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.

Nucleolar localization of aprataxin is dependent on interaction with nucleolin and on active ribosomal DNA transcription

The APTX gene, mutated in patients with the neurological disorder ataxia with oculomotor apraxia type 1 (AOA1), encodes a novel protein aprataxin. We describe here, the interaction and interdependence between aprataxin and several nucleolar proteins, including nucleolin, nucleophosmin and upstream binding factor-1 (UBF-1), involved in ribosomal RNA (rRNA) synthesis and cellular stress signalling. Interaction between aprataxin and nucleolin occurred through their respective N-terminal regions. In AOA1 cells lacking aprataxin, the stability of nucleolin was significantly reduced. On the other hand, down-regulation of nucleolin by RNA interference did not affect aprataxin protein levels but abolished its nucleolar localization suggesting that the interaction with nucleolin is involved in its nucleolar targeting. GFP-aprataxin fusion protein co-localized with nucleolin, nucleophosmin and UBF-1 in nucleoli and inhibition of ribosomal DNA transcription altered the distribution of aprataxin in the nucleolus, suggesting that the nature of the nucleolar localization of aprataxin is also dependent on ongoing rRNA synthesis. In vivo rRNA synthesis analysis showed only a minor decrease in AOA1 cells when compared with controls cells. These results demonstrate a cross-dependence between aprataxin and nucleolin in the nucleolus and while aprataxin does not appear to be directly involved in rRNA synthesis its nucleolar localization is dependent on this synthesis.

Symbiont diversity within the widespread Scleractinian coral Plesiastrea versipore across the northwestern Pacific

The molecular diversity of symbiotic dinoflagellates associated with the widespread western Pacific coral Plesiastrea versipora was explored in order to examine if associations between reef-building corals and symbiotic dinoflagellates change with environment. Several ribosomal DNA genes with different evolutionary rates were used.. including the large subunit (28S), the 5.8S region and the internal transcribed spacers (ITS). The phylogenetic analysis of the 28S and 5.8S rDNA regions indicated that a single endosymbiont species, highly related to one of the species of Symbiodinium in clade C (=Synbiodinium goreaui, Trench et Blank), associates with P. versipora along the Ryukyu Archipelago. The persistence of the same endosymbiont within P. versipora across this wide array of latitudes may be a result of such features as the Kuroshio Current, which brings tropical temperatures as far north as Honshu, Japan. Analysis of the faster evolving ITS rDNA region revealed significant genetic variability within endosymbionts from different populations. This variation was due to a high degree of interpopulation variability, based on the proportion of pairwise variation detected among the populations (0.95% approximately). By comparison with other studies, the results also indicate that some ITS1 haplotypes from P. versipora endosymbionts seem to be widely distributed within the western Pacific Ocean, ranging from the Great Barrier Reef to the northeast of the China Sea.

The life cycle of Paracardicoloides yamagutii Martin, 1974 (Digenea: Sanguinicolidae)

The sanguinicolids Paracardicoloides yamagutii Martin, 1974 and Plethorchis acanthus Martin, 1975 were obtained from their definitive hosts, Anguilla reinhardtii Steindachner and Mugil cephalus Linnaeus (respectively) in the tributaries of the Brisbane River, Queensland, Australia. Two putative sanguinicolid cercariae were collected from a hydrobiid gastropod, Posticobia brazieri Smith, in the same waters. The two cercariae differ markedly in size and the form of their sporocysts. Both putative cercariae develop in the digestive gland of Po. brazieri. The ITS2 rDNA region from these sanguinicolids and a Clinostomum species (utilised as an outgroup due to the close morphological similarities between the cercarial stages of the Clinostomidae and the Sanguinicolidae) were sequenced and aligned. Comparison of the ITS2 sequences showed one cercaria to be that of P. yamagutii. This is the first sanguinicolid life history determined by a molecular method. P. yamagutii is the fourth sanguinicolid known to utilise a freshwater hydrobiid gastropod as its intermediate host. ITS2 rDNA is effective in distinguishing sanguinicolids at the species level.

