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.

Desenvolvimento do sistema reprodutivo de Echinostoma paraensei (Trematoda: Digenea) de hamsters (Mesocricetus auratus) experimentalmente infectados, analisado por microscopia de luz de campo e microscopia laser confocal

O conhecimento da morfologia e ultraestrutura dos helmintos permite a correta classificação destes organismos, bem como fornece subsídios que poderão ser utilizados para diagnóstico e controle. A microscopia laser confocal é uma ferramenta para estudar a organização estrutural de várias espécies de helmintos, possibilitando acesso a detalhes morfológicos não evidenciados pela microscopia óptica. Echinostoma paraensei é um trematódeo, digenético, hermafrodita parasito de numerosos hospedeiros vertebrados. Neste trabalho foi investigado o desenvolvimento dos órgãos reprodutivo e a morfometria de E. paraensei, desde a fase jovem até a adulta, como contribuição ao conhecimento do desenvolvimento reprodutivo desta espécie. Os trematódeos foram recuperados aos 3, 4, 5, 6, 7, 10, 14 e 21 dias posterior à infecção (dpi) experimental em hamsters. Estes foram corados em carmim clorídrico, desidratados em série alcoólica e montados em lâmina permanente em bálsamo do Canadá, fotografados e medidos usando microscopia de luz de campo claro (MCC) e microscopia de varredura laser confocal (MVLC). Entre 3 e 4 dpi, os primórdios genitais estavam presentes e nenhuma organização do sistema reprodutivo foi visualizada por MCC e MVLC. Os primórdio do ovário, dos testículos e da bolsa do cirro foram visualizados por MCC aos 5 e 6 dpi, no entanto, MVLC dos helmintos aos 5dpi mostra que estes primórdios, o ootipo e o útero estavam presentes, como estruturas individualizadas. A bolsa do cirro apresenta metratermo e o ovário com primórdio do oótipo adjacente aos 7dpi por MVLC. A vesícula seminal, receptáculo seminal, células diferenciadas nos testículos, ducto e reservatório vitelínico e oviducto foram visualizados após 10 dias, enquanto os espermatozóides na vesícula seminal, ovos e oócitos, células vitelínicas, poro e canal de Laurer aos 14 dias. A morfometria evidencia um acelerado crescimento dos órgãos reprodutores a partir do 7 dia. Os testículos apresentam aumento significativo no comprimento do 7 ao 21 dia e o ovário durante o período de 7 à 10 dpi. Aos 21 dpi, todos os helmintos apresentaram glândulas vitelínicas, útero contendo ovos e espermatozóides no oviducto enquanto outros ovos estão sendo formados. As mudanças morfológicas acentuadas durante a gametogênese consistem no aumento do comprimento do helminto, maturação das gônadas, desenvolvimento e maturação das glândulas vitelínicas. O desenvolvimento do helminto como um todo está relacionado à maturação dos órgãos reprodutivo masculino e feminino indicando o investimento deste trematódeo em garantir a produção e eliminação dos ovos ao meio exterior.

A DNA-sequence based phylogeny for triculine snails (Gastropoda : Pomatiopsidae : Triculinae), intermediate hosts for Schistosoma (Trematoda : Digenea): phylogeography and the origin of Neotricula

Partial (DNA) sequences were examined for one nuclear (28S rRNA gene) and one mitochondrial (16S rRNA) locus for nine species of pomatiopsid snail (Gastropoda: Rissooidea: Pomatiopsidae) from south-east Asia and south-west China. Fresh field samples were

中华双腔吸虫（Dicrocoelium Chinensis Tang et Tang，1978）（Trematoda：Digenea）卵子发生的超微结构研究

利用透射电镜技术观察了中华双腔吸虫（ＤｉｃｒｏｃｏｅｌｉｕｍｃｈｉｎｅｎｓｉｓＴａｎｇｅｔＴａｎｇ，１９７８）的卵巢结构和卵子发生过程，同时提出了划分复殖吸虫卵子发生过程的标准，总结了复殖吸虫卵子发生的一般规律以及中华双腔吸虫卵子发生的特有特征。此外比较分析了不同复殖吸虫的皮质颗粒特征，指出它在分类上可能有重要意义。中华双腔吸虫卵巢和卵子发生的超微结构与大多数复殖吸虫相似，但其卵巢中的各生殖细胞间有网状片层结构及一些颗粒、膜状碎片等支持和营养结构，还有退化的成熟卵母细胞。中华双腔吸虫的卵子发生分为卵原细胞

A new species of Allocreadium (Trematoda : Allocreadiidae) from freshwater fishes in the Danjiangkou Reservoir in China

