Cestode genomics - progress and prospects for advancing basic and applied aspects of flatworm biology.

Characterization of the first tapeworm genome, Echinococcus multilocularis, is now nearly complete, and genome assemblies of E. granulosus, Taenia solium and Hymenolepis microstoma are in advanced draft versions. These initiatives herald the beginning of a genomic era in cestodology and underpin a diverse set of research agendas targeting both basic and applied aspects of tapeworm biology. We discuss the progress in the genomics of these species, provide insights into the presence and composition of immunologically relevant gene families, including the antigen B- and EG95/45W families, and discuss chemogenomic approaches toward the development of novel chemotherapeutics against cestode diseases. In addition, we discuss the evolution of tapeworm parasites and introduce the research programmes linked to genome initiatives that are aimed at understanding signalling systems involved in basic host-parasite interactions and morphogenesis.

Molecules and morphology reveal cryptic variation among digeneans infecting sympatric mullets in the Mediterranean.

We applied a combined molecular and morphological approach to resolve the taxonomic status of Saccocoelium spp. parasitizing sympatric mullets (Mugilidae) in the Mediterranean. Eight morphotypes of Saccocoelium were distinguished by means of multivariate statistical analyses: 2 of Saccocoelium obesum ex Liza spp.; 4 of S. tensum ex Liza spp.; and 2 (S. cephali and Saccocoelium sp.) ex Mugil cephalus. Sequences of the 28S and ITS2 rRNA gene regions were obtained for a total of 21 isolates of these morphotypes. Combining sequence data analysis with a detailed morphological and multivariate morphometric study of the specimens allowed the demonstration of cryptic diversity thus rejecting the hypothesis of a single species of Saccocoelium infecting sympatric mullets in the Mediterranean. Comparative sequence analysis revealed 4 unique genotypes, thus corroborating the distinct species status of Saccocoelium obesum, S. tensum and S. cephali and a new cryptic species ex Liza aurata and L. saliens recognized by its consistent morphological differentiation and genetic divergence. However, in spite of their sharp morphological difference the 2 morphotypes from M. cephalus showed no molecular differentiation and 4 morphotypes of S. tensum were genetically identical. This wide intraspecific morphological variation within S. tensum and S. cephali suggests that delimiting species of Saccocoelium using solely morphological criteria will be misleading.

Hox genes and the parasitic flatworms: new opportunities, challenges and lessons from the free-living.

Research into the roles played by Hox and related homeotic gene families in the diverse and complex developmental programmes exhibited by parasitic flatworms (Platyhelminthes) can hardly be said to have begun, and thus presents considerable opportunity for new research. Although featured in some of the earliest screens for homeotic genes outside Drosophila and mice, surveys in parasitic flatworms are few in number and almost nothing is yet known of where or when the genes are expressed during ontogeny. This contrasts sharply with a significant body of literature concerning Hox genes in free-living flatworms which have long served as models for the study of regeneration and the maintenance of omnipotent cell lines. Nevertheless, available information suggests that the complement of Hox genes and other classes of homeobox-containing genes in parasitic flatworms is typical of their free-living cousins and of other members of the Lophotrochozoa. Recent work on Schistosoma combined with information on Hox gene expression in planarians indicates that at least some disruption of the clustered genomic arrangement of the genes, as well as of the strict spatial and temporal colinear patterns of expression typical in other groups, may be characteristic of flatworms. However, available data on the genomic arrangement and expression of flatworm Hox genes is so limited at present that such generalities are highly tenuous. Moreover, a basic underlying pattern of colinearity is still observed in their spatial expression patterns making them suitable as cell or region-specific markers. I discuss a number of fundamental developmental questions and some of the challenges to addressing them in relation to each of the major parasitic lineages. In addition, I present newly characterized Hox genes from the model tapeworm Hymenolepis and analyze these by Bayesian inference together with >100 Hox and ParaHox homeodomains of flatworms and select lophotrochozoan taxa, providing a phylogenetic scaffold for their identification.

On the position of Archigetes and its bearing on the early evolution of the tapeworms.