Two new blood flukes (Digenea: Sanguinicolidae) from epinephelinae (Perciformes: Serranidae) of the Pacific Ocean

Pearsonellum pygmaeus n. sp. is described from Cromileptes altivelis (Serranidae), the Barramundi Cod, from Heron Island (southern Great Barrier Reef) and Lizard Island (northern Great Bat-Her Reef). This new species differs from Pearsonellum eorventum (type and only species) in the combination of smaller overall body size, the relative distance of the brain from the anterior end, the relative lengths of both the oesophagus and the testis, the degree to which the testis extends outside the intercaecal field, the shape of the testis, the shape and size of the ovary and the extent to which the uterzus loops around the ovary. There are in addition, 20 base pair differences between the ITS2 rDNA sequence of P. pygmaeus n. sp. and that of P corventum. Three new host records for P. corventum are reported. Adelomyllos teenae n. g., n. sp. is described from Epinephelus coioides (Serranidae), the Estuary Cod, from Moreton Bay, southeast Queensland. The new genus differs from the 22 other sanguinicolid genera in the combined possession of two testes, a cirrus-sac, separate genital pores, a post-ovarian uterus and an H-shaped intestine. A. teenae n. sp. is the third sanguinicolid described from the Epinephelinae. Sanguinicolids have now been reported from 11 species of Serranidae. (C) 2004 Elsevier Ireland Ltd. All rights reserved.

A preliminary phylogenetic analysis of the Capsalidae (Platyhelminthes : Monogenea : Monopisthocotylea) inferred from large subunit rDNA sequences

Phylogenetic relationships within the Capsalidae (Monogenea) were examined Using large subunit ribosomal DNA sequences from 17 capsalid species (representing 7 genera, 5 subfamilies), 2 outgroup taxa (Monocotylidae) plus Udonella caligorum (Udonellidae). Trees were constructed using maximum likelihood, minimum evolution and maximum parsimony algorithms. An initial tree, generated from sequences 315 bases long, Suggests that Capsalinae, Encotyllabinae, Entobdellinae and Trochopodinae are monophyletic, but that Benedeniinae is paraphyletic. Analyses indicate that Neobenedenia, currently in the Benedeniinae, should perhaps be placed in 2 separate subfamily. An additional analysis was made which omitted 3 capsalid taxa (for which only short sequences were available) and all outgroup taxa because of alignment difficulties. Sequence length increased to 693 bases and good branch support was achieved. The Benedeniinae was again paraphyletic. Higher-level classification of the Capsalidae, evolution of the Entobdellinae and issues of species identity in Neobenedenia are discussed.

Mutation scanning analysis of Marteilia sydneyi populations from different geographical locations in eastern Australia

Marteilia sydneyi (Paramyxea) is the causative agent of QX disease in oysters. In spite of the economic impact of this disease, its origin and the precise reason(s) for its apparent spread in Australian waters are not yet known. Given such knowledge gaps, investigating the population genetic structure(s) of M. sydneyi populations could provide insights into the epidemiology and ecology of the parasite and could assist in its prevention and control. In this study, single strand conformation polymorphism (SSCP)-based analysis of a region (195 bp) of the first internal transcribed spacer (ITS-1) of ribosomal DNA was employed to investigate genetic variation within and among five populations of M. sydneyi from oysters from five different locations in eastern Australia. The analysis showed the existence of a genetic variant of M. sydneyi common to the Great Sandy Strait, and the Richmond and Georges Rivers, as distinct from variants at the Pimpama and Clarence Rivers. Together with historical and other information relating to the QX disease outbreaks in eastern Australia, the molecular findings support the proposal that the parasite originated in the Great Sandy Strait and/or Richmond River and then extended southward along the coast. From a technical perspective, the study demonstrated the usefulness of SSCP as a tool to study the population genetics and epidemiology of M. sydneyi. (C) 2003 Elsevier Ltd. All rights reserved.

Systematics and evolution of ticks with a list of valid genus and species names

In recent years there has been much progress in our understanding of the phylogeny and evolution of ticks, in particular the hard ticks (Ixodidae). Indeed, a consensus about the phylogeny of the hard ticks has emerged which is quite different to the working hypothesis of 10 years ago. So that the classification reflects our knowledge of ticks, several changes to the nomenclature of ticks are imminent or have been made. One subfamily, the Hyalomminae, should be sunk, while another, the Bothriocrotoninae, has been created (Klompen, Dobson & Barker, 2002). Bothriocrotoninae, and its sole genus Bothriocroton, have been created to house an early-diverging ('basal') lineage of endemic Australian ticks that used to be in the genus Aponomma. The remaining species of the genus Aponomma have been moved to the genus Amblyomma. Thus, the name Aponomma is no longer a valid genus name. The genus Rhipicephalus is paraphyletic with respect to the genus Boophilus. Thus, the genus Boophilus has become a subgenus of the genus Rhipicephalus (Murrell & Barker, 2003). Knowledge of the phylogenetic relationships of ticks has also provided new insights into the evolution of ornateness and of their life cycles, and has allowed the historical zoogeography of ticks to be studied. Finally, we present a list of the 899 valid genus and species names of ticks as of February 2004.

