Determinants and consequences of interspecific body size variation in tetraphyllidean tapeworms

Tetraphyllidean cestodes are cosmopolitan, remarkably host specific, and form the most speciose and diverse group of helminths infecting elasmobranchs (sharks, skates and rays). They show substantial interspecific variation in a variety of morphological traits, including body size. Tetraphyllideans represent therefore, an ideal group in which to examine the relationship between parasite body size and abundance. The individual and combined effects of host size, environmental temperature, host habitat, host environment, host physiology, and host type (all likely correlates of parasite body size) on parasite length were assessed using general linear model analyses using data from 515 tetraphyllidean cestode species (182 species were included in analyses). The relationships between tetraphyllidean cestode length and intensity and abundance of infection were assessed using simple linear regression analyses. Due to the contrasting morphologies between shark and batoid hosts, and contrasting physiologies between sharks of the Lamnidae family and other sharks, analyses were repeated in different subsets based on host morphology and physiologies (“sharks” vs. batoids) to determine the influence of these variables on adult tetraphyllidean tapeworm body size. Results presented herein indicate that host body size, environmental temperature and host habitat are relatively important variables in models explaining interspecific variations in tetraphyllidean tapeworm length. In addition, a negative relationship between tetraphyllidean body size and intensity of infection was apparent. These results suggest that space constraints and ambient temperature, via their effects on metabolism and growth, determine adult tetraphyllidean cestode size. Consequently, a trade-off between size and numbers is possibly imposed by external forces influencing host size, hence limiting physical space or other resources available to the parasites.     

The taxonomic status of Spatulifer cf. maringaensis Pavanelli & Rego, 1989 (Eucestoda: Proteocephalidea) from Sorubim lima (Bloch & Schneider) (Pisces: Siluriformes), and the use of the microthrix pattern in the discrimination of Spatulifer spp

de Chambrier & Vaucher (1999) compared the proteocephalidean cestode Spatulifer maringaensis Pavanelli & Rego, 1989 from Hemisorubim platyrhynchos (Valenciennes) with similar specimens, which they described as S. cf. maringaensis, parasitising Sorubim lima (Bloch & Schneider) collected in the Paraná and Paraguay Rivers. No remarkable differences between these worms were found by these authors, except for the scolex diameter and a different infection rate in each fish host. In order to elucidate the taxonomic status of the fish cestode Spatulifer cf. maringaensis from Sorubim lima, type and voucher material of S. maringaensis from H. platyrhynchos, and voucher and new material recently collected from Sorubim lima in Argentina are described in terms of their internal morphology and tegumental surface. During the study, mature and gravid worms with smaller metascoleces were found among voucher specimens from both hosts and in the new material from S. lima. The number of testes in the type-material was difficult to assess, but it could be determined in the voucher material from the type-host as being within the range of the specimens from S. lima. Both taxa are morphologically identical and can therefore be considered as conspecific. The data on their parasitological indices support the idea that S. lima is the principal final host and H. platyrhynchos is a secondary final host. Spatulifer maringaensis is widespread throughout the Paraná basin. A comparison of the microthrix pattern of S. maringaensis with that of S. rugosa (Woodland, 1935) revealed that they have the same type of microtriches, but with a different distribution, size and density. Tumuli were observed for the first time in a Neotropical taxon. Some studies have shown that the microthrix pattern is useful for characterising and discriminating species of the Proteocephalidea, and it was used herein as a tool for determining the conspecificity of S. cf. maringaensis with S. maringaensis.     

Identification of life cycle stages of Cyathocephalus truncatus (Cestoda: spathebothriidea) using molecular techniques

Morphological identification of tapeworm species at larval stages (procercoids and cysticercoids) is often difficult because few diagnostic characters are available. In the present study, a molecular approach (sequencing of partial 18S rDNA gene) was used to evaluate the genetic similarity between adult specimens of Cyathocephalus truncatus (Pallas, 1871) (Cestoda: Spathebothriidea) found in fish, its definitive host, and procercoids of the same species recovered from amphipod, Echinogammarus stammeri (Karaman, 1931). Furthermore, cestode cysticercoids of uncertain species were found in the amphipod's hemocoel. The sequences obtained from adults and procercoids were identical, and even very similar to those of C. truncatus available in GenBank, whereas the sequences obtained from cysticercoids differed significantly from those of adults and procercoids, indicating that these larvae belong to another species; later it was demonstrated that they were cysticercoids of Microsomacanthus pachycephala (Linstow, 1972), a cestode of the Hymenolepididae (Cyclophyllidea). The results of this investigation show that the comparison of nucleotide sequence data may avoid misclassification of developmental stages of parasites, which use the same intermediate host.     