The tapeworm Archigetes sieboldi Leuckart, 1878 (Platyhelminthes: Cestoda: Caryophyllidea) has been cited as a likely representative of the "protocestode" condition, owing to its lack of segmentation and ability to attain sexual maturity in the invertebrate host (aquatic oligochaetes). The idea has been variously amplified or rejected in the literature, although the actual phylogenetic position of the species has not been investigated until now. New collections of Archigetes sp. from both its vertebrate and invertebrate hosts provided the opportunity to estimate its phylogenetic position with the use of molecular systematics, while prompting new analyses aimed at assessing the early diversification of the Cestoda. Additional collections representing the Amphilinidea, Caryophyllidea, and Gyrocotylidea were combined with published gene sequences to construct data sets of complete 18S (110 taxa) and partial (D1-D3) 28S (107 taxa) rDNA sequences, including 8 neodermatan outgroup taxa. Estimates resulting from Bayesian inference, maximum likelihood, and maximum parsimony analyses of the separate and combined data sets supported a derived position of the genus within the Caryophyllidea, and thus reject the idea that Archigetes sp. may exemplify a "primitive" condition. Topological constraint analyses rejected the hypothesis that Archigetes represents the most basal lineage of the Eucestoda, but did not rule out that it could represent the earliest branching taxon of the Caryophyllidea. In all analyses, the Eucestoda were monophyletic and supported basal positions of the nonsegmented Caryophyllidea and Spathebothriidea relative to other major lineages of the Eucestoda, implying that segmentation is a derived feature of the common ancestor of the di- and tetrafossate eucestodes. However, constraint analyses could not provide unequivocal evidence as to the precise branching patterns of the cestodarian, spathebothriidean, and caryophyllidean lineages. Phylogenetic analyses favor the interpretation that sexual maturity of Archigetes sp. in the invertebrate host, and similar examples in members of the Spathebothriidea, are the result of progenesis and have little if any bearing on understanding the protocestode condition.

Insight into the role of cetaceans in the life cycle of the tetraphyllideans (Platyhelminthes: Cestoda).

Four types of tetraphyllidean larvae infect cetaceans worldwide: two plerocercoids differing in size, 'small' (SP) and 'large' (LP), and two merocercoids referred to as Phyllobothrium delphini and Monorygma grimaldii. The latter merocercoid larvae parasitize marine mammals exclusively and exhibit a specialised cystic structure. Adult stages are unknown for any of the larvae and thus the role of cetaceans in the life cycle of these species has been a long-standing problem. The SP and LP forms are thought to be earlier stages of P. delphini and M. grimaldii that are presumed to infect large pelagic sharks that feed on cetaceans. A molecular analysis of the D2 variable region of the large subunit ribosomal DNA gene based on several individuals of each larval type collected from three Mediterranean species of cetaceans showed consistent and unique molecular signatures for each type regardless of host species or site of infection. The degree of divergence suggested that LP, P. delphini and M. grimaldii larvae may represent separate species, whereas SP may be conspecific with M. grimaldii. In all host species, individuals of SP accumulated in the gut areas in which the lymphoid tissue was especially developed. We suggest therefore that these larvae use the lymphatic system to migrate to the abdominal peritoneum and mesenteries where they develop into forms recognizable as M. grimaldii. The plerocercoid stage of P. delphini remains unknown. In a partial phylogenetic tree of the Tetraphyllidea, all larvae formed a clade that included a representative of the genus Clistobothrium, some species of which parasitize sharks such as the great white which is known to feed on cetaceans. A bibliographic examination of tetraphyllidean infections in marine mammals indicated that these larvae are acquired mostly offshore. In summary, the evidence suggests that cetaceans play a significant role in the life cycle of these larvae. In addition, it seems clear that cetaceans act as natural intermediate hosts for P. delphini and M. grimaldii, as within these hosts they undergo development from the plerocercoid stage to the merocercoid stage. Because tetraphyllidean species use fish, cephalopods and other marine invertebrates as intermediate hosts, the inclusion of cetaceans in the life cycle would have facilitated their transmission to apex predators such as the large, lamnid sharks. The biological significance of infections of LP in cetaceans is unclear, but infections do not seem to be accidental as such larvae show high prevalence and abundance as well as a high degree of site specificity, particularly in the anal crypts and bile ducts.

Evidence for host-specific clades of tetraphyllidean tapeworms (Platyhelminthes: Eucestoda) revealed by analysis of 18S ssrDNA.