A new species of Allocreadium, Allocreadium danjiangensis n. sp., is described from the intestine of several species of freshwater fish, including Abbottina rivularis (Basilewsky, 1855), Sarcocheilichthys nigripinnis nigripinns (Gunther, 1873), Gnathopogon argentatus (Sauvage et Dabry 1874), Opsariichthys uncirostris bidens (Gunther, 1873), and Erythroculter mongolicus mongolicus (Basilewsky, 1855) (Cyprinidae) from the Danjiangkou Reservoir in central China. The main morphological characters of the new species are as follows: vitelline follicles numerous, extending from the level of acetabulum to posterior extremity, distributed over both sides around the ceca; cirrus sac relatively large, developed, lying obliquely anterior to the acetabulum, extending from the level of the intestinal bifurcation to the central level of acetabulum, and overlapping left or right cecal; and ovary much smaller than testes, generally close to or even overlapping the anterior border of anterior testis. Observation by scanning electron microscopy shows only 2 kinds of tegumental formations, i.e., papillae and tubercles, instead of 3 types of tegumental formations, i.e., papillae, bosses, and minute sensor receptors observed on other species of the Allocreadiidae. The tegumental striations of the present species vary on the different parts of the body. In addition, a new structure, identified as the "groove" with a tonguelike tubercle, was observed on the inner wall of acetabulum.

Seasonal dynamics and distribution of the digenean Phyllodistomum pawlovskii (Trematoda : Gorgoderidae) in the bullhead catfish, Pseudobagrus fulvidraco, in a lake of China

Seasonal population dynamics of the digenean Phyllodistomum pawlovskii in the urinary bladder of the bullhead catfish, Pseudobagrus fulvidraco, were investigated in Liangzi Lake in the flood plain of the Yangtze River in China from February 2001 to July 2002. The overall prevalence of the parasite was high, 41.5% (n = 1,476), while the mean abundance was relatively low, 1.24 +/- 2.11. The parasite exhibited evident seasonality in changes of prevalence and abundance. In brief, prevalence and abundance were very low in midwinter (January), but increased and remained relatively high in other seasons and months. The distribution pattern of this parasite in the fish was overdispersed, with a variance to mean ratio > 1, but its frequency distribution could not be described by the negative binomial model. There were positive correlations between the number of the parasites per fish and the age and length of the fish; a peaked age-parasite abundance curve was not detected in the parasite-host association. It is suggested that the parasite P. pawlovskii has little effect on the population structure of the bullhead catfish.

Observations on two species of phyllodistomes (Trematoda : Gorgoderidae) from fishes of the Hubei Province, central China, with an erection of Neophyllodistomum gen. n.

Two rare trematode species so far reported in the genus Phyllodistomum Braun, 1899, P. pawlovskii (Zmeev, 1936) and P. serrispatula Chin, 1963, were found in the urinary system the yellow catfish Pelteobagrus fulvidraco (Richardson) of Bao'an Lake (prevalence 8 %) and the swamp eel Monopterus albus (Zouiev) of Liangzi Lake (prevalence 6 9/6), respectively, from the Hubei Province, central China. In contrast to the original description, P. pawlovskii showed a considerable morphological variability particularly in the shape, size and topography of testes. A unique morphological feature of P. serrispatula is the presence of many conspicuous lateral outgrowths on the hindbody and, principally based on this character, a new genus Neophyl-lodistomum is erected (type species N. serrispatula (Chin, 1963) comb. n.)) to accommodate this species. Both trematode species are briefly redescribed. P. pawlovskii and N. serrispatula are reported for the first time from the Hubei Province and the former from the Yangtze River drainage system.

Gross anatomy of the muscle systems and associated innervation of Apatemon cobitidis proterorhini metacercaria (Trematoda: Strigeidea), as visualized by confocal microscopy.:Strigeidea), as visualized by confocal microscopy

The major muscle systems of the metacercaria of the strigeid trematode, Apatemon cobitidis proterorhini have been examined using phalloidin as a site-specific probe for filamentous actin. Regional differences were evident in the organization of the body wall musculature of the forebody and hindbody, the former comprising outer circular, intermediate longitudinal and inner diagonal fibres, the latter having the inner diagonal fibres replaced with an extra layer of more widely spaced circular muscle. Three orientations of muscle fibres (equatorial, meridional, radial) were discernible in the oral sucker, acetabulum and paired lappets. Large longitudinal extensor and flexor muscles project into the hindbody where they connect to the body wall or end blindly. Innervation to the muscle systems of Apatemon was examined by immunocytochemistry, using antibodies to known myoactive substances: the flatworm FMRFamide-related neuropeptide (FaRP), GYIRFamide, and the biogenic amine, 5-hydroxytryptamine (5-HT). Strong immunostaining for both peptidergic and serotoninergic components was found in the central nervous system and confocal microscopic mapping of the distribution of these neuroactive substances revealed they occupied separate neuronal pathways. In the peripheral nervous system, GYIRFamide-immunoreactivity was extensive and, in particular, associated with the innervation of all attachment structures; serotoninergic fibres, on the other hand, were localized to the oral sucker and pharynx and to regions along the anterior margins of the forebody.

