermatozoa and the tegument in Atriaster sp. (Platyhelminthes, Monogenea, Polyopisthocotylea, Microcotylidae)

Atriaster (Atriaster) sp., a gill parasite in the fish Diplodus cervinus caught near Dakar, Senegal, was studied by transmission electron microscopy. Spermiogenesis is similar to the general scheme in the Cercomeridae and particularly the polyopisthocotylean monogeneans, but shows two remarkable characteristics: (a) the zone of differentiation for each spermatid is separated from the common spermatid cytoplasmic mass by a region without longitudinal microtubules but with many mitochondria, herein termed the pre-zone of differentiation; (b) transverse sections of nuclei in late spermatids are polygonal with concave sides. These two characteristics are provisionally interpreted as autapomorphies, since observations in other species are lacking. The outer layer of the tegument is similar to that of other polyopisthocotylean monogeneans, and has microvilli and three kinds of cytoplasmic inclusions: granules with fibrous contents, granules with homogeneous contents, and elongate electron-dense bodies.     

Phylogenetic relationships between the families of recent Thysanoptera (Insecta)

One family, the Phlaeothripidae, is recognized in the suborder Tubulifera, whereas extant species of Terebrantia are classified into seven families: Uzelothripidae, Merothripidae, Aeolothripidae, Adiheterothripidae, Fauriellidae (stat. n.), Heterothripidae and Thripidae. A phylogenetic analysis of the relationships between these families is given, based on consideration of 35 imaginal characters; however, the relationships of Uzelothripidae and Phlaeothripidae to the rest of the Thysanoptera remain equivocal. The Phlaeothripidae are either derived independently from Protothysanoptera, or else are the sister-group of the Thripidae, the most specialized family of Terebrantia.
Diagnostic characters, diversity, distribution and relationships of each family are discussed. Keys to family and, in Fauriellidae, to genus are provided. Holarthrothrips Bagnall (= Adiheterothrips Ramakrishna, syn. n.) and Oligothrips Moulton are removed from Heterothripidae to Adiheterothripidae and Fauriella Hood, Opisthothrips Hood and Ropotamothrips Pelikan (= Osmanothrips Priesner, syn. n.) from Heteromripidae to Fauriellidae (stat. n.). These transfers leave Aulacothrips Hood, Heterothrips Hood and Scutothrips Stannard as the only genera in Heterothripidae.     

Hox genes from the Polystomatidae (Platyhelminthes, Monogenea)

Hox genes form a multigenic family that play a fundamental role during the early stages of development. They are organised in a single cluster and share a 60 amino acid conserved sequence that corresponds to the DNA binding domain, i.e. the homeodomain. Sequence conservation in this region has allowed investigators to explore Hox diversity in the metazoan lineages. Within parasitic flatworms only homeobox sequences of parasite species from the Cestoda and Digenea have been reported. In the present study we surveyed species of the Polyopisthocotylea (Monogenea) in order to clarify Hox identification and diversification processes in the neodermatan lineage. From cloning of degenerative PCR products of the central region of the homeobox, we report one ParaHox and 25 new Hox sequences from 10 species of the Polystomatidae and one species of the Diclidophoridae, which extend Hox gene diversity from 46 to 72 within Neodermata. Hox sequences from the Polyopisthocotylea were annotated and classified from sequence alignments and Bayesian inferences of 178 Hox, ParaHox and related gene families recovered from all available parasitic platyhelminths and other bilaterian taxa. Our results are discussed in the light of the recent Hox evolutionary schemes. They may provide new perspectives to study the transition from turbellarians to parasitic flatworms with complex life-cycles and outline the first steps for evolutionary developmental biological approaches within platyhelminth parasites.     

Phylogenetic relationships among families of the Scaphopoda (Mollusca)

Phylogenetic relationships among families in the molluscan class Scaphopoda were analysed using morphological characters and cladistic parsimony methods. A maximum parsimony analysis of 34 discrete characters, treated as unordered and equally weighted, from nine ingroup terminal taxa produced a single most parsimonious tree; supplementary analyses of tree length frequency distribution and Bremer support indices indicate a strong phylogenetic signal from the data and moderate to minimally supported clades. The traditional major division of the class, the orders Dentaliida and Gadilida, is supported as both taxa are confirmed as monophyletic clades. Within the Dentaliida, two clades are recognized, the first comprised of the families Dentaliidae and Fustiariidae, the second of the Rhabdidae and Calliodentaliidae; together, these groups comprise a third clade, which has the Gadilinidae as sister. Within the Gadilida, a nested series of relationships is found among [Entalinidae, [Pulsellidae, [Wemersoniellidae, Gadilidae]]]. These results lend cladistic support to earlier hypotheses of shared common ancestry for some families, but are at variance with other previous hypotheses of evolution in the Scaphopoda. Furthermore, analysis of constituent Gadilinidae representatives provide evidence for paraphyly of this family. The relationships supported here provide a working hypothesis that the development of new characters and greater breadth of taxonomic sampling can test, with a suggested primary goal of establishing monophyly at the family level.     

