Interplay of host specificity and biogeography in the population structure of a cosmopolitan endoparasite: microsatellite study of Ligula intestinalis (Cestoda).

Organisms with wide geographical or phenotypic diversity often constitute assemblages of genetically distinct species or lineages. Within parasites, an emergence of host-specific lineages is assumed to create such cryptic variability; however, empirical evaluation of these processes is scarce. Here, we analyse populations of a parasite with a complex life cycle, wide host spectrum and global distribution, with the aim to reveal factors underlying the evolution of host- or geography-dependent lineages. Using 15 microsatellite loci, deep genetic distances were observed between populations from distant geographical areas. On the local scale, host-mediated genetic structure was found among sympatric samples. Two lineages differing in the spectrum of infected hosts co-occurred in the Euro-Mediterranean area, and two distinct lineages were recovered from Lake Tana in Ethiopia. Although sampled across several host taxa and multiple localities, a lack of marked genetic structure was seen in the populations belonging to one of the European lineages. Only weak genetic differentiation between sympatric samples from two host species was found. Complexity of the parasite life-cycle contributed to such a stratified pattern. Differences in the immune response between fish hosts were suggested as the factor diversifying the populations locally; conversely, high mobility of the parasite due to migration with its bird (definitive) host were assessed to homogenize populations across the area of distribution. However, despite the high mobility, large bodies of salt water prevent the parasite from long-distance migrations, as was demonstrated in an example of the Mediterranean Sea which represented an effective barrier to gene flow.

Phylogenetic relationships among Eimeria spp. (Apicomplexa, Eimeriidae) infecting rabbits: evolutionary significance of biological and morphological features.

Monophyly of all 11 valid Eimeria species from rabbits (Oryctolagus cuniculus Linnaeus, 1758) was revealed based on nuclear 18S rDNA sequence data. This finding implies that these species, which vary considerably in terms of their morphology and biology, diversified on a single host or several closely related species. Phylogenetic analysis divided rabbit Eimeria species into 2 sister lineages, corresponding to the presence/absence of the oocyst residuum. Other morphological or biological traits (oocyst shape and size, presence/absence of oocyst inner structures, pathogenicity, infection site, pre-patent and patent periods, sporulation time, and number of asexual generations) do not explicitly correlate with the phylogeny of rabbit coccidia.

Phylogeny, evolution and host-parasite relationships of the order Proteocephalidea (Eucestoda) as revealed by combined analysis and secondary structure characters.

In a manner similar to many other groups of organisms, the tapeworm order Proteocephalidea poses a difficult phylogenetic problem if treated on the basis of single-gene analysis. Since the biogeography and host distribution of proteocephalideans make these tapeworms a potentially interesting model for evolutionary and co-evolutionary studies, we tried to resolve their phylogenetic relationships by applying a multi-gene approach. The ITS2 sequences and V4 hypervariable loop of 18S rRNA were obtained for 43 and 35 proteocephalidean taxa, respectively, and combined with other sequences available in the GenBank. The phylogenetic analysis of the combined DNA set was confronted with characters derived from ITS2 secondary structures. Using this approach, a species-rich Neotropical lineage of proteocephalideans could be reliably resolved. The phylogenetic relationships within this group show a high degree of phylogeny-independent host distribution. The reconstruction of ITS2 secondary structure revealed a universal 4-domain arrangement, which is conserved across a wide range of Neodermata. Several motifs of the secondary structure could be mapped to the phylogenetic tree as possible clade synapomorphies.

Phylogenetic analysis of European species of Proteocephalus (Cestoda: Proteocephalidea): compatibility of molecular and morphological data, and parasite-host coevolution.

The phylogeny of European species of the tapeworm genus Proteocephalus was studied, based on partial 18S rDNA and morphological data. The group was found to be monophyletic. The analysis showed a low informative value of available morphological characters in comparison with molecular data. The morphological matrix resulted in a poorly resolved tree which is, however, compatible with the topology (Proteocephalus osculatus (Proteocephalus torulosus (Proteocephalus macrocephalus, Proteocephalus filicollis) (Proteocephalus tetrastomus, Proteocephalus percae, Proteocephalus longicollis))) based on the 18S rDNA data. A comparison performed by the program TreeMap showed a lack of significant congruency between parasite and host phylogenies. Therefore, the distribution of species in their hosts appears to be independent of the phylogeny and it is likely to be a result of host-switching, rather than co-speciation events.

Molecular phylogeny of Calyptratae (Diptera: Brachycera): the evolution of 18S and 16S ribosomal rDNAs in higher dipterans and their use in phylogenetic inference.

