Scott C. Cutmore

Molecular approaches to trematode systematics: 'best practice' and implications for future study.

To date, morphological analysis has been the cornerstone to trematode systematics. However, since the late-1980s we have seen an increased integration of genetic data to overcome problems encountered when morphological data are considered in isolation. Here, we provide advice regarding the ‘best molecular practice’ for trematode taxonomy and systematic studies, in an attempt to help unify the field and provide a solid foundation to underpin future work. Emphasis is placed on defining the study goals and recommendations are made regarding sample preservation, extraction methods, and the submission of molecular vouchers. We advocate generating sequence data from all parasite species/host species/geographic location combinations and stress the importance of selecting two independently evolving loci (one ribosomal and one mitochondrial marker). We recommend that loci should be chosen to provide genetic variation suitable to address the question at hand and for which sufficient ‘useful’ comparative sequence data already exist. Quality control of the molecular data via using proof-reading Taq polymerase, sequencing PCR amplicons using both forward and reverse primers, ensuring that a minimum of 85% overlap exists when constructing consensus sequences, and checking electropherograms by eye is stressed. We advise that all genetic results are best interpreted using a holistic biological approach, which considers morphology, host identity, collection locality, and ecology. Finally, we consider what advances next-generation sequencing holds for trematode taxonomy and systematics.

Trematodes of fishes of the Indo-west Pacific: told and untold richness

The Indo-west Pacific is a marine bioregion stretching from the east coast of Africa to Hawaii, French Polynesia and Easter Island. An assessment of the literature from the region found reports of 2,582 trematode species infecting 1,485 fish species. Reports are concentrated in larger fishes, undoubtedly reflecting the tendency for larger hosts to be infected by more species of parasites as well as a collecting bias. Many hundreds of fish species, including many from families known to be rich in trematodes, have yet to be reported as hosts. Despite some areas (the Great Barrier Reef, Hawaii and the waters off China, India and Japan) receiving sustained attention, none can be considered to be comprehensively known. Several regions, most importantly in East Africa, French Polynesia and the Coral Triangle, are especially poorly known. The fauna of the Indo-west Pacific has been reported so unevenly that we consider it impossible to predict the true trematode richness for the region. We conclude that the greatest gap in our understanding is of the geographical distribution of species in the Indo-west Pacific. This is highlighted by the fact that 87% of trematodes in the region have been reported no more than five times. The reliable recognition of species is a major problem in this field; molecular approaches offer prospects for resolution of species identification but have been little adopted to date.

Trematodes of the Great Barrier Reef, Australia: emerging patterns of diversity and richness in coral reef fishes

The Great Barrier Reef holds the richest array of marine life found anywhere in Australia, including a diverse and fascinating parasite fauna. Members of one group, the trematodes, occur as sexually mature adult worms in almost all Great Barrier Reef bony fish species. Although the first reports of these parasites were made 100 years ago, the fauna has been studied systematically for only the last 25 years. When the fauna was last reviewed in 1994 there were 94 species known from the Great Barrier Reef and it was predicted that there might be 2,270 in total. There are now 326 species reported for the region, suggesting that we are in a much improved position to make an accurate prediction of true trematode richness. Here we review the current state of knowledge of the fauna and the ways in which our understanding of this fascinating group is changing. Our best estimate of the true richness is now a range, 1,100-1,800 species. However there remains considerable scope for even these figures to be incorrect given that fewer than one-third of the fish species of the region have been examined for trematodes. Our goal is a comprehensive characterisation of this fauna, and we outline what work needs to be done to achieve this and discuss whether this goal is practically achievable or philosophically justifiable.

Phylogenetic relationships of the Gorgoderidae (Platyhelminthes: Trematoda), including the proposal of a new subfamily (Degeneriinae n. subfam.)

Phylogenetic analyses of a range of gorgoderid trematodes based on ITS2 and partial 28S rDNA data lead us to propose the Degeneriinae n. subfam. for the genus Degeneria in recognition of its phylogenetic isolation and distinctive morphology and biology. The current concepts of the subfamilies Anaporrhutinae and Gorgoderinae were supported. Within the Gorgoderinae, the large genus Phyllodistomum is shown to be paraphyletic relative to Pseudophyllodistomum and Xystretrum. Notably, the clade of marine Phyllodistomum does not form a clade with the other marine genus, Xystretrum. Distinct clades within the Gorgoderinae correspond variously to identity of first intermediate host, form of cercaria and their marine or freshwater habitat. We are not yet in a position to propose separate genera for these clades.

