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Current phylogenies of the intracellular bacteria belonging to the genus Wolbachia identify six major clades (A-F), termed 'supergroups', but the branching order of these supergroups remains unresolved. Supergroups A, B and E include most of the wolbachiae found thus far in arthropods, while supergroups C and D include most of those found in filarial nematodes. Members of supergroup F have been found in arthropods (i.e. termites), and have previously been detected in the nematode Mansonella ozzardi, a causative agent of human filariasis. To resolve the phylogenetic positions of Wolbachia from Mansonella spp., and other novel strains from the flea Ctenocephalides felis and the filarial nematode Dipetalonema gracile, the authors generated new DNA sequences of the Wolbachia genes encoding citrate synthase (gltA), heat-shock protein 60 (groEL), and the cell division protein ftsZ. Phylogenetic analysis confirmed the designation of Wolbachia from Mansonella spp. as a member of the F supergroup. In addition, it was found that divergent lineages from Dip. gracile and Cte. felis lack any clear affiliation with known supergroups, indicating further genetic diversity within the Wolbachia genus. Finally, although the data generated did not permit clear resolution of the root of the global Wolbachia tree, the results suggest that the transfer of Wolbachia spp. from arthropods to nematodes (or vice versa) probably occurred more than once.

Wolbachia pipientis is a bacterial endosymbiont associated with arthropods and filarial nematodes. In filarial nematodes, W. pipientis has been shown to play an important role in the biology of the host and in the immuno-pathology of filariasis. Several species of filariae, including the most important parasites of humans and animals (e.g. Onchocerca volvulus, Wuchereria bancrofti and Dirofilaria immitis) have been shown to harbour these bacteria. Other filarial species, including an important rodent species (Acanthocheilonema viteae), which has been used as a model for the study of filariasis, do not appear to harbour these symbionts. There are still several open questions about the distribution of W. pipientis in filarial nematodes. Firstly the number of species examined is still limited. Secondly, it is not clear whether the absence of W. pipientis in negative species could represent an ancestral characteristic or the result of a secondary loss. Thirdly, several aspects of the phylogeny of filarial nematodes are still unclear and it is thus difficult to overlay the presence/absence of W. pipientis on a tree representing filarial evolution. Here we present the results of a PCR screening for W. pipientis in 16 species of filariae and related nematodes, representing different families/subfamilies. Evidence for the presence of W. pipientis is reported for five species examined for the first time (representing the genera Litomosoides, Litomosa and Dipetalonema); original results on the absence of this bacterium are reported for nine species; for the remaining two species, we have confirmed the absence of W. pipientis recently reported by other authors. In the positive species, the infecting W. pipientis bacteria have been identified through 16S rDNA gene sequence analysis. In addition to the screening for W. pipientis in 16 species, we have generated phylogenetic reconstructions based on mitochondrial gene sequences (12S rDNA; COI), including a total of 28 filarial sp

A 12-year-old, 13 kg, mixed-breed male dog was referred for anorexia and depression. The dog showed discomfort on abdominal palpation. Abdominal ultrasound examination revealed multiple, small, round anechoic cystic structures. Cystic fluid obtained with fine needle aspiration contained several 2-4 mm white motile flecks. Microscopic examination of the fluid revealed numerous irregularly shaped organisms measuring several hundred microns to 3 mm, the morphology of which was suggestive of intact and fragmented acephalic metacestodes of the genus Mesocestoides sp. Molecular analysis confirmed that the peritoneal infection was caused by Mesocestoides sp.

Universal bacterial primers are often used in PCR-coupled sequencing approaches to investigate environmental and host-associated bacterial communities. Some of these primers can also amplify eukaryotic DNA. This is leading to the submission of datasets to public databases which are erroneously annotated as prokaryotic sequences. The present note sends a message about the risk of submitting incorrectly annotated sequence data and suggests a reliable approach for the sequencing of 16S rRNA genes and identification of bacteria within complex communities.