Sequences for nearly complete 18S rRNA and partial 16S rRNA genes were determined for sixteen species representing twelve calyptrate families. Two unique insertions are present in expansion regions of the 18S rDNA in nycteribiids. Alignments containing other dipteran rRNA genes provided good resolution at higher taxonomic level: monophyly of Calyptratae is well supported. While both 16S and 18S rDNA matrices produce unstable topologies within Calyptratae when analysed separately, their combination results in a tree with several robust and well supported nodes. Of three superfamilies recognized in recent classifications, the Hippoboscoidea is well supported by 16S rDNA and by combined matrices. The representatives of Muscoidea, Musca sp. and Antipoda sp., display a tendency to cluster within Oestroidea. The comparison of secondary structures of two variable regions indicates that Sarcophagidae are related to Calliphoridae rather than to Tachinidae.

On the phylogenetic positions of the Caryophyllidea, Pseudophyllidea and Proteocephalidea (Eucestoda) inferred from 18S rRNA.

A phylogenetic analysis of tapeworms (Eucestoda) based on complete sequences of the 18S rRNA genes of 43 taxa (including new sequences of 12 species) was carried out, with the emphasis on the groups parasitising teleost fish and reptiles. Spathebothriidea and Trypanorhyncha (the latter group being paraphyletic) appeared as basal groups of the Eucestoda but their position was not stable. The tetrafossate orders (Litobothriidea, Lecanicephalidea, Tetraphyllidea, Proteocephalidea, Nippotaeniidea, Tetrabothriidea and Cyclophyllidea) were well separated from the remaining groups. Results supported polyphyly of the Pseudophyllidea formed by two distinct clades: one with diphyllobothriids (Diphyllobothrium, Schistocephalus, Spirometra and Duthiersia) and another including Abothrium, Probothriocephalus, Eubothrium and Bothriocephalus. The former pseudophyllidean clade formed a separate branch with the Caryophyllidea (Khawia and Hunterella) and Haplobothriidea (Haplobothrium), the latter taxon being closely related to either caryophyllideans or diphyllobothriids in different analyses. Proteocephalideans formed a monophyletic group in all analyses and constituted a clade within the Tetraphyllidea thus rendered paraphyletic. Within the Proteocephalidea, the Acanthotaeniinae (Acanthotaenia from reptiles in Africa) and Gangesiinae (Gangesia and Silurotaenia from silurid fish in the Palearctic Region) were separated from parasites of freshwater fish and mammals. The family Proteocephalidae was found to be paraphyletic due to the placement of a monticelliid species, Monticellia sp., in a clade within the former family. The genus Proteocephalus appeared as an artificial assemblage of unrelated taxa which is congruent with previous molecular analyses.

Phylogenetic analysis of Sarcocystis spp. of mammals and reptiles supports the coevolution of Sarcocystis spp. with their final hosts.

Sequences of the small subunit rRNA genes were obtained for two coccidians, Sarcocystis dispersa and an unnamed Sarcocystis sp. which parasitise the European barn owl and an African viperid snake as their final host, respectively, and share mouse as their intermediate host. Phylogenetic analysis of the sequence data showed that Sarcocystis sp. from the viperid snake is most closely related to another Sarcocystis sp. isolated from an American crotalid snake, while S. dispersa grouped with other bird-transmitted species. The available dataset failed to resolve the evolutionary relationships among four major branches into which all Sarcocystidae and Isospora spp. were split. However, within these branches, the phylogenetic relationships of the majority of analysed members of the genus Sarcocystis reflected coevolution with their final, rather than intermediate hosts.

Molecular phylogenetic relatedness of Frenkelia spp. (Protozoa, Apicomplexa) to Sarcocystis falcatula Stiles 1893: is the genus Sarcocystis paraphyletic?

The coccidians Frenkelia microti and F. glareoli (Apicomplexa: Sarcocystidae) form tissue cysts in the brain of small rodents (intermediate hosts) while oocysts are formed in the intestine of final hosts, buzzards of the genus Buteo. The inclusion of the small subunit ribosomal RNA gene sequences (SSU rRNA) of both Frenkelia species into the SSU rRNA trees of other, tissue cyst-forming coccidia strongly supports paraphyly of the genus Sarcocystis. Frenkelia spp. exhibit close relatedness to Sarcocystis falcatula Stiles 1893, a bird-opossum parasite, recognized under its junior synonym S. neurona Dubey et al. 1991, as the causative agent of equine protozoan myeloencephalitis on the American continent. As the definition of the genus Frenkelia is based on a plesiomorphic character (affinity to the neural tissue) of supposedly low phylogenetic value, the synonymization of the genus Frenkelia with Sarcocystis is proposed. This renders the genus Sarcocystis monophyletic.

In vitro culture and phylogenetic analysis of "Candidatus Arsenophonus triatominarum," an intracellular bacterium from the triatomine bug, Triatoma infestans.