Multiple origins of parasitism in lice: phylogenetic analysis of SSU rDNA indicates that the Phthiraptera and Psocoptera are not monophyletic

The Paraneoptera (Hemipteroid Assemblage) comprises the orders Thysanoptera (thrips), Hemiptera (bugs), Phthiraptera (lice) and Psocoptera (booklice and barklice). The phylogenetic relationships among the Psocodea (Phthiraptera and Psocoptera), Thysanoptera and Hemiptera are unresolved, as are some relationships within the Psocodea. Here, we present phylogenetic hypotheses inferred from SSU rDNA sequences; the most controversial of which is the apparent paraphyly of the Phthiraptera, which are parasites of birds and mammals, with respect to one family of Psocoptera, the Liposcelididae. The order Psocoptera and the suborder that contains the Liposcelididae, the Troctomorpha, are also paraphyletic. The two remaining psocopteran suborders, the Psocomorpha and the Trogiomorpha, are apparently monophyletic. The Liposcelididae is most closely related to lice from the suborder Amblycera. These results suggest that the taxonomy of the Psocodea needs revision. In addition, there are implications for the evolution of parasitism in insects; parasitism may have evolved twice in lice or have evolved once and been subsequently lost in the Liposcelididae.

Molecular characterisation, pathogenesis and fungicide sensitivity of Pythium spp. from table beet (Beta vulgaris var. vulgaris) grown in the Lockyer Valley, Queensland

Table beet production in the Lockyer Valley of south-eastern Queensland is known to be adversely affected by soilborne root disease from infection by Pythium spp. However, little is known regarding the species or genotypes that are the causal agents of both pre- and post-emergence damping off. Based on RFLP analysis with HhaI, HinfI and MboI of the PCR amplified ITS region DNA from soil and diseased plant samples, the majority of 130 Pythium isolates could be grouped into three genotypes, designated LVP A, LVP B and LVP C. These groups comprised 43, 41 and 7% of all isolates, respectively. Deoxyribonucleic acid sequence analysis of the ITS region indicated that LVP A was a strain of Pythium aphanidermatum, with greater than 99% similarity to the corresponding P. aphanidermatum sequences from the publicly accessible databases. The DNA sequences from LVP B and LVP C were most closely related to P. ultimum and P. dissotocum, respectively. Lower frequencies of other distinct isolates with unique RFLP patterns were also obtained with high levels of similarity (> 97%) to P. heterothallicum, P. periplocum and genotypes of P. ultimum other than LVP B. Inoculation trials of 1- and 4-week-old beet seedlings indicated that compared with isolates of the LVP B genotype, a higher frequency of LVP A isolates caused disease. Isolates with the LVP A, LVP B and LVP C genotypes were highly sensitive to the fungicide Ridomil MZ, which suppressed radial growth on V8 agar between approximately four and thirty fold at 5 mu g/mL metalaxyl and 40 mu g/mL mancozeb, a concentration far lower than the recommended field application rate.

Application of nox-restriction fragment length polymorphism for the differentiation of Brachyspira intestinal spirochetes isolated from pigs and poultry in Australia

Sixty-nine intestinal spirochetes isolated from pigs and poultry in eastern Australia were selected to evaluate the effectiveness of a species-specific PCR-based restriction fragment length polymorphism (RFLP) analysis of the Brachyspira nox gene. For comparative purposes, all isolates were subjected to species-specific PCRs for the pathogenic species Brachyspira hyodysenteriae and Brachyspira pilosicoli, and selected isolates were examined further by sequence analysis of the nox and 16S ribosomal RNA genes. Modifications to the original nox-RFLP method included direct inoculation of bacterial cells into the amplification mixture and purification of the PCR product, which further optimized the nox-RFLP for use in a veterinary diagnostic laboratory, producing sufficient product for both species identification and future comparisons. Although some novel profiles that prevented definitive identification were observed, the nox-RFLP method successfully classified 45 of 51 (88%) porcine and 15 of 18 (83%) avian isolates into 5 of the 6 recognized species of Brachyspira. This protocol represents a significant improvement over conventional methods currently used in veterinary diagnostic laboratories for rapid specific identification of Brachyspira spp. isolated from both pigs and poultry.