In modern taxonomy, DNA barcoding is particularly useful where biometric parameters are difficult to determine or useless owing to the poor quality of samples. These situations are frequent in parasitology. Here, we present an integrated study, based on both DNA barcoding and morphological analysis, on cestodes belonging to the genus Taenia, for which biodiversity is still largely underestimated. In particular, we characterized cestodes from Italian wildcats (Felis silvestris silvestris), free-ranging domestic cats (Felis silvestris catus) and hybrids populations. Adult taeniids were collected by post-mortem examinations of the hosts and morphologically identified as Taenia taeniaeformis. We produced cox1 barcode sequences for all the analysed specimens, and we compared them with reference sequences of individuals belonging to the genus Taenia retrieved from GenBank. In order to evaluate the performance of a DNA barcoding approach to discriminate these parasites, the strength of correlation between species identification based on classical morphology and the molecular divergence of cox1 sequences was measured. Our study provides clear evidence that DNA barcoding is highly efficient to reveal the presence of cryptic lineages within already-described taeniid species. Indeed, we detected three well-defined molecular lineages within the whole panel of specimens morphologically identified as T. taeniaeformis. Two of these molecular groups were already identified by other authors and should be ranked at species level. The third molecular group encompasses only samples collected in Italy during this study, and it represents a third candidate species, still morphologically undescribed.

Background: Wolbachia are intriguing symbiotic endobacteria with a peculiar host range that includes arthropods and a single nematode family, the Onchocercidae encompassing agents of filariases. This raises the question of the origin of infection in filariae. Wolbachia infect the female germline and the hypodermis. Some evidences lead to the theory that Wolbachia act as mutualist and coevolved with filariae from one infection event: their removal sterilizes female filariae; all the specimens of a positive species are infected; Wolbachia are vertically inherited; a few species lost the symbiont. However, most data on Wolbachia and filaria relationships derive from studies on few species of Onchocercinae and Dirofilariinae, from mammals. Methodology/Principal Findings: We investigated the Wolbachia distribution testing 35 filarial species, including 28 species and 7 genera and/or subgenera newly screened, using PCR, immunohistochemical staining, whole mount fluorescent analysis, and cocladogenesis analysis. (i) Among the newly screened Onchocercinae from mammals eight species harbour Wolbachia but for some of them, bacteria are absent in the hypodermis, or in variable density. (ii) Wolbachia are not detected in the pathological model Monanema martini and in 8, upon 9, species of Cercopithifilaria. (iii) Supergroup F Wolbachia is identified in two newly screened Mansonella species and in Cercopithifilaria japonica. (iv) Type F Wolbachia infect the intestinal cells and somatic female genital tract. (v) Among Oswaldofilariinae, Waltonellinae and Splendidofilariinae, from saurian, anuran and bird respectively, Wolbachia are not detected. Conclusions/Significance: The absence of Wolbachia in 63% of onchocercids, notably in the ancestral Oswaldofilariinae estimated 140 mya old, the diverse tissues or specimens distribution, and a recent lateral transfer in supergroup F Wolbachia, modify the current view on the role and evolution of the endosymbiont and their hosts. Further

Background: We compared here the suitability and efficacy of traditional morphological approach and DNA barcoding to distinguish filarioid nematodes species (Nematoda, Spirurida). A reliable and rapid taxonomic identification of these parasites is the basis for a correct diagnosis of important and widespread parasitic diseases. The performance of DNA barcoding with different parameters was compared measuring the strength of correlation between morphological and molecular identification approaches. Molecular distance estimation was performed with two different mitochondrial markers (coxI and 12S rDNA) and different combinations of data handling were compared in order to provide a stronger tool for easy identification of filarioid worms. Results: DNA barcoding and morphology based identification of filarioid nematodes revealed high coherence. Despite both coxI and 12S rDNA allow to reach high-quality performances, only coxI revealed to be manageable. Both alignment algorithm, gaps treatment, and the criteria used to define the threshold value were found to affect the performance of DNA barcoding with 12S rDNA marker. Using coxI and a defined level of nucleotide divergence to delimit species boundaries, DNA barcoding can also be used to infer potential new species. Conclusion: An integrated approach allows to reach a higher discrimination power. The results clearly show where DNA-based and morphological identifications are consistent, and where they are not. The coherence between DNA-based and morphological identification for almost all the species examined in our work is very strong. We propose DNA barcoding as a reliable, consistent, and democratic tool for species discrimination in routine identification of parasitic nematodes.