An intracellular symbiotic bacterium was isolated from the hemolymph of Triatoma infestans and cultured in an Aedes albopictus cell line. 16S ribosomal DNA sequence analysis revealed that the bacterium was a member of the gamma-3 subgroup of the class Proteobacteria, having 96.2% sequence identity with the most closely related bacterium, Arsenophonus nasoniae, the causative agent of the son-killer trait in the parasitoid wasp Nasonia vitripennis. These bacteria share morphological features and a common tissue distribution and transmission mode. The A. nasoniae-T. infestans symbiont branch represents a lineage of insect symbionts which may be capable of horizontal transmission between phylogenetically distant host insects. We propose that the intracellular symbiont from T. infestans be classified as "Candidatus Arsenophonus triatominarum." The bacterium found in the hemocytes of T. infestans is designated the type strain of this species.

Phylogenetic characterization of two transovarially transmitted endosymbionts of the bedbug Cimex lectularius (Heteroptera:Cimicidae).

Two different inherited bacterial symbionts from ovary tissue of the bedbug Cimex lectularius were characterized by gene amplification and sequencing analysis of their 16S rDNA gene. The first bacterium belongs to the Wolbachia subgroup of the alpha-Proteobacteria, the second is a member of the gamma-subdivision, and is closely related to the bacterial parasite of the leafhopper Euscelidius variegatus (BEV) which was shown to be capable of transovarial transmission. The high similarity (> 97%) between the C. lectularius symbiont and BEV indicates that these two microorganisms belong to the same lineage and share the ability to invade distant insect hosts to fulfill their symbiotic functions and to establish transovarial transmission to future generations.

Phylogeny and potential transmission routes of midgut-associated endosymbionts of tsetse (Diptera:Glossinidae).

Many tsetse species (Diptera: Glossinidae) harbour two morphologically different intracellular endosymbiotic microorganisms associated with gut tissue: primary (P) and secondary (S) endosymbionts. The P-endosymbionts of tsetse (Wigglesworthia glossinidia) are sequestered in specialized epithelial cells, bacteriocytes, which form a structure (bacteriome) in the anterior portion of the gut. Phylogenetic characterization of P-endsymbionts from the three subgenera of genus Glossina has shown that these organisms constitute a distinct lineage within the gamma-subdivision of Proteobacteria and have evolved concordantly with their insect host species, suggesting an evolutionarily ancient association for this symbiosis. The S-endosymbiont is a smaller (1-2 micron) gram-negative rod and is harboured in midgut epithelial cells. Its phylogenetic characterization from Glossina morsitans morsitans had shown that it is a member of the family Enterobacteriaceae within the gamma-3 subdivision of the Proteobacteria, closely related to enteric bacteria. Some tsetse species harbour a third bacterium in their reproductive tissue, which was shown phylogenetically to belong to to the Wolbachia pipientis assemblage of microorganisms. Here, we show that S-endosymbionts from five tsetse species, representing all three subgenera, form a cluster of closely related microorganisms, based on their almost identical 16S rRNA gene sequences. The high similarity provides strong evidence of recent independent acquisition of S-endosymbionts by individual tsetse species, unlike Wigglesworthia which displays concordant evolution with host insect species. A PCR-based assay and restriction fragment length polymorphism (RFLP) analysis was developed to localize the S-endosymbionts and Wigglesworthia in ovary, egg, milk-gland and spermatheca tissues in order to investigate the potential routes for the vertical transmission of these symbionts to the intrauterine larvae. Only S-endosymbionts were found to infect milk gland tissue, suggesting that milk gland secretions represent a route of transmission for these symbionts into the developing larva. The ovary tissue was found to harbour only Wolbachia, confirming its transovarial transmission, whereas the mode of transmission of Wigglesworthia remains unknown.

Two hemocyte populations in Triatoma infestans: ultrastructural and lectin-binding characterization.

Two distinct hemocyte populations are determined in the hemolymph of the triatomine bug Triatoma infestans Klug, oenocytoids and plasmatocytes, and their independent origin from separate stem cells is shown. Both hemocyte populations differ considerably in their morphology, ultrastructure and lectin-binding properties. While oenocytoids are quite uniform with easily definable cells which do not to bind any assayed lectin, the plasmatocytes are a very polymorphic population possessing several morphological types and displaying a positive reactivity with lectins.

Lectins (hemagglutinins) in the gut of the important disease vectors.

The review is devoted to the gut lectins/hemagglutinins of the following representatives of important disease vectors: ticks, kissing-bugs, mosquitoes, sandflies and tsetse flies. The paper surveys the recent knowledge on these carbohydrate binding factors with respect to their structural and functional properties, and their significance for pathogen/parasite transmission by the blood-sucking arthropods. Recent results suggest that in most vectors the gut lectin activities are blood-meal enhanced, might participate in blood-meal processing and digestion and could serve as antibacterial and antiparasitic agents.