Sequence data from the V4 and V7-V9 variable regions of the 18S small subunit ribosomal DNA (ssrDNA) gene were used to examine relationships among 26 tetraphyllidean and two lecanicephalidean taxa. Newly collected specimens of 21 of the tetraphyllidean species were used to generate ssrDNA sequences that were combined with sequences previously available, including those of two diphyllidean taxa used for outgroup rooting. The sequences were aligned by eye according to secondary structural motifs of the conserved core of the molecule. Of the 1520 sites in the alignment, 874 (58%) were excluded from analysis due to alignment gaps and lack of positional homology as inferred by manual inspection. Genetic variability of the ssrDNA gene regions compared was greater than would be expected, based on the present taxonomy of the ingroup species, and the genetic divergences among tetraphyllidean 'families' and genera were comparable to that among tapeworm orders. Phylogenetic hypotheses were generated by the methods of maximum parsimony and maximum likelihood (GTR + I + Gamma nucleotide substitution model). Four most parsimonious trees resulted from analysis by maximum parsimony. Strict consensus of the four trees supported the monophyly of the Tetraphyllidea, with the lecanicephalidean taxa forming a sister lineage. Among the tetraphyllidean taxa included in the analysis were three major clades: a basal clade including species of the phyllobothriid genera Anthocephalum, Echeneibothrium, Rhinebothrium, Rhodobothrium and Spongiobothrium; a clade uniting the phyllobothriids of the genus Duplicibothrium with the dioecotaeniid genus Dioecotaenia; and a larger sister clade to the Duplicibothrium + Dioecotaenia clade that included the phyllobothriid genera Caulohothrium, Ceratobothrium, Clistobothrium, Paraoryigmatobothrium and Prosobothrium, the litobothriid genus Litobothrium and the onchobothriid genera Acanthobothrium, Calliobothrium, Phoreiobothrium and Platybothrium. Maximum likelihood analysis resulted in a topology that was congruent where nodes were strongly supported by parsimony analysis, but differed in the relative positions of the well-supported clades. In addition,maximum likelihood analysis grouped the lecanicephalidean taxa among the tetraphyllidean taxa, indicating paraphyly of the order Tetraphyllidea as currently defined. Relationships suggested by both methods of analysis reflected common host associations of the taxa better than their current classification, suggesting that coevolution has had a significant role in the evolution of the group.

Phylogeny and classification of the Digenea (Platyhelminthes: Trematoda).

Complete small subunit ribosomal RNA gene (ssrDNA) and partial (D1-D3) large subunit ribosomal RNA gene (lsrDNA) sequences were used to estimate the phylogeny of the Digenea via maximum parsimony and Bayesian inference. Here we contribute 80 new ssrDNA and 124 new lsrDNA sequences. Fully complementary data sets of the two genes were assembled from newly generated and previously published sequences and comprised 163 digenean taxa representing 77 nominal families and seven aspidogastrean outgroup taxa representing three families. Analyses were conducted on the genes independently as well as combined and separate analyses including only the higher plagiorchiidan taxa were performed using a reduced-taxon alignment including additional characters that could not be otherwise unambiguously aligned. The combined data analyses yielded the most strongly supported results and differences between the two methods of analysis were primarily in their degree of resolution. The Bayesian analysis including all taxa and characters, and incorporating a model of nucleotide substitution (general-time-reversible with among-site rate heterogeneity), was considered the best estimate of the phylogeny and was used to evaluate their classification and evolution. In broad terms, the Digenea forms a dichotomy that is split between a lineage leading to the Brachylaimoidea, Diplostomoidea and Schistosomatoidea (collectively the Diplostomida nomen novum (nom. nov.)) and the remainder of the Digenea (the Plagiorchiida), in which the Bivesiculata nom. nov. and Transversotremata nom. nov. form the two most basal lineages, followed by the Hemiurata. The remainder of the Plagiorchiida forms a large number of independent lineages leading to the crown clade Xiphidiata nom. nov. that comprises the Allocreadioidea, Gorgoderoidea, Microphalloidea and Plagiorchioidea, which are united by the presence of a penetrating stylet in their cercariae. Although a majority of families and to a lesser degree, superfamilies are supported as currently defined, the traditional divisions of the Echinostomida, Plagiorchiida and Strigeida were found to comprise non-natural assemblages. Therefore, the membership of established higher taxa are emended, new taxa erected and a revised, phylogenetically based classification proposed and discussed in light of ontogeny, morphology and taxonomic history.

Cestodes of cestodes of Peruvian freshwater stingrays.

Adult tetraphyllidean tapeworms (Platyhelminthes: Eucestoda) from the spiral intestines of 3 species of potamotrygonid stingrays (Paratrygon aiereba, Potamotrygon castexi, and Portamotrygon motoro) in the Madre de Dios river in Peru were found to host numerous cysts embedded in their parenchymal tissues. Histological sections of the cysts revealed the presence of a scolex bearing 4 suckers and an unarmed apical organ consistent with larval stages of both Cyclophyllidea and Proteocephalidea. To further elucidate their identities, partial 28S ribosomal DNA (rDNA) sequences were characterized from 3 cysts and 4 adult Rhinebothrium spp. 'host' worms and screened against all available cestode 28S rDNA data. Initial BLAST screening and subsequent alignment ruled out the possibility that the cysts were cyclophyllidean, and the cyst and adult sequences were thus aligned together with all available lecanicephalidean, litobothriidean, proteocephalidean, and tetraphyllidean sequences. Sequences from all 3 cysts were identical, and phylogenetic analysis clearly placed them among derived members of the Proteocephalidea, although no exact match was found. Sequences from the adult host worms formed 2 identical pairs and grouped together with other tetraphyllidean species from rays. These results are compared with records of hyperparasites of South American catfish cestodes. This is the first confirmed record of a proteocephalidean cestode parasitizing a tetraphyllidean cestode.