Natural hybridization between Paragonimus bangkokensis and Paragonimus harinasutai

Paragonimus bangkokensis and Paragonimus harinasutai, which are morphologically distinguishable species, often co-infect in the same crab intermediate hosts. Recent molecular phylogenetic analyses revealed that these two species are genetically close to each other and are considered as the sister species. While we have been studying Paragonimus adult worms obtained from the lungs of a cat experimentally infected with Paragonimus metacercariae which were morphologically identified as P. harinasutai collected from central Viet Nam, one out of 6 adult worms has grouped cuticular spines, which is a feature of P. bangkokensis. By molecular analyses, the CO1 sequence of this specimen was identical with that of P. bangkokensis, but the ITS2 and the D2 region of 28S rDNA sequences showed a two peak pattern. Then, PCR products of the ITS2 and the D2 region of 28S rDNA sequences were ligated to TOPO vector and subcloned to determine the heterozygosity. Two types of sequences were obtained from each ITS2 and D2 region of 28S; one was identical with P. harinasutai and the other with P. bangkokensis. Taking all these morphological and molecular data together, we identified this adult worm as a hybrid specimen of P. bangkokensis and P. harinasutai.     

Reinvestigation of spermiogenesis in the proteocephalidean cestode Proteocephalus longicollis (Zeder, 1800)

The reinvestigation of spermiogenesis in the proteocephalidean cestode Proteocephalus longicollis (Zeder, 1800) provides new details that were not described previously for this species. The 2 centrioles with roots are oriented at the first tangential to the long axis of the nucleus in a differentiation zone. They come to be arranged in the same plane only when a single intercentriolar body (ICB) appears between them. The ICB had been described previously as consisting of a number of dense elements (Swiderski, 1985) but, in the present study, appeared as a single, electron-dense plate. Also new for this species is the observation of 2 arching membranes that appear at the base of the differentiation zone only when the 2 flagella rotate toward the median cytoplasmic protrusion. Moreover, the formation of a crested body is described for the first time in P. longicollis. The present data support the suggestion that the process of spermiogenesis in proteocephalidean tapeworms corresponds in most features to that observed in some members of the unrelated tetraphyllidean clades, the Onchobothriidae and Phyllobothriidae. This may represent more evidence regarding the close relationship between tetraphyllidean and proteocephalidean cestodes, but existing information is fragmentary and controversial in some characters.     

Molecular systematic analysis of the order Proteocephalidea (Eucestoda) based on mitochondrial and nuclear rDNA sequences

Two ribosomal DNA sequences were used to infer phylogenetic relationships among the Eucestoda order Proteocephalidea. A 437 bp portion of the 16S mitochondrial and a 1149 bp 5' portion of the nuclear large sub-unit rRNA molecule were sequenced for 53 proteocephalidean cestodes (representing nine subfamilies and 22 genera) and for one outgroup species. Parsimony and distance-based analyses of the two databases, alone and combined, failed to support the monophyly of the two traditionally accepted families, of numerous subfamilies (with the exception of the Rudolphielliinae and Othinoscolescinae which were validated in our analysis) and of various genera, including the genus Nomimoscolex (Woodland), Ophiotaenia (La Rue) as well as the type genus Proteocephalus (Weinland). Palaearctic Proteocephalus species nevertheless constituted a well-defined clade. The two genes globally yielded compatible results; however, the nuclear ribosomal gene provided a better resolution of relations among Proteocephalidea.     