The musculature and associated innervation of adult and intramolluscan stages of Echinostoma caproni (Trematoda) visualised by confocal microscopy

Gross anatomy of muscle and sensory/motor innervation of adult and intramolluscan developmental stages of Echinostoma caproni have been investigated to ascertain the organisation and the functional correlates of any stage-specific patterns of staining. Using indirect immunocytochemistry to demonstrate neuroactive substances and the phalloidin-fluorescence technique for staining myofibril F-actin, the muscle systems and aminergic and peptidergic innervation of daughter rediae, cercariae, metacercariae, and pre- and post-ovigerous adults were examined and compared using confocal scanning laser microscopy. A complex arrangement of specific muscle fibre systems occurs within the body wall (composed of circular, longitudinal and diagonal fibres), suckers (radial, equatorial, meridional), pharynx (radial, circular), gut caeca (mainly circular), cercarial tail (circular, pseudo-striated longitudinal), and ducts of the reproductive system (circular, longitudinal), presumed to serve locomotor, adhesive, alimentary and reproductive functions. Immunostaining for serotonin (5-HT) and FMRFamide-related peptides (FaRPs) was evident throughout the central (CNS) and peripheral (PNS) nervous systems of all stages, and use of dual-labelling techniques demonstrated separate neuronal pathways for 5-HT and FaRP in both CNS and PNS. FaRP expression in the innervation of the ootype wall was demonstrated only in post-ovigerous worms and not in pre-ovigerous worms, suggesting an involvement of FaRP neuropeptides in the process of egg assembly. Comparison of the present findings with those recorded for other digeneans suggests that muscle organisation and innervation patterns in trematodes are highly conserved.

Neuroactive substances and associated major muscle systems in Bucephaloides gracilescens (Trematoda : Digenea) metacercaria and adult

Cholinergic, serotoninergic and neuropeptidergic components of the nervous system were examined and compared in the progenetic metacercaria and adult gasterostome trematode, Bucephaloides gracilescens in order to provide baseline information on neuronal control of the musculature involved in egg-assembly. Enzyme cytochemistry and indirect immunocytochemical techniques interfaced with confocal scanning laser microscopy demonstrated all three classes of neuroactive substance throughout the central and peripheral nervous systems. A comparable orthogonal arrangement of the central nervous system (CNS) and peripheral array of nerve plexuses was observed in both metacercaria and adult. Staining patterns for cholinergic and peptidergic substances showed significant overlap, while the serotoninergic system was confined to a separate set of neurons. Immunostaining for FMRFamide-related peptides (FaRPs) was strong in the CNS and peripheral innervation to the attachment apparatus of metacercaria and adult but was only found in the innervation of the ootype in ovigerous adults, implicating FaRPs in neuronal control of the muscle of the female reproductive tract during egg-assembly.

Multiple origins of European populations of giant liver fluke Fascioloides magna (Trematoda: Fasciolidae), an invasive liver parasite of ruminants

The giant liver fluke, Fascioloides magna, liver parasite of free-living and domestic ruminants of Europe and North America, was analysed in order to determine the origin of European populations and to reveal the biogeography of this originally North American parasite on the European continent. The previously selected variable fragments of the mitochondrial cytochrome c oxidase subunit I (cox1; 384 bp) and nicotinamid dehydrogenase subunit I (nad1; 405 bp) were applied as a tool. The phylogenetic trees and haplotype networks were constructed and the level of genetic structuring was evaluated using population genetic tools. In F. magna individuals originating from all European natural foci (Italy, Czech Republic, Danube floodplain forests) and from four of five major North American enzootic areas, 16 cox1 and 18 nad1 haplotypes were determined. The concatenated sequence set produced 22 distinct haplotypes. The European fluke populations were less diverse than those from North America in that they contained proportionately fewer haplotypes (8), while more substantial level of genetic diversity and higher number of haplotypes (15) were recorded in North America. Only one haplotype was shared between the European (Italy) and North American (USA/Oregon and Canada/Alberta) flukes supporting a western North American origin of the Italian F. magna population. Haplotypes found in Italy were distinct from those determined in the remaining European localities what indicates that introduction of F. magna onto the European continent is a result of more than one event. In Czech focus, a south-eastern US origin of giant liver fluke was revealed. Identical haplotypes, common for parasites from Czech Republic and from expanding focus of Danube floodplain forests, implies introduction of F. magna to the Danube region from an already established Czech focus.