Inference of phylogeny and taxonomy within the Didymozoidae (Digenea) from the second internal transcribed spacer (ITS2) of ribosomal DNA

DNA sequences of the second internal transcribed spacer (ITS2) of ribosomal DNA (rDNA) were determined for 11 species from four genera of Didymozoinae (Indodidymozoon, Helicodidymozoon, Rhopalotrema and Neometadidymozoon) and a species of the Lecithasteridae, Lecithaster stellatus. Sequences were used to test the validity of species recognised on morphological criteria and to infer phylogenetic relationships. Sequences of the 11 didymozoids differed by 0.5% to 19%. Our phylogenetic analyses: (i) indicate that species in the genera Helicodidymozoon and Rhopalotrema are a monophyletic group; (ii) support separation of the genus Helicodidymozoon from the genera Indodidymozoon and Neometadidymozoon; and (iii) support recognition of Rhopalotrema as a genus distinct from Neometadidymozoon. We found the gonochoristic species, I. pearsoni and I. suttiei, to be genetically similar to the hermaphroditic species in the genus Indodidymozoon and found no evidence to indicate that they belong in a separate genus.

Spermatogenesis and spermiogenesis in Didymocystis wedli Ariola, 1902 (Didymozoidae, Digenea)

The ultrastructure of the male reproductive system of Didymocystis wedli was studied for the first time, demonstrating spermiogenesis and spermatogenesis at different cell stages. The spermatozoa morphology was compared with that of other Digenea species. It was observed that the different cells of the spermatogenesis process follow the classic pattern reported for the majority of the parasitic platyhelminthes. During spermiogenesis, rootlet fibers, electrondense bodies and median cytoplasmic process were not observed. The mature spermatozoa of D. wedli were filiform, presenting nucleus, mitochondrion and two 9+1 axonemes, with a biflagellate distal extremity.

The use and implications of ribosomal DNA sequencing for the discrimination of digenean species

In just over a decade, the use of molecular approaches for the recognition of parasites has become commonplace. For trematodes, the internal transcribed spacer region of ribosomal DNA (ITS rDNA) has become the default region of choice. Here, we review the findings of 63 studies that report ITS rDNA sequence data for about 155 digenean species from 19 families, and then review the levels of variation that have been reported and how the variation has been interpreted. Overall, complete ITS sequences (or ITS1 or ITS2 regions alone) usually distinguish trematode species clearly, including combinations for which morphology gives ambiguous results. Closely related species may have few base differences and in at least one convincing case the ITS2 sequences of two good species are identical. In some cases, the ITS1 region gives greater resolution than the ITS2 because of the presence of variable repeat units that are generally lacking in the ITS2. Intraspecific variation is usually low and frequently apparently absent. Information on geographical variation of digeneans is limited but at least some of the reported variation probably reflects the presence of multiple species. Despite the accepted dogma that concerted evolution makes the individual representative of the entire species, a significant number of studies have reported at least some intraspecific variation. The significance of such variation is difficult to assess a posteriori, but it seems likely that identification and sequencing errors account for some of it and failure to recognise separate species may also be significant. Some reported variation clearly requires further analysis. The use of a yardstick to determine when separate species should be recognised is flawed. Instead, we argue that consistent genetic differences that are associated with consistent morphological or biological traits should be considered the marker for separate species. We propose a generalised approach to the use of rDNA to distinguish trematode species.

Report of Didymocystis wedli Ariola, 1902 (Digenea; Didymozoidae) from Thunnus albacares in Brazil

Didymocystis wedli a parasite from the gills of Thunnus albacares from the coast of the State of Rio de Janeiro, is described by use of light and scanning electron microscopy. This is the first report of this species in Brazil and South America. New data are presented on the surface topography as demonstrated by scanning electron microscopy.

First record in South America of Didymosulcus palati and Didymosulcus philobranchiarca (Digenea, Didymozoidae) with new hosts records and pathological alterations

Two species of Didymozoidae, Didymosulcus palati (Yamaguti 1970) and Didymosulcus philobranchiarca (Yamaguti 1970) were reported for the first time in South America, Atlantic Ocean, parasitizing three different tuna species from the coast of Rio de Janeiro, Brazil: Thunnus atlanticus (Lesson), Thunnus albacares (Bonnaterre) and Thunnus obesus (Lowe). Pairs of D. philobranchiarca were found on gill arches of T. albacares and T. obesus, in longitudinal rows of yellow cysts located inside grooves in the hard denticle palate (new site) of the three hosts species studied, and as disperse groups of cysts in the operculum (new site) and gill arches of T. atlanticus (new host record). D. palati occurred as disperse groups of encysted worm pairs in the gill arches of T. albacares and T. obesus and in gill arches and operculum of T. atlanticus (new host record). The pathological alterations induced by D. philobranchiarca in the palate of T. atlanticus are described for the first time. Original measurements and figures are presented.