Sequence comparisons of 28S ribosomal DNA and mitochondrial cytochrome c oxidase subunit I of Metagonimus yokogawai, M. takahashii and M. miyatai

We compared the DNA sequences of the genus Metagonimus: M. yokogawai, M. takahashii, and M. miyatai. We obtained 28S D1 ribosomal DNA (rDNA) and mitochondrial cytochrome c oxidase subunit I (mtCOI) fragments from the adult worms by PCR, that were cloned and sequenced. Phylogenetic relationships inferred from the nucleotide sequences of the 28S D1 rDNA and mtCOI gene. M. takahashii and M. yokogawai are placed in the same clade supported by DNA sequence and phylogenic tree analysis in 28S D1 rDNA and mtCOI gene region. The above findings tell us that M. takahashii is closer to M. yokogawai than to M. miyatai genetically. This phylogenetic data also support the nomination of M. miyatai as a separate species.     

Sandonella sandoni (Lynsdale, 1960), an enigmatic and morphologically unique cestode parasitic in the osteoglossiform fish Heterotis niloticus in Africa

Sandonella sandoni (Lynsdale, 1960) is the type and only species of the Sandonellinae, a cestode subfamily of unclear phylogenetic position. It is redescribed here on the basis of a re-examination of its syntypes, voucher specimens from museum collections, and freshly collected material from the intestine of Heterotis niloticus (Osteoglossiformes: Arapaimidae) from Benin, Nigeria, Senegal, and the Sudan. The species possesses several unique morphological characters, such as (1) a vitellarium formed by 2 compact, but deeply lobulated, postovarian masses near the posterior margin of proglottids; (2) a scolex with a highly modified apical structure formed by 4 muscular retractile lappets; (3) a well-developed circular musculature, which is external to the inner longitudinal muscles; (4) a dilated, vesicle-like proximal part of the external sperm duct; (5) the unique morphology of the uterus and its development, which represents an intermediate form between the 2 basic types recognized in the Proteocephalidea; (6) the growth of eggs during their development within the uterus; and (7) the complex proglottization with intermingled smaller and larger (wider) proglottids. The morphology of S. sandoni, including the form and distribution of microtriches, was studied by scanning electron microscopy for the first time, and the lectotype and paralectotypes of S. sandoni are designated. Sequences of the 28S rRNA gene of 4 specimens (2 from the Sudan and 2 from Senegal) were identical, which confirms conspecificity of geographically distant samples. Sandonella sandoni sequences have also shown that it actually belongs among the Proteocephalidea, being a sister taxon of a relatively derived clade of Palaearctic proteocephalideans, containing Glanitaenia osculata and Paraproteocephalus parasiluri from catfish and Palaearctic species of the Proteocephalus aggregate.     

Phylogenetic position of the parasitoid nanoflagellate Pirsonia inferred from nuclear-encoded small subunit ribosomal DNA and a description of Pseudopirsonia n. gen. and Pseudopirsonia mucosa (Drebes) comb. nov

Sequences of the nuclear encoded small subunit (SSU) rRNA were determined for Pirsonia diadema, P. guinardiae, P. punctigerae, P. verrucosa, P. mucosa and three newly isolated strains 99-1, 99-2, 99-S. Based on phylogenetic analysis all Pirsonia strains, except P. mucosa, clustered together in one clade, most closely related to Hyphochytrium catenoides within the group of stramenopiles. However, P. mucosa was most closely related to Cercomonas sp. SIC 7235 and Heteromita globosa and belongs to the heterogenic group of Cercozoa. In addition to the SSU rDNA sequences, P. mucosa differs from the stramenopile Pirsonia species in some characteristics and was therefore redescribed in this paper as Pseudopirsonia mucosa. The three newly isolated strains 99-1, 99-2, and 99-S differed by 28 bp in their SSU rDNA sequences from their closest neighbour P. diadema and only 1 to 3 bp among themselves. These base differences and a host range similar to P. formosa were sufficient to assign them as new strains of P. formosa.     