Comparison of Leptorhynchoides thecatus (Acanthocephala) recruitment into green sunfish and largemouth bass populations.

The degree to which host suitability is a reflection of host community structure in generalist parasites was studied experimentally in the common fish acanthocephalan Leptorhynchoides thecatus. Previous study has shown that green sunfish (Lepomis cyanellus) are required, and largemouth bass (Micropterus salmoides) are suitable (but not required) hosts, where they occur sympatrically in natural communities. The present study examined populations of L. cyanellus and M. salmoides held separately in mesocosms and exposed to L. thecatus cystacanths via laboratory-infected Hyalella azteca (Amphipoda). Recruitment, maturation, and transmission of worms were examined over a 17-wk period and compared between fish species. Infections with L. thecatus were found as early as 2 wk after the introduction of cystacanth-infected amphipods, and by week 11 fishes of both species harbored gravid worms. Immature worms were observed in both host species by week 17 and were presumed to be a result of natural egg production and release resulting in infections of amphipods and the subsequent reinfection of fish. No significant difference in the prevalence, abundance, percentage of worms gravid, or time of parasite maturation was found between host populations. Results indicate that the different roles played by these host species in the maintenance of L. thecatus supra-populations in natural systems are not due to intrinsic factors but rather to differences in host autecology and community structure.

Evolution of the major lineages of tapeworms (Platyhelminthes: Cestoidea) inferred from 18S ribosomal DNA and elongation factor-1alpha.

The interrelationships of the tapeworms (Platyhelminthes: Cestoidea) were inferred by analysis of complete gene sequences (approximately 2,200 bp) of 18S small subunit ribosomal DNA (18S) and partial gene sequences (approximately 900 bp) of elongation factor-1alpha (Ef-1alpha). New collections were made of 23 species representing each of the 14 currently recognized orders of tapeworms, including the Amphilinidea, Gyrocotylidea, and the 12 orders of the Eucestoda. Sequences were determined directly from polymerase chain reaction (PCR) products by either manual or automated methods. Nucleotide sequences of platyhelminth species outside of the Cestoidea were obtained for rooting the resulting trees. The 18S sequences were aligned with reference to the secondary structural features of the gene and the Ef-1alpha sequences were aligned with reference to their corresponding amino acid residues. Significant length variation among taxa was observed in the V2, V4, and V7 variable regions of the 18S gene. Such positions where sequences could not be aligned confidently were excluded from the analyses. Third codon positions of the Ef-1alpha gene were inferred to be saturated at an ordinal level of comparison. In addition, a short (approximately 35 bp) intron region of the Ef-1alpha gene was found to be shared only among the eucestode taxa, with the exception of Spathebothrium simplex (Spathebothriidea), which lacked the intron. Complete alignments showing structural features of the genes and sites excluded from analysis are provided as appendices. The sequence data were partitioned into 7 data sets in order to examine the effects of analyses on different subsets of the data. Analyses were conducted on the 2 genes independently, different codon positions of Ef-1alpha, amino acid sequences of Ef-1alpha, and combinations thereof. All subsets of the data were analyzed under the criterion of maximum parsimony as well as minimum evolution using both maximum-likelihood estimated, and LogDet-transformed distances. Results varied among the different data partitions and methods of analysis. Nodes with strong character support, however, were consistently recovered, and a general pattern of evolution was observed. Monophyly of the Cestoidea (Amphilinidea + Gyrocotylidea + Eucestoda) and Eucestoda and the traditionally accepted positions of the Amphilinidea and Gyrocotylidea as sister lineages to the Eucestoda were supported. Within the Eucestoda, the Spathebothriidea was found to be the sister of all other eucestodes. The remaining orders generally formed a diphyletic pattern of evolution consisting of separate difossate and tetrafossate lineages. This pattern was not universally observed among the analyses, primarily because the trypanorhynch and diphyllidean taxa showed instability in their phylogenetic position. Additional relationships that showed high levels of nodal support included a sister relationship between the Pseudophyllidea and Haplobothriidea and a clade uniting the Cyclophyllidea, Nippotaeniidea, and Tetrabothriidea. The Tetraphyllidea, as currently defined, was found to be paraphyletic without the inclusion of the orders Proteocephalidea and, possibly, Lecanicephalidea. Ordinal status of a monophyletic Litobothriidea, currently classified within the Tetraphyllidea, was found to be supported from a phylogenetic perspective.