Molecules and morphology in phylogenetic studies of the Hemiuroidea (Digenea : Trematoda : Platyhelminthes)

Phylogenies of trematodes based on characters derived from morphology and life cycles have been controversial. Here, we add molecular data to the phylogenetic study of a group of trematodes, members of the superfamily Hemiuroidea Looss, 1899. DNA sequences from the V4 domain of the nuclear small subunit (18S) rRNA gene and a matrix of morphological characters modified from a previous study were used. There was no significant incongruence between the molecular and the morphological data. However, this was probably due largely to the limited resolving power of the morphological data. Analyses support a monophyletic Hemiuroidea containing at least the families Accacoeliidae, Derogenidae, Didymozoidae, Hirudinellidae, Sclerodistomidae, Syncoeliidae, Isoparorchiidae, Lecithasteridae, and Hemiuridae. These families fall into two principal clades. One contains the first six families and the other the Hemiuridae and lecithasterine lecithasterids. The positions of the hysterolecithine lecithasterids and the Isoparorchiidae were poorly resolved. The Ptychogonimidae may be the sister group of the remaining Hemiuroidea, but there was no support from the molecular data for the placement of the Azygiidae within the superfamily. (C) 1998 Academic Press.

Lesions caused by cardiovascular flukes (Digenea : Spirorchidae) in stranded green turtles (Chelonia mydas)

Evidence of infection with spirorchid flukes (Digenea: Spirorchidae) was sought at necropsy of 96 stranded green turtles, Chelonia mydas, that were examined during the course of a survey of marine turtle mortality in southeastern Queensland, Australia. Three species of spirorchid (Hapalotrema mehrai, H. postorchis, and Neospirorchis schistosomatoides) were identified. Severe disease due to spirorchid fluke infection (spirorchidiasis) was implicated as the principal cause of mortality in 10 turtles (10%), and appeared to be one of multiple severe problems in an additional 29 turtles (30%). Although flukes were observed in only 45% of stranded C. mydas in this study, presumed spirorchid fluke infection was diagnosed in an additional 53% of turtles, based principally on characteristic necropsy lesions and to a lesser extent on the histopathological detection of spirorchid eggs. Characteristic necropsy lesions included miliary spirorchid egg granulomas, which were observed most readily on serosal surfaces, particularly of the small intestine. Cardiovascular lesions included mural endocarditis, arteritis, and thrombosis, frequently accompanied by aneurysm formation. Resolution of thrombi was observed to occur via a combination of granuloma formation about indigestible components (spirorchid fluke egg shells) and exteriorization through the vessel wall, which resulted in granulomatous nodules on the adventitial surface. Septic aortic thrombosis complicated by disseminated bacterial infection, observed in five turtles, was recorded for the first time. Egg granulomas were ubiquitous in turtle tissues throughout this study. Although they generally appeared to be mild or incidental lesions, they were occasionally associated with severe multifocal granulomatous pneumonia or meningitis.

Pholeohedra overstreeti n.g., n.sp. (Digenea : Haploporidae) from Girella zebra (Kyphosidae) in south Australia

Pholeohedra overstreeti n. g., n. sp. (Digenea: Haploporidae) is described from Girella zebra (Kyphosidae) in South Australia. The new genus is compared with all genera of Haploporidae sensu late (including Atractotrematidae, Megasolenidae and Waretrematidae) and has a unique bell-shaped concavity at its posterior end. The genus otherwise resembles Hapladena in the arrangement of the testis, vitellarium and gut but also resembles Megasolena, Metamegasolena and Vitellibaculum except in having a single testis. This is the first haploporid reported from kyphosid fishes in Australia.