The ghost of parasites past: eggs of the blood fluke Cardicola chaetodontis (Aporocotylidae) trapped in the heart and gills of butterflyfishes (Perciformes: Chaetodontidae) of the Great Barrier Reef

We explored the distribution of Cardicola chaetodontis in chaetodontid fishes from the Great Barrier Reef. We found just four infections of adult worms in 238 individuals of 26 chaetodontid species. By contrast, eggs were present in hearts of 75 fishes (31·5%) and 19 of 26 chaetodontid species (all Chaetodon species). In 10 cases eggs contained moving miracidia; all the others were dead and degenerating. Eggs were sought in the gills of 51 individual fish. There were 17 cases of eggs being present in gills while present in the heart, but also 13 cases where eggs were absent from gills but present in the heart, suggesting that eggs remain longer in heart tissue than in gills. ITS2 rDNA sequences from two adult worms and eggs extracted from gills of five fishes (all different species) were identical to previously reported sequences of C. chaetodontis except for a single base-pair difference in two samples. We conclude that aporocotylid eggs trapped in fish heart tissues may inform understanding of the distributions and host ranges of aporocotylids, especially where adult prevalence is low. The low host-specificity of C. chaetodontis contrasts with higher specificity of trematodes of chaetodontids that have trophic transmission.

Pseudobacciger cheneyae n. sp. (Digenea: Gymnophalloidea) from Weber’s chromis (Chromis weberi Fowler & Bean) (Perciformes: Pomacentridae) at Lizard Island, Great Barrier Reef, Australia

A new species of digenean, Pseudobacciger cheneyae n. sp., is described from the intestines of Weber’s chromis (Chromis weberi Fowler & Bean) from off Lizard Island, Great Barrier Reef, Australia. This species differs from the three described species of Pseudobacciger Nahhas & Cable, 1964 [P. cablei Madhavi, 1975, P. harengulae Yamaguti, 1938 and P. manteri Nahhas & Cable, 1964] in combinations of the size of the suckers and the length of the caeca. The host of the present species is a perciform (Family Pomacentridae) which contrasts with previous records of the genus which are almost exclusively from clupeiform fishes. The genus Pseudobacciger is presently recognised within the family Faustulidae (Poche, 1926) but phylogenetic analyses of 28S and ITS2 rDNA show that the new species bears no relationship to species of four other faustulid genera (Antorchis Linton, 1911, Bacciger Nicoll, 1924, Paradiscogaster Yamaguti, 1934 and Trigonocryptus Martin, 1958) but that instead it is nested within the Gymnophalloidea (Odhner, 1905) as sister to the Tandanicolidae (Johnston, 1927). This result suggests that the Faustulidae is polyphyletic.

Biogeography of tropical Indo-West Pacific parasites: A cryptic species of Transversotrema and evidence for rarity of Transversotrematidae (Trematoda) in French Polynesia

We sought transversotrematid trematodes from French Polynesian fishes by examining 304 individual scaled fishes of 53 species from seven families known to harbour the family elsewhere. A single species was found at two locations in the Tuamotus Archipelago on two species of Chaetodontidae (Chaetodon auriga and Chaetodon ephippium) and one species of Lutjanidae (Lutjanus gibbus). The species closely resembles Transversotrema borboleta Hunter & Cribb, 2012 from chaetodontids and lutjanids of the northern Great Barrier Reef (GBR) but differs from it consistently in 8 base positions of ITS2 rDNA. This level of variation exceeds that between some clearly morphologically distinct pairs of species of Transversotrema and the form from French Polynesia is thus interpreted as a distinct, though cryptic, species and named Transversotrema polynesiae n. sp. The new species forms part of a complex of species, here characterised as the T. borboleta complex, associated with chaetodontids and lutjanids in the tropical Indo-West Pacific. Most of the putative species within this complex are yet to be described. Comparison of identical numbers of matched samples of fishes from French Polynesia, Heron Island (southern GBR) and Lizard Island (northern GBR) revealed 1, 4 and 10 species of Transversotrema respectively suggesting that the French Polynesian fauna is depauperate for this family. In addition to those species apparently missing from suitable hosts in French Polynesia, several species from further west infect fishes (especially Nemipteridae) that are themselves absent from French Polynesia. This dramatic east-west decline in richness contrasts strongly with what is known for monogeneans, which appear to maintain their richness over the same scale, and is more precipitate than is known for other groups of trematodes. The decline might be explained in part by the absence of the as yet unknown first intermediate hosts in French Polynesia. However, we predict that it is explained by other life cycle traits. We hypothesise that the characters of large short-lived cercariae, short-lived miracidia, the absence in the life-cycle of second intermediate hosts that are capable of transporting the species, and definitive and first intermediate hosts that have limited vagility combine to give marine Transversotrematidae limited dispersal capacity and a propensity for localised speciation.