Digenean trematodes infecting the tropical abalone Haliotis asinina have species-specific cercarial emergence patterns that follow daily or semilunar spawning cycles

Approximately 1-2% of the tropical abalone Haliotis asinina inhabiting Heron Island Reef are infected with opecoelid digeneans. These largely inhabit the haemocoel surrounding the cerebral ganglia and digestive gland-gonad complex, and infected abalone typically have significantly reduced or ablated gonads. Observations of infected abalone reveal two distinct cercarial emergence patterns, one which correlates tightly with the abalone's highly regular and synchronous fortnightly spawning cycle, and the other which occurs in a circadian pattern. The former appears to be a novel emergence strategy not previously observed in digeneans. While the cercariae in all abalone are morphologically indistinguishable, comparison of sequences from the internal transcribed spacer 2 (ITS 2) region of the ribosomal DNA reveals a 5.7% difference between cercariae displaying different emergence patterns, indicating these are two distinct species that probably belong to the same genus. The ITS 2 sequences of the species with the daily emergence pattern are identical to that of an undescribed adult opecoelid from the gut of the barramundi cod, Cromileptes altivelis. Combined molecular, morphological and emergence data suggest that while these opecoelid cercariae use the same first intermediate host and are closely related species-members of the genus Allopodocotyle-they fill different ecological niches that are likely to include different definitive hosts.

Immunocytochemistry and metamorphic fate of the larval nervous system of Triphyllozoon mucronatum (Ectoprocta : Gymnolaemata : Cheilostomata)

The development of gymnolaemate Ectoprocta includes a larval stage of either the coronate or the cyphonautes type. Herein, we provide the first description of the larval neural anatomy of a coronate larva using immunocytochemical methods. We used antibodies against the neurotransmitters serotonin and FMRFamide and followed the fate of immunoreactive cells through metamorphosis. The larval serotonergic nervous system of Triphyllozoon mucronatum consists of an apical commissure, one pair of lateral axons, a coronate nerve net, an internal nerve mesh, and one pair of axons innervating the frontal organ. FMRFamide is only found in the larval commissure and in the lateral axons. The entire serotonergic and FMRFamidergic nervous system is lost during metamorphosis and the adult neural structures form independent of the larval ones. In the postlarval zooid, both neurotransmitters are detected in the cerebral commissure, in cell bodies located at the base of the lophophore, and in neurites connecting these somata to the cerebral commissure. These findings differ significantly from that observed in other lophotrochozoans, where certain larval neural features are either incorporated in the adult nervous system and/or have inductive functions during its ontogeny. The occurrence of a larval commissure and the lack of a serotonergic or FMRFamidergic apical organ in T. mucronatum are unique among lophotrochozoan larvae, which usually have a distinct apical organ containing serotonergic cells. Our data show that the larval neuroanatomy and the processes that underlie the reorganization of larval organ systems during metamorphosis may vary much more among lophotrochozoan taxa than previously thought.

An exceptionally rich complex of Sanguinicolidae von Graff, 1907 (Platyhelminthes : Trematoda) from Siganidae, Labridae and Mullidae (Teleostei : Perciformes) from the Indo-West Pacific Region

We describe an unprecedented radiation of sanguinicolid blood flukes ( Digenea: Sanguinicolidae) from two species of Labridae (Choerodon venustus and C. cauteroma), seven species of Mullidae (Mulloidichthys vanicolensis, Parupeneus barberinoides, P. barberinus, P. bifasciatus, P. cyclostomus, P. indicus and P. multifasciatus) and ten species of Siganidae (Siganus argenteus, S. corallinus, S. doliatus, S. fuscescens, S. lineatus, S. margaritiferus, S. puellus, S. punctatus, S. virgatus and S. vulpinus) from sites off Australia and Palau. The flukes were morphologically similar in having the combination of a long thread-like body, tegumental spines in lateral transverse rows, a vestigial oral sucker bearing concentric rows of fine spines, an H-shaped intestine, a cirrussac, a notch level with the male genital pore, a lateral or post-ovarian uterus, a uterine chamber and separate genital pores. These species are divided into two genera on the basis of testis number. Sanguinicolids from Siganus fuscescens have a single large testis between the intestinal bifurcation and the ovary and are placed in Ankistromeces Nolan & Cribb, 2004. Species from the remaining nine species of Siganidae, Labridae and Mullidae are placed in Phthinomita n. g.; these species have two testes, the anterior testis being large and between the intestinal bifurcation and the ovary whereas the small posterior testis is at the posterior end of the body and appears rudimentary or degenerate and probably non-functional. The second internal transcribed spacer (ITS2) of ribosomal DNA ( rDNA) from 29 host/parasite/location combinations (h/p/l) was sequenced together with that of Ankistromeces mariae Nolan & Cribb, 2004 for comparison. From 135 samples we found 19 distinct genotypes which were interpreted as representing at least that many species. Replicate sequences were obtained for 25 of 30 h/p/l combinations ( including A. mariae); there was no intraspecific variation between replicates sequences for any of these. Interspecific variation ranged from 1 - 41 base differences (0.3 - 12.7% sequence divergence). The 19 putative species were difficult to recognise by morphological examination. We describe 13 new species; we do not describe (= name) six species characterised solely by molecular sequences and three putative species for which morphological data is available but for which molecular data is not. We have neither morphological nor molecular data for sanguinicolids harboured in five hosts species ( Siganus margaritiferus, S. puellus, Choerodon cauteroma, Parupeneus indicus and P. multifasciatus) in which we have seen infections. Where host species were infected in different localities they almost always harboured distinct species. Some host species ( for example, S. argenteus and S. lineatus from Lizard Island) harboured two or three species in a single geographical location. This suggests that, for parts of this system, parasite speciation has outstripped host speciation. Distance analysis of ITS2 showed species from each host family ( Siganidae, Mullidae and Labridae) did not form monophyletic clades to the exclusion of species from other host families. However, a host defined clade was formed by the sequences from sanguinicolids from S. fuscescens.