Hapalotrema (Digenea : Spirorchidae) in the green turtle (Chelonia mydas) in Australia

Hapalotrema mehrai Rao, 1976 and Hapalotrema postorchis Rao, 1976 (Digenea: Spirorchidae) are redescribed from the heart and pulmonary arteries of the green turtle, Chelonia mydas, from Moreton Bay in south-eastern Queensland. Hapalotrema pambanensis Gupta and Mehrotra, 1981 from C. mydas in India is made a synonym of H. mehrai. Hapalotrema dorsopora Dailey, Fast and Balazs, 1993 from C. mydas from Hawaii was described with a dorsally opening uterine pore, but this is found to be the opening of Laurer's canal; therefore H. dorsopora is also made a synonym of H. mehrai. In addition to differences in the numbers of testes and general dimensions, H. mehrai and H. postorchis differ in the development of Laurer's canal and in the absence of a canalicular seminal receptacle in H. postorchis.

A phylogenetic study of Lepidapedoides Yamaguti, 1970 (Digenea) with a key and descriptions of two new species from western Australia

Two new species of the genus Lepidapedoides are described from the aulopodid teleost Aulopus purpurissatus from south-western Australia. Both are distinguished from other Lepidapedoides spp. by their pedunculate ventral sucker. Lepidapedoides pistoris n. sp. and L. elongatrium n. sp. are distinguished by the possession of a narrow, elongate form, a long ventral sucker to ovary distance: the vitellarium reaching only to the posterior level of the cirrus-sac, the cirrus-sac length and the deep genital atrium with the metraterm entering distally to its base in L. elongatrium. A key to species of the genus is given. A character matrix is included for the genus. Poorly resolved phylogenetic trees indicate two main lineages in the genus. The two new species described here are resolved as sister taxa. The new combination Lepidapedoides freitasi (Kohn gr Fernandes, 1970) is formed for Acanthocolpoides freitasi.

Digenetic trematodes of the genus Clinostomum Leidy, 1856 (Digenea : Clinostomidae) from birds of Queensland, Australia, including C. wilsoni n. sp. from Egretta intermedia

Two species of Clinostomum previously described from Australia, C. hornum from Botaurus poiciloptilus (Australian bittern) and Nycticorax caledonicus (Nankeen night heros) and C. australiense from Pelecanus conspicillatus (Australian pelican), which have previously been synonymised with C. complanatum, are redescribed and recognised as valid species. In addition, C. complanatum is recorded from Egretta alba (large egret), E. garzetta (little egret), E. intermedia (plumed egret), N. caledonicus and Ardea novaehollandiae (white-faced heron). C. wilsoni n. sp. is described from E. intermedia from Queensland. C. wilsoni differs from the other three species in size and shape of the body and in the oral collar, oral sucker, intestinal caeca, caecal diverticula and position of testes. Taxonomic problems within the genus Clinostomum are discussed.

Two new genera, Provitellus and Ovipusillus, and four new species of Monorchiidae (Digenea) from carangid fishes of queensland, Australia

Two new genera and four new species of monorchiid digeneans are described from the Great Barrier Reef and Moreton Bay, Queensland. Provitellus turrum n. g., n. sp. from Pseudocaranx dentex and Trachinotus coppingeri is characterised by the presence of vitelline follicles in the forebody, a single testis, a unipartite terminal organ and filamented eggs. Ovipusillus mayu n. g., n. sp. from Gnathanodon speciosus is characterised by the presence of two testes, vitelline follicles overlapping the ventral sucker and a large, complex cirrus-sac that contains a coiled eversible ejaculatory duct joined by the pars prostatica halfway along its length. Paramonorcheides pseudocaranxi n. sp. from Pseudocaranx dentex differs from other species described in this genus in the longer flatter forebody, entire ovary and the well-developed cirrus-sac. Chrisomon gaigai n. sp. from Trachinotus coppingeri and T botla is characterised by the unflattened forebody and transversely oval pharynx. Chrisomon is redefined to include species of Lasiotocus with a vitellarium composed of clusters of tubular acini, creating the following new combinations: C. albulae n. comb. for L. albulae Overstreet, 1969, C. ulua n. comb, for L. ulua Yamaguti, 1970 and C. weke n. comb, for L. weke Yamaguti, 1970. The diagnosis of Lasiotocus is amended accordingly and the new combinations, L. polynemi n. comb. and L. sunderbanensis n. comb., are created for C.polynemi Dutta, Hafeezullah & Manna, 1994 and C. sunderbanensis Dutta, Hafeezullah B Manna, 1994, respectively. Extrapolation of our collection data suggests that there may be as many as 80 species of monorchiids infecting carangid fishes in Australia and 180 species infecting carangids in all oceans of the world. The latter figure greatly exceeds the number of monorchiids described from all host families to date.