A ray of venom: Combined proteomic and transcriptomic investigation of fish venom composition using barb tissue from the blue-spotted stingray (Neotrygon kuhlii)

UNLABELLED Fish venoms remain almost completely unstudied despite the large number of species. In part this is due to the inherent nature of fish venoms, in that they are highly sensitive to heat, pH, lyophilisation, storage and repeated freeze-thawing. They are also heavily contaminated with mucus, which makes proteomic study difficult. Here we describe a novel protein-handling protocol to remove mucus contamination, utilising ammonium sulphate and acetone precipitation. We validated this approach using barb venom gland tissue protein extract from the blue-spotted stingray Neotrygon kuhlii. We analysed the protein extract using 1D and 2D gels with LC-MS/MS sequencing. Protein annotation was underpinned by a venom gland transcriptome. The composition of our N. kuhlii venom sample revealed a variety of protein types that are completely novel to animal venom systems. Notably, none of the detected proteins exhibited similarity to the few toxin components previously characterised from fish venoms, including those found in other stingrays. Putative venom toxins identified here included cystatin, peroxiredoxin and galectin. Our study represents the first combined survey of gene and protein composition from the venom apparatus of any fish and our novel protein handling method will aid the future characterisation of toxins from other unstudied venomous fish lineages. BIOLOGICAL SIGNIFICANCE These results show an efficient manner for removing mucus from fish venoms. These results are the first insights into the evolution of proteins present on stingrayvenom barbs.

Three new species of blood flukes (Digenea: Aporocotylidae) infecting pufferfishes (Teleostei: Tetraodontidae) from off Bali, Indonesia.

We describe three new species of blood flukes (Aporocotylidae) and propose their classification within the genus Psettarium Goto & Ozaki, 1929. All three species were collected from the circulatory systems of pufferfishes caught off Bali, central Indonesia. Psettarium pulchellum n. sp. was found in the gills of both the narrow-lined puffer (Arothron manilensis de Procé) and the spiny blaasop (Tylerius spinosissimus Regan), while P. ogawai n. sp. and P. jimbaranense n. sp. were found in the gills of the reticulated puffer (Arothron reticularis Bloch & Schneider). The morphological characteristics of these taxa necessitated emendation of the diagnosis for the genus Psettarium, to accommodate the presence of an oral sucker, multiple or entirely post-caecal testes and a degenerate posterior testis. Features such as proportion of body length occupied by the oesophagus, and posterior caeca being ≥7× the length of anterior caeca, are no longer regarded as useful genus-level characters. Additionally, Sasala nolani is reassigned to this genus as Psettarium nolani n. comb. In phylogenetic analyses of the 28S and ITS2 rDNA regions, all three new taxa form a well-supported clade, together with Psettarium sinense and Psettarium nolani n. comb., the two other species of tetraodontid-infecting aporocotylids for which comparative rDNA data were available. The short branch lengths within this clade, despite dramatic morphological differences between the five species, suggest that rapid morphological diversification has occurred among the tetraodontid-infecting aporocotylids. The genus Psettarium has long been considered problematic. Further commentary is given on the history of this genus and how the issues presented might be resolved.

Staphylorchis cymatodes (Gorgoderidae: Anaporrhutinae) from carcharhiniform, orectolobiform and myliobatiform elasmobranchs of Australasia: low host specificity, wide distribution and morphological plasticity.

Anaporrhutine gorgoderids (Digenea: Gorgoderidae: Anaporrhutinae) found in the body cavity of six species of elasmobranchs from the orders Carcharhiniformes, Myliobatiformes and Orectolobiformes from Australian waters were found to belong to the genus Staphylorchis. Although these specimens were morphologically variable, sequences of ITS2 and 28S ribosomal DNA from specimens from three host families and two host orders were identical. Based on morphological and molecular data these specimens were identified as the type-species of the genus, Staphylorchis cymatodes. New measurements are provided for S. cymatodes, and for the first time genetic data are presented for this species. In addition to providing new morphological and molecular data for S. cymatodes, the previously described species S. gigas, S. parisi and S. scoliodonii, are here synonymised with S. cymatodes. This implies that S. cymatodes, as conceived here, has remarkably low host-specificity, being recorded from eight elasmobranch species from four families and three orders, has a wide geographical distribution in the Indo-west Pacific from off India, in the Bay of Bengal, to Moreton Bay in the Coral Sea, and is morphologically plastic, with body size, size of specific organs and body shape differing dramatically between specimens from different host species. The genus Staphylorchis now contains only two valid species, S. cymatodes and S. pacifica.