Dionchus postoncomiracidia (Monogenea: Dionchidae) from the skin of blacktip sharks, Carcharhinus limbatus (Carcharhinidae)

Postoncomiracidia of Dionchus sp. are described from specimens collected from the skin of 2 blacktip sharks Carcharhinus limbatus, captured in the northern Gulf of Mexico. The parasites resemble nonciliated oncomiracidia hatched from eggs laid by Dionchus sp. on gills of a cobia Rachycentron canadum and adults of Dionchus remorae that were collected from gills of a common sharksucker Echeneis naucrates, captured in association with a third blacktip shark. The hamuli of the postoncomiracidia were morphologically similar to those of adult D. remorae. This is the first report of dionchids from an elasmobranch and from a location other than the gills. These findings support the idea that some dionchid oncomiracidia colonize the skin of sharks or other aquatic vertebrates that sponsor remoras, prior to transferring to other remoras and maturing.     

Sperm ultrastructure in Myxinidocotyle and Acanthocotyle (Platyhelminthes, Monogenea, Acanthocotylidae)

Spermatozoa of Myxinidocotyle californica from the hagfish Eptatretus stoutii and of Acanthocotyle lobianchi from the skate Raja clavata show a similar ultrastructure: two axoncmes of the 9 + 1 type in parallel with the nucleus-and one mitochondrion. In the released Acanthocotyle spermatozoa nucleus and mitochondrion both have a triangular cross-section. No cortical microtubules are present. The ultrastructure of these two acanthocotylid spermatozoa thus corresponds to sperm pattern 2 according to Justine et al. (1985). This pattern is derived from the more primitive pattern 1, which in the Monogenea is found only in the Oligonchoinea Bychowsky, 1937 or the Polyopisthocotylea sensu Justine et al.     

Speciation and host-parasite relationships in the parasite genus Gyrodactylus (Monogenea, Platyhelminthes) infecting gobies of the genus Pomatoschistus (Gobiidae, Teleostei)

Using species-level phylogenies, the speciation mode of Gyrodactylus species infecting a single host genus was evaluated. Eighteen Gyrodactylus species were collected from gobies of the genus Pomatoschistus and sympatric fish species across the distribution range of the hosts. The V4 region of the ssrRNA and the internal transcribed spacers encompassing the 5.8S rRNA gene were sequenced; by including published sequences a total of 30 species representing all subgenera were used in the data analyses. The molecular phylogeny did not support the morphological groupings into subgenera as based on the excretory system, suggesting that the genus needs systematic revisions. Paraphyly of the total Gyrodactylus fauna of the gobies indicates that at least two independent colonisation events were involved, giving rise to two separate groups, belonging to the subgenus Mesonephrotus and Paranephrotus, respectively. The most recent association probably originated from a host switching event from Gyrodactylus arcuatus, which parasitises three-spined stickleback, onto Pomatoschistus gobies. These species are highly host-specific and form a monophyletic group, two possible ‘signatures’ of co-speciation. Host specificity was lower in the second group. The colonising capacity of these species is illustrated by a host jump from gobiids to another fish order (Anguilliformes), supporting the hypothesis of a European origin of Gyrodactylus anguillae and its intercontinental introduction by the eel trade. Thus, allopatric speciation seems to be the dominant mode of speciation in this host–parasite system, with a possible case of sympatric speciation.     

Spermatogenesis and spermiogenesis in Microcotyle sp. (Microcotylidae, Monogenea)

Ultrastructural observations of spermatogenesis and spermiogenesis in Microcotyle sp. a microcotylid monogenean parasite from the gills of Hypostomus sp., are described. The spermatogonia were irregularly shaped, forming a peripheral layer of cells; spermatocytes were larger than spermatogonia and a nuclear synaptonemal complex was observed; young spermatids were joined by a central cytophore forming rosettes. Spermiogenesis was characterized by the outgrowth of a cytoplasmic protuberance, the zone of differentiation, containing the basal bodies, separated by an intercentriolar body, from which flagella grow out and fuse posteriorly with the median process. Cross sections of the anterior and the middle regions of spermatozoa revealed nuclei, mitochondria, peripheral microtubules, and paired axonemes each with a 9+1 pattern.     