Orders out of chaos – molecular phylogenetics reveals the complexity of shark and stingray tapeworm relationships

Novel molecular data are presented to resolve the long-standing issue of the non-monophyly of the elasmobranch-hosted tapeworm order Tetraphyllidea relative to the other acetabulate eucestode orders. Bayesian inference analyses of various combinations of full ssrDNA, and full or partial lsrDNA (D1–D3), sequence data, which included 134 species representing 97 genera across the 15 eucestode orders, were conducted. New ssrDNA data were generated for 82 species, partial lsrDNA data for 53 species, and full lsrDNA data for 29 species. The monophyly of each of the elasmobranch-hosted orders Cathetocephalidea, Litobothriidea, Lecanicephalidea and Rhinebothriidea was confirmed, as was the non-monophyly of the Tetraphyllidea. Two relatively stable groups of tetraphyllidean taxa emerged and are hereby designated as new orders. The Onchoproteocephalidea n. ord. is established to recognise the integrated nature of one undescribed and 10 described genera of hook-bearing tetraphyllideans, previously placed in the family Onchobothriidae, with the members of the order Proteocephalidea. The Phyllobothriidea n. ord. is established for a subset of 12 non-hooked genera characterised by scoleces bearing four bothridia each with an anterior accessory sucker; most parasitise sharks and have been assigned to the Phyllobothriidae at one time or another. Tentative ordinal placements are suggested for eight additional genera; placements for the remaining tetraphyllidean genera have not yet emerged. We propose that these 17 genera remain in the “Tetraphyllidea”. Among these, particularly labile across analyses were Anthobothrium, Megalonchos, Carpobothrium, Calliobothrium and Caulobothrium. The unique association of Chimaerocestus with holocephalans, rather than with elasmobranchs, appears to represent a host-switching event. Both of the non-elasmobranch hosted clades of acetabulate cestodes (i.e. Proteocephalidea and Cyclophyllidea and their kin) appear to have had their origins with elasmobranch cestodes. Across analyses, the sister group to the clade of “terrestrial” cestode orders was found to be an elasmobranch-hosted genus, as was the sister to the freshwater fish- and tetrapod-hosted Proteocephalidea. Whilst further data are required to resolve outstanding nomenclatural and phylogenetic issues, the present analyses contribute significantly to an understanding of the evolutionary radiation of the entire Cestoda. Clearly, elasmobranch tapeworms comprise the backbone of cestode phylogeny.     

Comparative study on DNA sequences of ribosomal DNA and cytochrome c oxidase subunit 1 of mitochondrial DNA among five species of gnathostomes

The nucleotide sequences of partial 18S, complete internal transcribed spacer region 1 (ITS1), complete 5.8S, complete ITS2 and partial 28S of ribosomal DNA (rDNA) and cytochrome c oxidase subunit 1 of mitochondrial DNA (MCOI) from five species of gnathostomes (G. spinigerum, G. doloresi, G. nipponicum, G. hispidum and G. binucleatum with the former four species being distributed in Japan and Asia) that cause human gnathostomiasis were compared by direct polymerase chain reaction cycle-sequencing. The nucleotide sequences of each region of the18S (613 bp), 5.8S (158 bp) and 28S (598 bp) rDNA from the five species were almost identical. The ITS1 region was different in length for the five species. The nucleotide sequences of each region of ITS2 and partial MCO1 regions were different among the five species. Therefore, these two regions can be used as genetic markers for identification of worms.     

Infracommunity structure of parasites of Hemigymnus melapterus (Pisces: Labridae) from Lizard Island, Australia: the importance of habitat and parasite body size

This study describes the community of all metazoan parasites from 14 individuals of thicklip wrasse, Hemigymnus melapterus, from Lizard Island, Australia. All fish were parasitized, and 4,649 parasite individuals were found. Twenty-six parasite species were identified although only 6 species were abundant and prevalent: gnathiid isopods, the copepod Hatschekia hemigymni, the digenean Callohelmis pichelinae, and 3 morphotypes of tetraphyllidean cestode larvae. We analyzed whether the body size and microhabitat of the parasites and size of the host affected understanding of the structure of the parasite community. We related the abundance, biovolume, and density of parasites with the host body size and analyzed the abundances and volumetric densities of some parasite species within microhabitats. Although the 2 most abundant species comprised 75% of all parasite individuals, 4 species, each in similar proportion, comprised 85% of the total biovolume. Although larger host individuals had higher richness, abundance, and biovolume of parasites than smaller individuals, overall parasite volumetric density actually decreased with the host body size. Moreover, parasites exhibited abundances and densities significantly different among microhabitats; some parasite species depended on the area available, whereas others selected a specific microhabitat. Parasite and habitat size exhibited interesting relationships that should be considered more frequently. Considerations of these parameters improve understanding of parasite community structure and how the parasites use their habitats.     

