Michela Barbuto

Integrated taxonomy: traditional approach and DNA barcoding for the identification of filarioid worms and related parasites (Nematoda)

BackgroundWe 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.ResultsDNA 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.ConclusionAn 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.

New Insights into the Evolution of Wolbachia Infections in Filarial Nematodes Inferred from a Large Range of Screened Species

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 genomic analyses on some of the newly sampled species are welcomed to decipher the open questions.

A DNA Barcoding Approach to Characterize Pollen Collected by Honeybees

In the present study, we investigated DNA barcoding effectiveness to characterize honeybee pollen pellets, a food supplement largely used for human nutrition due to its therapeutic properties. We collected pollen pellets using modified beehives placed in three zones within an alpine protected area (Grigna Settentrionale Regional Park, Italy). A DNA barcoding reference database, including rbcL and trnH-psbA sequences from 693 plant species (104 sequenced in this study) was assembled. The database was used to identify pollen collected from the hives. Fifty-two plant species were identified at the molecular level. Results suggested rbcL alone could not distinguish among congeneric plants; however, psbA-trnH identified most of the pollen samples at the species level. Substantial variability in pollen composition was observed between the highest elevation locality (Alpe Moconodeno), characterized by arid grasslands and a rocky substrate, and the other two sites (Cornisella and Ortanella) at lower altitudes. Pollen from Ortanella and Cornisella showed the presence of typical deciduous forest species; however in samples collected at Ortanella, pollen of the invasive Lonicera japonica, and the ornamental Pelargonium x hortorum were observed. Our results indicated pollen composition was largely influenced by floristic local biodiversity, plant phenology, and the presence of alien flowering species. Therefore, pollen molecular characterization based on DNA barcoding might serve useful to beekeepers in obtaining honeybee products with specific nutritional or therapeutic characteristics desired by food market demands.

A new type F Wolbachia from Splendidofilariinae (Onchocercidae) supports the recent emergence of this supergroup.

Wolbachia are vertically transmitted endosymbiotic bacteria of arthropods and onchocercid nematodes. It is commonly accepted that they co-evolved with their filarial hosts, and have secondarily been lost in some species. However, most of the data on the Wolbachia/Onchocercidae relationship have been derived from studies on two subfamilies, the Dirofilariinae and the Onchocercinae, which harbour parasites of humans and domestic animals. Within the last few years, analyses of more diverse material have suggested that some groups of Onchocercidae do not have Wolbachia, such as recently studied Splendidofilariinae from birds. This study takes advantage of the analysis of additional Splendidofilariinae, Rumenfilaria andersoni from a Finnish reindeer and Madathamugadia hiepei from a South African gecko, using PCR, immunohistochemical staining and whole-mount fluorescent analysis to detect Wolbachia and describe its strains. A DNA barcoding approach and phylogenetic analyses were used to investigate the symbiosis between Wolbachia and the Onchocercidae. A new supergroup F Wolbachia was demonstrated in M. hiepei, representing the first filarial nematode harbouring Wolbachia described in a non-mammalian host. In the adult, Wolbachia infects the female germline but not the hypodermis, and intestinal cells are also infected. The phylogenetic analyses confirmed a recent emergence of supergroup F. They also suggested several events of horizontal transmission between nematodes and arthropods in this supergroup, and the existence of different metabolic interactions between the filarial nematodes and their symbionts.

Rhabdias rhampholeonis n. sp. and Rhabdias mariauxi n. sp. (Nematoda, Rhabdiasoidea), first lung worms from leaf chameleons : Description, molecular evidence and notes on biology

Rhabdias rhampholeonis n. sp. from Rhampholeon (Rh.) spectrum, Cameroon, and Rhabdias mariauxi n. sp. from Rieppeleon brevicaudatus, Tanzania, are the first lung worms from leaf chameleons. The new species are similar to the majority of species parasitic in chamaeleonids by having a long (>or=10 mm) and thick body (>or=500 microm), long oesophagus (>or=800 microm), wide buccal capsule (>or=40 microm) and low buccal ratio (<0.5). They most closely resemble Rhabdias chamaeleonis and Rhabdias cristati parasitic in Trioceros spp. from East Africa and Cameroon, respectively. Main distinctive characters are a buccal capsule composed of two segments and the head shape. The dorso-ventrally flattened buccal capsule of R. mariauxi n. sp. is unique in Rhabdias parasitising Chamaeleonidae. Sequences of the 12S rDNA and mitochondrial cytochrome c oxidase subunit 1 (coxI) genes were obtained and compared to those of Rhabdias okuensis, the only sequences published for chamaeleonid lung worms. The smallest nucleotide interspecific distances were found between R. mariauxi n. sp. and the former species of Trioceros from Cameroon. Hermaphroditism in females in the lungs, and R. mariauxi n. sp. free-living stages are like in other species from Chamaeleonidae, but the number of infective larvae produced per free-living female (one or two) was not fixed.

Ocular dirofilariosis by Dirofilaria immitis in a dog: first case report from Europe

A five-year-old, entire female mixed-breed dog was presented with corneal oedema and episcleral hyperaemia in the left eye. The ophthalmological examination revealed the presence of a free-swimming nematode in the anterior chamber. Circulating microfilariae were not observed by a modified Knott test nor were adult antigens detected in serum by a commercial ELISA. The parasite was surgically removed from the dogs eye, but its anterior end was damaged during the surgery. Based on the morphology of the posterior end, the nematode was preliminarily identified as a male Dirofilaria immitis. The species identification was confirmed by PCR amplification and sequencing of the mitochondrial coxI and 12S rDNA genes, using a DNA barcoding approach. Although other cases of ocular dirofilariosis by D. immitis have been previously recorded in Australia and the United States, the case reported herein is the first in a dog from Europe.

Toward a resolution of a taxonomic enigma: First genetic analyses of Paradoxornis webbianus and Paradoxornis alphonsianus (Aves: Paradoxornithidae) from China and Italy

Paradoxornis webbianus and Paradoxornis alphonsianus naturally occur in South-East Asia. Due to a recent introduction, a mixed population currently occurs in northern Italy. A preliminary phylogeographic analysis using samples from Italy and China found little genetic differentiation between the two taxa and revealed the existence of two molecular lineages, sympatric in some part of China, that do not correspond to the morphological classification. Possible taxonomic changes and preliminary inferences on the relationships between Chinese and the Italian populations and on the likely provenance of the founders introduced in Italy are also discussed.

Cutidiplogaster manati n. gen., n. sp. (Nematoda: Diplogastridae) from skin lesions of a West Indian manatee (Sirenia) from the Okinawa Churaumi Aquarium

A new species of the nematode taxon Diplogastridae is described on morphology and additional molecular data. The new species cannot be grouped in any of the currently accepted diplogastrid genera. Therefore, we introduce the new genus Cutidiplogaster n. gen. along with the description of our new species Cutidiplogaster manati n. gen., n. sp. Cutidiplogaster manati n. gen., n. sp. was isolated from skin lesions of manatees, Trichechus manatus manatus , kept in Okinawa Churaumi Aquarium, Japan. The specimens were found anchored with their extraordinarily long tails to the small, thick epidermal bumps within thread algae that form part of the epizoic community on the manatee skin. Cutidiplogaster manati n. gen., n. sp. is characterised by the tube-shaped and finely annulated postdental part of the stegostom that makes it distinct from all other Diplogastridae so far known, and the coiled long tail that makes it unique in comparison with all known diplogastrids. On the basis of detail in stoma morphology in C. manati n. gen., n. sp., we discuss implications in homologising diplogastrid mouthparts and their evolution.