Ultrastructure of the protonephridial system, of Anoplodiscus cirrusspiralis (Monogenea Monopisthocotylea).

The flame bulb is formed by a terminal cell and a proximal canal cell. The weir consists of interdigitating ribs all of which form one circle, i.e. alternating ribs do not have distinctly 'internal' or 'external' positions. Cytoplasmic cords are absent and all ribs, i.e. those continuous with the proximal canal cell and those continuous with the terminal cell, form external leptotriches. At least some external leptotriches have interconnected branches extending along the flame bulb. Internal leptotriches are not branched and arise from the basal perikaryon of the terminal cell. In the cytoplasmic cylinder at the tip of the flame bulb, structures resembling incomplete septate junctions were seen. However, neither the cytoplasmic cylinder nor the small protonephridial capillaries contain complete septate junctions as found in all other Monogenea Polyopisthocotylea, Monogenea Monopisthocotylea, Trematoda Aspidogastrea and Trematoda Digenea examined to date. In the lack of a septate junction, Anoplodiscus resembles Udonella, Amphilinidea, Gyrocotylidea and Eucestoda. However, the presence in this species of rudimentary septate junctions in the small capillaries and of complete junctions in larger ones indicates that complete junctions have been secondarily lost. Anoplodiscus resembles the Monogenea and Trematoda in the presence of lamellae in the larger protonephridial ducts. For the first time in a monogenean, the ultrastructure of the excretory bladder is described. A nucleated convoluted duct opens through a narrow connecting duct into the bladder, which in turn opens through a narrow connecting duct into the excretory pore lined by tegument. Convoluted duct, connecting ducts and bladder are lined by a lamellated epitheliu.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ultrastructure of Spermiogenesis in Acanthocotyle and Myxinidocotyle (Platyhelminthes,Monogenea, Acanthocotylidae)

Abstract Spermiogenesis was studied by transmission electron microscopy in the acanthocotylid monogeneans Myxinidocotyle californica (from Eptatretus stoutii) and Acanthocotyle lobianchi (from Raja clavata). In Myxinidocotyle and Acanthocotyle, the zone of differentiation shows two 9+‘1’ axonemes, the elongating nucleus and mitochondrion, and a single cortical cytoplasmic microtubule. This single microtubule is found in the mature spermatozoon of both species and was also noted in capsalids. This requires a modified definition of ‘pattern 2’ of spermatozoa which becomes: ‘spermatozoa with two axonemes and no cortical microtubules, except one single element much shorter than the spermatozoon’. A very unusual structure was found in Myxinidocotyle, but not in Acanthocotyle: the centriolar derivative of one of the 9+‘1’ axonemes is made up of 18 diverging singlets of unequal length associated with electron-dense cytoplasm. This seems to be the first case of a centriolar derivative without nine-fold symmetry associated with an axoneme with nine-fold symmetry.     

Reproductive Strategies of the Insidious Fish Ectoparasite,Neobenedenia sp. (Capsalidae: Monogenea)

Fish monogeneans are lethal parasites in aquaculture. We provide the first experimental evidence that a notorious fish monogenean, Neobenedenia sp., can produce viable eggs in isolation for three consecutive generations. We infected individual, isolated, farmed barramundi, Lates calcarifer (Bloch) with a single oncomiracidium (larva) of the hermaphroditic monogenean Neobenedenia sp. Isolated parasites reached sexual maturity at day 10 post-hatch (24°C, 35‰) and laid ∼3,300 embryonated eggs over 17 days. Egg production rapidly increased following sexually maturity on day 10 (58±15 eggs) and peaked on day 15 (496±68 eggs) before gradually decreasing. Neobenedenia sp. exhibited egg laying and egg hatching rhythms. Parasites laid eggs continuously, but egg production increased in periods of darkness (64.3%), while the majority of oncomiracidia (81%) emerged from eggs in the first three hours of light. Eggs laid by isolated ‘parent’ parasites hatched and individual emerging oncomiracidia were used to infect more individual, isolated fish, with three consecutive, isolated, parasite generations (F1, F2 and F3) raised in the laboratory. Infection success and egg hatching success did not differ between generations. Our data show that one parasite, in the absence of a mate, presents a severe threat to captive fish populations.     