Phylogeny of Steinernema travassos, 1927 (Cephalobina: Steinernematidae) inferred from ribosomal DNA sequences and morphological characters

Entomopathogenic nematodes in Steinernema, together with their symbiont bacteria Xenorhabdus, are obligate and lethal parasites of insects that can provide effective biological control of some important lepidopteran, dipteran, and coleopteran pests of commercial crops. Phylogenetic relationships among 21 Steinernema species were estimated using 28S ribosomal DNA (rDNA) sequences and morphological characters. Sequences of the rDNA internal transcribed spacers were obtained to provide additional molecular characters to resolve relationships among Steinernema carpocapsae, Steinernema scapterisci, Steinernema siamkavai, and Steinernema monticolum. Four equally parsimonious trees resulted from combined analysis of 28S sequences and 22 morphological characters. Clades inferred from analyses of molecular sequences and combined datasets were primarily reliably supported as assessed by bootstrap resampling, whereas those inferred from morphological data alone were not. Although partially consistent with some traditional expectations and previous phylogenetic studies, the hypotheses inferred from molecular evidence, and those from combined analysis of morphological and molecular data, provide a new and comprehensive framework for evaluating character evolution of steinernematids. Interpretation of morphological character evolution on 6 trees inferred from sequence data and combined evidence suggests that many structural features of these nematodes are highly homoplastic, and that some structures previously used to hypothesize relationships represent ancestral character states.     

基于28S rDNA D1-D2基因片段与16S rDNA部分序列联合分析的叶肢介系统发育研究

叶肢介(Conchostraca)的系统发育问题一直是甲壳动物研究中颇具争议的一个课题.本研究测定了我国2种叶肢介(Eocyzicus mongolianus,Eoc yzicus orientalis)的28S rDNA D1-D2区基因序列和16S rDNA E-G区序列,并与GenBank中的20种叶肢介序列一起...     

The evolution of the Proteocephalidea (Platyhelminthes,Eucestoda) based on an enlarged molecular phylogeny,with comments on their uterine development

We present a molecular phylogeny of the Proteocephalidea based on 28S rDNA sequence data that is a follow-up to the paper by Zehnder & Mariaux (1999). Twenty-three new sequences, including three outgroups are added in our new data-set. The Gangesiinae Mola, 1929 and the Acanthotaeniinae Freze, 1963 appear to be the most primitive clades. They are followed by a robust clade comprising the Palaearctic Proteocephalinae Mola, 1929 from freshwater fishes. The structure of the more derived clades, comprising most Neotropical and Nearctic species, is less resolved. At the nomenclatural level, we erect a new genus, Glanitaenia n. g. for G. osculata (Goeze, 1782) n. comb., previously Proteocephalus osculatus, and define an aggregate for the Palaearctic Proteocephalus Weinland, 1858. After a re-examination of all of the studied taxa, we identify two types of uterine development and show the importance of this character for the systematics of the order. Our phylogeny does not support the classical view of a Neotropical origin for the Proteocephalidea but rather favours an Old World origin of the group either in saurians or Palaearctic Siluriformes.     

Molecular evidence of natural hybridization between Fasciola hepatica and F. gigantica.