Cardicola Short, 1953 and Braya n. gen. (Digenea : Sanguinicolidae) from five families of tropical Indo-Pacific fishes

A survey of Pacific coral reef fishes for sanguinicolids revealed that two species of Lutjanidae (Lutjanus argentimaculatus, L. bohar), six species of Siganidae (Siganus corallinus, S. fuscescens, S. lineatus, S. margaritiferus, S. punctatus, S. vulpinus), seven species of Chaetodontidae (Chaetodon aureofasciatus, C. citrinellus, C. flavirostris, C. lineolatus, C. reticulatus, C. ulietensis, C. unimaculatus), three species of Scombridae (Euthynnus affinis, Scomberomorus commerson, S. munroi) and three species of Scaridae (Chlorurus microrhinos, Scarus frenatus, S. ghobban) were infected with morphologically similar sanguinicolids. These flukes have a flat elliptical body, a vestigial oral sucker, a single testis, separate genital pores and a post-ovarian uterus. However, these species clearly belong in two genera based on the position of the testis and genital pores. Sanguinicolids from Lutjanidae, Siganidae, Chaetodontidae and Scombridae belong in Cardicola Short, 1953; the testis originates anteriorly to, or at the anterior end of, the intercaecal field and does not extend posteriorly to it, the male genital pore opens laterally to the sinistral lateral nerve chord and the female pore opens near the level of the ootype ( may be anterior, lateral or posterior to it) antero-dextral to the male pore. Those from Scaridae are placed in a new genus, Braya; the testis originates near the posterior end of the intercaecal field and extends posteriorly to it, the male pore opens medially at the posterior end of the body and the female pore opens posterior to the ootype, antero-sinistral to the male pore. The second internal transcribed spacer (ITS2) of ribosomal DNA from these sanguinicolids and a known species, Cardicola forsteri Cribb, Daintith & Munday, 2000, were sequenced, aligned and analysed to test the distinctness of the putative new species. Results from morphological comparisons and molecular analyses suggest the presence of 18 putative species; 11 are described on the basis of combined morphological and molecular data and seven are not because they are characterised solely by molecular sequences or to few morphological specimens (n= one). There was usually a correlation between levels of morphological and genetic distinction in that pairs of species with the greatest genetic separation were also the least morphologically similar. The exception in this regard was the combination of Cardicola tantabiddii n. sp. from S. fuscescens from Ningaloo Reef ( Western Australia) and Cardicola sp. 2 from the same host from Heron Island ( Great Barrier Reef). These two parasite/ host/location combinations had identical ITS2 sequences but appeared to differ morphologically ( however, this could simply be due to a lack of morphological material for Cardicola sp. 2). Only one putative species ( Cardicola sp. 1) was found in more than one location; most host species harboured distinct species in each geographical location surveyed ( for example, S. corallinus from Heron and Lizard Islands) and some ( for example, S. punctatus, S. fuscescens and Chlorurus microrhinos) harboured two species at a single location. Distance analysis of ITS2 showed that nine species from siganids, three from scombrids and five from scarids formed monophyletic clades to the exclusion of sanguinicolids from the other host families. Cardicola milleri n. sp. and C. chaetodontis Yamaguti, 1970 from lutjanids and chaetodontids, respectively, were the only representatives from those families that were sequenced. Within the clade formed by sanguinicolids from Siganidae there wasa further division of species; species from the morphologically similar S. fuscescens and S. margaritiferus formed a monophyletic group to the exclusion of sanguinicolids from all other siganid species.