Emanuele Ferri

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.

Phylogenomic evidence for the presence of a flagellum and cbb3 oxidase in the free-living mitochondrial ancestor

The initiation of the intracellular symbiosis that would give rise to mitochondria and eukaryotes was a major event in the history of life on earth. Hypotheses to explain eukaryogenesis fall into two broad and competing categories: those proposing that the host was a phagocytotic proto-eukaryote that preyed upon the free-living mitochondrial ancestor (hereafter FMA), and those proposing that the host was an archaebacterium that engaged in syntrophy with the FMA. Of key importance to these hypotheses are whether the FMA was motile or nonmotile, and the atmospheric conditions under which the FMA thrived. Reconstructions of the FMA based on genome content of Rickettsiales representatives-generally considered to be the closest living relatives of mitochondria-indicate that it was nonmotile and aerobic. We have sequenced the genome of Candidatus Midichloria mitochondrii, a novel and phylogenetically divergent member of the Rickettsiales. We found that it possesses unique gene sets found in no other Rickettsiales, including 26 genes associated with flagellar assembly, and a cbb(3)-type cytochrome oxidase. Phylogenomic analyses show that these genes were inherited in a vertical fashion from an ancestral α-proteobacterium, and indicate that the FMA possessed a flagellum, and could undergo oxidative phosphorylation under both aerobic and microoxic conditions. These results indicate that the FMA played a more active and potentially parasitic role in eukaryogenesis than currently appreciated and provide an explanation for how the symbiosis could have evolved under low levels of oxygen.

Integrated operational taxonomic units (IOTUs) in echolocating bats: a bridge between molecular and traditional taxonomy.

Background Nowadays, molecular techniques are widespread tools for the identification of biological entities. However, until very few years ago, their application to taxonomy provoked intense debates between traditional and molecular taxonomists. To prevent every kind of disagreement, it is essential to standardize taxonomic definitions. Along these lines, we introduced the concept of Integrated Operational Taxonomic Unit (IOTU). IOTUs come from the concept of Operational Taxonomic Unit (OTU) and paralleled the Molecular Operational Taxonomic Unit (MOTU). The latter is largely used as a standard in many molecular-based works (even if not always explicitly formalized). However, while MOTUs are assigned solely on molecular variation criteria, IOTUs are identified from patterns of molecular variation that are supported by at least one more taxonomic characteristic. Methodology/Principal Findings We tested the use of IOTUs on the widest DNA barcoding dataset of Italian echolocating bats species ever assembled (i.e. 31 species, 209 samples). We identified 31 molecular entities, 26 of which corresponded to the morphologically assigned species, two MOTUs and three IOTUs. Interestingly, we found three IOTUs in Myotis nattereri, one of which is a newly described lineage found only in central and southern Italy. In addition, we found a level of molecular variability within four vespertilionid species deserving further analyses. According to our scheme two of them (i.e. M. bechsteinii and Plecotus auritus) should be ranked as unconfirmed candidate species (UCS). Conclusions/Significance From a systematic point of view, IOTUs are more informative than the general concept of OTUs and the more recent MOTUs. According to information content, IOTUs are closer to species, although it is important to underline that IOTUs are not species. Overall, the use of a more precise panel of taxonomic entities increases the clarity in the systematic field and has the potential to fill the gaps between modern and traditional taxonomy.

Analytical approaches for DNA barcoding data – how to find a way for plants?

Abstract Identification of living beings is a core problem in biology. Identification can be achieved by several methods, but in recent decades, molecular techniques have become more and more common. DNA barcoding is an initiative that was launched less than 10 years ago, but which has already gained a reputation in biological studies. The method was originally applied to metazoans, but it was rapidly used to discriminate plants. However, in plants there were several problems, including the choice of the right markers, their universality and the discrimination power. On the whole, DNA barcoding in plants is slightly behind when compared to animals, but it is likely that the gap will be filled in a short period of time. In a DNA barcoding approach, molecular and bioinformatics are deeply linked to generate the identification system. In this paper, we summarize the recent advances in DNA barcoding bioinformatics, by reviewing the principal approaches and critically analyzing the procedures.