Nucleotide sequences of two regions, cytochrome c-oxidase subunit 1 (CO1) and NADH dehydrogenase subunit 1 (ND1) of the mitochondrial DNA and two regions, internal-transcribed spacer 2 (ITS2) and the D2 region in the 28S rDNA (28S) of the nuclear DNA were obtained from five Korean worms of the genus Fasciola in order to elucidate their taxonomic status. The CO1 and ND1 regions are all monomorphic in the Korean worms and similar to those of F. gigantica. On the other hand, the ITS2 and D2 regions were found to be polymorphic; that is, out of five worms, two possessed a F. gigantica-type sequence, one, a F. hepatica-type sequence and two possessed sequences of both types indicating an existence of different alleles at the loci. It should be noted that these variations of the ITS2 and D2 regions co-occur at the same individual worms. This was confirmed by sequencing five to six cloned PCR products for each worm. The present study strongly suggests interspecific cross-hybridization between the two species coexisting in Korea.     

A new genus and species of proteocephalidean (Cestoda) from Clarias catfishes (Siluriformes: Clariidae) in Africa

A new proteocephalidean cestode is described from 2 catfishes, Clarias gariepinus (type host) and C. cf. anguillaris (Siluriformes: Clariidae), from Ethiopia (type locality), Sudan, Tanzania, and Zimbabwe, and a new genus, Barsonella, is proposed to accommodate it. The genus belongs to the Proteocephalinae because its genital organs (testes, ovary, vitellarium, and uterus) are situated in the medulla. Barsonella lafoni, the type and only species of the new genus, is characterized mainly by the possession of an additional opening of each sucker; circular musculature on the anterior margin of suckers, serving as a sphincter; a small thin-walled glandular apical organ; absence of well-developed osmoregulatory canals in mature, pregravid, and gravid proglottids; and a large strobila, up to 173 mm long and 3.2 mm wide. Species of Marsypocephalus Wedl, 1861 (Marsypocephalinae), other large-sized proteocephalidean tapeworms occurring sympatrically in African catfishes (Clarias and Heterobranchus) and also possessing a sphincter-like, circular musculature on the anterior part of suckers, differ from B. lafoni in the absence of an additional sucker opening and glandular apical organ, the cortical position of the testes, well-developed osmoregulatory canals throughout the strobila, and a large cirrus sac. Proteocephalus glanduligerus (Janicki, 1928), another cestode parasitic in Clarias spp. in Africa, is much smaller than B. lafoni (maximum length 15 mm), has suckers without additional opening and circular musculature on the suckers, a large-sized glandular organ, much larger than suckers, and well-developed osmoregulatory canals. Comparison of partial sequences of the 28S rRNA gene for 7 samples of B. lafoni from 2 different hosts and 4 localities in Ethiopia, Sudan, and Tanzania has shown a very low genetic variability. In a limited phylogenetic analysis, B. lafoni formed a clade with Corallobothrium solidum Fritsch, 1886 (Proteocephalidae: Corallobothriinae), an African electric catfish parasite. This clade was the sister group of almost all Neotropical taxa from pimelodid and other catfishes.     

Confirmation and identification of parasitic stages of obligate endobionts (Harpellales) in blackflies (Simuliidae) by means of rRNA sequence data

Over the last 35 y, the life cycle of endosymbiotic gut fungi (Harpellales) has been expanded to include cyst stages associated with the developing ovaries. Ovarian cysts (chlamydospore stages) have been identified after germination and production of asexual trichospores in vitro, but germination is not always successful, and spores exhibit morphological variation. Sequence data (for partial 18S and 28S rRNA genes) were generated for these putative stages of Harpellales using ungerminated cysts from adult blackflies and germinated cysts associated with field-collected blackfly egg masses. Cladistic analyses of the 18S and 28S rRNA sequences confirmed that ovarian cysts are stages in the life cycle of Harpellales. Ungerminated cysts, from a blackfly collected from New York state were identified as Pennella simulii and two samples from Newfoundland Prosimulium mixtum adults were identified as Harpella melusinae. Cysts with bipolar germ tubes, associated with field-collected Simulium egg masses from Newfoundland, were also identified as H. melusinae. Two other samples of cysts could not be matched with available sequences of gut fungi from larval hosts. The potential use of this approach to identify pathogenic stages associated with adult ovaries or field-collected egg masses among other host groups is highlighted and promoted as a tool to test the hypothesis that ovarian cysts are a dispersal stage common to all genera of Harpellales.