Evidence of complement-mediated killing of Discocotyle sagittata (Platyhelminthes, Monogenea) oncomiracidia

Discocotyle sagittata oncomiracidia were rapidly killed when incubated in na?ve plasma and immune sera from both rainbow trout (Oncorhynchus mykiss) and brown trout (Salmo trutta), the killing proceeding at a faster rate with blood material from the latter fish species. The lethal activity of na?ve plasma and immune sera was comparable. This was abolished after incubation at 45 degrees C for 30 min and by the addition of EDTA but not EGTA supplemented with Mg(2+), indicating that complement acting via the alternative pathway is responsible for the parasiticidal effect observed. Scanning electron micrographs showed varying degrees of surface disruption in larvae exposed to fish plasma, suggesting that complement acts by breaching the oncomiracidial tegument. Control (untreated) oncomiracidia showed no damage. Ultrastructural damage was more extensive in oncomiracidia exposed to brown trout plasma than to rainbow trout plasma for equal periods, suggesting that the complement cascade may be involved in mediating host susceptibility.     

Chaetotaxy of members of the Gyrodactylidae (Monogenea), with comments upon their systematic relationships with the Monopisthocotylea and Polyopisthocotylea

This study investigates intra- and inter-generic variation in the chaetotaxy pattern of members of the Gyrodactylidae and compares these data with those from other monogenean groups elucidated by other workers. The staining of surface sensillary structures has revealed areas of homology between the two genera Gyrodactylus and Ooegyrodactylus, which have markedly contrasting modes of reproduction. Although species specific maps have been elucidated for members of the genus Gyrodactylus, no evolutionary trends in their sensillary patterns could be determined. Members of the family Gyrodactylidae are shown to possess a cluster of 3 × 2 sensilla, similar in their size and arrangement to the post-ocular (spo) cluster of sensilla in polyopisthocotyleans, although on the opposite surface. A comparable cluster of sensilla is lacking in monopisthocotyleans. This information gathered from chaetotaxy supports the proposal that the order Gyrodactylidea is close to the polyopisthocotylean line of monogenean evolution.     

Phylogenetic relationships within the polyopisthocotylean monogeneans (Platyhelminthes) inferred from partial 28S rDNA sequences

Recent studies based on molecular data (18S rDNA and partial 28S rDNA) and morphology did not resolve a terminal polytomy within the Polyopisthocotylea. Here, we have used sequences from the full domain D2 of the 28S rDNA for 24 species (18 new sequences) with three phylogenetic methods, maximum parsimony, neighbour-joining and maximum likelihood, to infer the relationships among the Polyopisthocotylea. The analysis of the domain D2 of the 28S rDNA has been performed on two data sets. The first one, complete, included the Polystomatidae as the outgroup in order to infer general relationships, and the second one, reduced, excluded the Polystomatidae and the polyopisthocotylean parasites of chondrichthyans, but used the Mazocraeidae as the outgroup in order to resolve the relationships between the terminal groups. The topology found, sustained by high bootstrap and decay index value, is: (outgroup (Chimaericolidae (Mazocraeidae (Gastrocotylinea, other Polyopisthocotylea)))). The polyopisthocotylean parasites of chondrichthyans are the sister-group of the polyopisthocotylean parasites of teleosts. In the latter, the Mazocraeidae, essentially parasites of Clupeidae, have a basal position. The polytomy between Gastrocotylinea, Discocotylinea and Microcotylinea is partially resolved in this study for the first time: the Gastrocotylinea are the sister-group of an unresolved group including the Microcotylinea, Discocotylinea and Plectanocotylidae. Inclusion of the Plectanocotylidae in the suborder Mazocraeinea is rejected. Monophyly of the Microcotylinea and Plectanocotylidae is confirmed, but monophyly of the Discocotylinea is questioned by the exclusion of Diplozoon.     

Phylogenetic systematic assessment of the Aspidobothrea (Platyhelminthes, Neodermata, Trematoda)

Phylogenetic systematic analysis of 20 aspidobothrean taxa using 33 transformation series based in comparative morphology yields three most parsimonious trees with a consistency index of 62%. The trees agree with familial-level relationships of (Rugogastridae (Stichocotylidae (Multicalycidae + Aspidogastridae))) supported by previous phylogenetic systematic assessments, which were based on only 10 transformation series. The analysis does not support completely the current subfamilial classification of the Aspidogastridae: both the Aspidobothriinae [as ( Aspidogaster + Lobatostoma )] and the Cotylaspinae [as Cotylogasteroides + Cotylogaster basiri (( Cotylaspis + Lissemysia ) ( Rohdella ( Lophotaspis ( Multicotyle + Sychnocotyle )))))] are supported as monophyletic groups. Recognizing Rohdellinae, however, would make the Cotylaspinae paraphyletic. The trees support a basal trichotomy of Cotylogaster michaelis + Aspidobothriinae + Cotylaspinae. Within the Aspidogastrinae, Aspidogaster conchicola , type species of the genus, is the sister group of all other species currently placed in the genus + Lobatosoma spp., rendering Aspidogaster paraphyletic.