The diversity of the Digenea of Australian animals

The Digenea is one of five major helminth assemblages represented in Australian animals. History of the study of digeneans in Australia is reviewed briefly to show that it has never been subjected to the kind of sustained study needed to reach an understanding of it. The Australian vertebrate fauna comprises over 5500 species. These have so far been shown to harbour just over 70 families, about 306 genera and 566 species of digeneans. Digeneans occur in all classes of vertebrates in Australia but are distributed very unevenly; aquatic hosts are generally most heavily infected, but many terrestrial species are also infected. Particular weaknesses in knowledge of the fauna concern the bats, cetaceans and teleosts. Another weakness is in knowledge of life-cycles; representative life-cycles are known for only about 20 of the 70 families known in Australia. Estimates of the overall size of the fauna are dependent on an understanding of sampling strategies, the heterogeneity of distribution of the fauna, and the nature of host-specificity. These subjects are reviewed briefly and an estimate of the total fauna is made. There may be as many as 6000 species of digeneans in Australia. (C) 1998 Australian Society for Parasitology. Published by Elsevier Science Ltd.

A DNA-based demonstration of a three-host life-cycle for the Bivesiculidae (Platyhelminthes : Digenea)

Immature bivesiculid trematodes collected from the intestine of Thalassoma lunare (Labridae) are shown to be morphologically consistent with adults of Bivesicula claviformis from Epinephelus fasciatus (Serranidae). In addition, the immature bivesiculids have the same sequence for the second internal transcribed spacer of the ribosomal DNA. Comparison with three other species of Bivesiculidae showed differences of between 23% and 30%. These results show that bivesiculids may have three-host life-cycles in addition to the two-host life-cycles that have been demonstrated previously. The three-host life-cycle enables bivesiculids to infect large carnivorous fishes. (C) 1998 Australian Society for Parasitology. Published by Elsevier Science Ltd. All rights reserved.

Lepocreadiidae (Digenea) of Australian coastal fishes: new species of Opechona Looss, 1907, Lepotrema Ozaki, 1932 and Bianium Stunkard, 1930 and comments on other species reported for the first time or poorly known in Australian waters

Opechona austrobacillaris n, sp. is described from Pomatomus saltatrix from marine sites off Western Australia and New South Wales, Australia. It differs from O. bacillaris in its elongate outline, small ventral sucker, longer pseudoesophagus (relative to the oesophagus), relatively shorter ventral sucker to ovary distance and the relatively longer post-testicular region. Lepotrema monile n. sp. is described from Pomacentrus wardi from Heron Island, Queensland. It differs from its congeners in the sphincter around the distal metraterm and the more-or-less oval ovary. Bianium spongiosum n. sp, is described from Ostracion cubicus from Lizard Island, Queensland. It differs from its congeners in lacking lateral flaps in the forebody, but in having large, internal spongiform patches in the lateral forebody. The following species are redescribed from Australian sites: Lepocreadium oyabitcha from Abudefduf whitleyi, Lizard Island; Clavogalea trachinoti from Trachinotus botla, Heron Island and T. coppingeri, New South Wales, Stradbroke Island, Queensland and Heron Island; Myzoxenus insolens from Notolabrus parilus, Western Australia; Bulbocirrus aulostomi from Aulostomus chinensis, Heron Island; Lepocreadioides orientalis [new synonyms: Bicaudum interruptum Bilqees, 1973; Lepocreadioides interruptum (Bilqees, 1973) Madhavi, Narasimhulu & Shameem, 1986; Lepocreadioides discum Wang, 1986; Lepocreadioides sp. of Karyakarte & Yadav (1976)] from Cynoglossus bilineata, Moreton Bay, Queensland; Hypocreadium patellare from Sufflamen chrysopterus, Heron Island; Echeneidocoelium indicum from Echeneis naucrates, Heron Island; Multitestis pyriformis from Epinephelus cyanopodus, Heron Island; Pseudopisthogonoporus vitellosus from Naso brevirostris, Heron Island; and Bianium hispidum from Torquigener whitleyi and T. pleurogramma, southern Queensland. Only M. solens and M. pyriformis have been reported from Australian waters before; both are new host records.