Scaphoideus titanus is the insect vector of flavescence dorée (FD), a yellow disease of grapevines. Observations on adult females and nymphs of S. titanus showed that this insect is associated with a complex microbial community. Ultrastructural analysis showed that the fat body, salivary glands and ovary of the insect harbour microorganisms showing the brush-like structure typically observed in the genus Cardinium. In particular, it has been shown that these symbiotic bacteria are present both in the follicular cells and in the eggs. In addition, cells resembling bacteriocytes, harbouring numerous Cardinium symbionts in the cytoplasm, were observed in the apical portion of the ovary in adult females. These cells are likely responsible for bacterial transmission to the ovary. Optical microscopy showed that the fat body harbours an enormous population of yeast-like symbionts (YLSs). Ultrastructural observations showed that these symbionts are enclosed within specialized cells of the fat body and are also present in the ovary, where they are found in both the follicular cells and the eggs. There is thus evidence that both Cardinium and the YLSs are transovarially transmitted to the offspring. To our knowledge, S. titanus is the sole insect known to transmit two different kinds of symbionts to the eggs, a prokaryote and an eukaryote. Gene sequence analysis and in situ hybridization led to the identification of YLSs as members of the class Sordariomycetes (=Pyrenomycetes). Finally, ultrastructural observation of the midgut content revealed the presence, in both adult females and nymphs, of a complex microbial community, which include a phytoplasma-like microorganism, likely the agent of FD.

A recent survey on the microflora associated with the leafhopper Scaphoideus titanus Ball (Hemiptera Cicadellidae), the vector of “flavescence dorée” (FD), showed the presence of bacteria belonging to the genus Asaia. These bacteria are symbionts of insects of the genus Anopheles, malarial mosquito vectors. We focused on the association between Asaia sp. and S. titanus with the aim to evaluate a possible role of this microorganism as a symbiotic control agent. After development of artificial feeding systems for S. titanus, quantitative PCR on insects or feeds DNA was performed with specific primers. Results underlined that Asaia sp. was dominant in the insect microflora. A strain of Asaia expressing a green fluorescent protein was used to perform recolonization of the body of S. titanus, showing the colonization by the bacterium of salivary glands, guts, female and male reproductive organs of the insect. In situ hybridization with specific probes was performed on insect dissections, confirming the presence of the bacterium in spermatic bundles and in Malpighian tubules. Easy cultivability, dominance within the bacterial population in the insect body, cryogenic preservability and easiness for genetic manipulation demonstrated by the overall data make Asaia sp. an optimal candidate for carrying factors for FD control.

Here, we show that an alpha-proteobacterium of the genus Asaia is stably associated with larvae and adults of Anopheles stephensi, an important mosquito vector of Plasmodium vivax, a main malaria agent in Asia. Asaia bacteria dominate mosquito-associated microbiota, as shown by 16S rRNA gene abundance, quantitative PCR, transmission electron microscopy and in situ-hybridization of 16S rRNA genes. In adult mosquitoes, Asaia sp. is present in high population density in the female gut and in the male reproductive tract. Asaia sp. from An. stephensi has been cultured in cell-free media and then transformed with foreign DNA. A green fluorescent protein-tagged Asaia sp. strain effectively lodged in the female gut and salivary glands, sites that are crucial for Plasmodium sp. development and transmission. The larval gut and the male reproductive system were also colonized by the transformed Asaia sp. strain. As an efficient inducible colonizer of mosquitoes that transmit Plasmodium sp., Asaia sp. may be a candidate for malaria control.

Woodroaches of the genus Cryptocercus are subsocial and xylophagous cockroaches, distributed in North America and Asia. Studies on male chromosome number in Nearctic species have shown that diploid numbers vary from 2n=37 to 2n=47; numbers from Palearctic species were heretofore unknown. Two hypotheses have been proposed to explain the varying number of chromosomes among Nearctic species: the serial reduction hypothesis, and the parallel scenario. We performed phylogenetic analyses of the COII gene in these species and found evidence for the topology (47(45(43(39,37), which is congruent with the serial reduction hypothesis. We also determined chromosome numbers for the first time in Palearctic species, and found Cryptocercus primarius and Cryptocercus relictus to have relatively low chromosome numbers (2n=17-21) compared to their Nearctic relatives. Finally, our study determined the phylogenetic position of Cryptocercus primarius among other Asian taxa.

This collection of articles provides an account of six presentations delivered at the 19th International Conference of the World Association for the Advancement of Veterinary Parasitology (WAAVP) (held in New Orleans, Louisiana, USA, from 10 to 14 August 2003) in a symposium session on Molecular Systematics and Diagnosis, organised and chaired by R.B. Gasser and D.S. Zarlenga. The focus was on recent advances in molecular tools for specific and genotypic identification, diagnosis, systematics and population genetics, with special emphasis on investigations of parasitic nematodes and protists.