Plasma Levels of Bacterial DNA in HIV Infection: The Limits of Quantitative Polymerase Chain Reaction

To the Editor—In a recent article, Jiang et al [1] showed that blood levels of bacterial DNA, as determined by quantitative polymerase chain reaction (PCR) on 16S ribosomal DNA fragments, were significantly higher in human immunodefi-ciency virus (HIV)–infected patients than were those in uninfected control patients. Moreover, the level of bacterial DNA in the blood was shown to decrease after an-tiretroviral treatment. Blood levels of 16S ribosomal DNA could be regarded as an indicator of the translocation of microbial molecules from the gut lumen to other body compartments. This could be responsible for chronic activation of the immune system and could have a role in AIDS progression.

A Randomized, Double-Blind, Placebo-Controlled Trial: The Efficacy of Multispecies Probiotic Supplementation in Alleviating Symptoms of Irritable Bowel Syndrome Associated with Constipation

Background and Aim. The efficacy of supplementation treatment with two multispecies probiotic formulates on subjects diagnosed with IBS-C and the assessment of their gut microbiota were investigated. Methods. A randomized, double-blind, three-arm parallel group trial was carried out on 150 IBS-C subjects divided into three groups (F_1, F_2, and F_3). Each group received a daily oral administration of probiotic mixtures (for 60 days) F_1 or F_2 or placebo F_3, respectively. Fecal microbiological analyses were performed by species-specific qPCR to assess the different amount of probiotics. Results. The percentage of responders for each symptom was higher in the probiotic groups when compared to placebo group during the treatment period (t60) and was maintained quite similar during the follow-up period (t90). Fecal analysis demonstrated that probiotics of the formulations increased during the times of treatment only in fecal DNA from subjects treated with F_1 and F_2 and not with F_3, and the same level was maintained during the follow-up period. Conclusions. Multispecies probiotic supplementations are effective in IBS-C subjects and induce a different assessment in the composition of intestinal microbiota. This clinical study is registered with the clinical study registration number ISRCTN15032219.

Towards a better understanding of Apis mellifera and Varroa destructor microbiomes: introducing ‘phyloh’ as a novel phylogenetic diversity analysis tool

The study of diversity in biological communities is an intriguing field. Huge amount of data are nowadays available (provided by the innovative DNA sequencing techniques), and management, analysis and display of results are not trivial. Here, we propose for the first time the use of phylogenetic entropy as a measure of bacterial diversity in studies of microbial community structure. We then compared our new method (i.e. the web tool phyloh) for partitioning phylogenetic diversity with the traditional approach in diversity analyses of bacteria communities. We tested phyloh to characterize microbiome in the honeybee (Apis mellifera, Insecta: Hymenoptera) and its parasitic mite varroa (Varroa destructor, Arachnida: Parasitiformes). The rationale is that the comparative analysis of honeybee and varroa microbiomes could open new perspectives concerning the role of the parasites on honeybee colonies health. Our results showed a dramatic change of the honeybee microbiome when varroa occurs, suggesting that this parasite is able to influence host microbiome. Among the different approaches used, only the entropy method, in conjunction with phylogenetic constraint as implemented in phyloh, was able to discriminate varroa microbiome from that of parasitized honeybees. In conclusion, we foresee that the use of phylogenetic entropy could become a new standard in the analyses of community structure, in particular to prove the contribution of each biological entity to the overall diversity.

Errors in ribosomal sequence datasets generated using PCR-coupled 'panbacterial' pyrosequencing, and the establishment of an improved approach.

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.

Poisonous or non-poisonous plants? DNA-based tools and applications for accurate identification

Plant exposures are among the most frequently reported cases to poison control centres worldwide. This is a growing condition due to recent societal trends oriented towards the consumption of wild plants as food, cosmetics, or medicine. At least three general causes of plant poisoning can be identified: plant misidentification, introduction of new plant-based supplements and medicines with no controls about their safety, and the lack of regulation for the trading of herbal and phytochemical products. Moreover, an efficient screening for the occurrence of plants poisonous to humans is also desirable at the different stages of the food supply chain: from the raw material to the final transformed product. A rapid diagnosis of intoxication cases is necessary in order to provide the most reliable treatment. However, a precise taxonomic characterization of the ingested species is often challenging. In this review, we provide an overview of the emerging DNA-based tools and technologies to address the issue of poisonous plant identification. Specifically, classic DNA barcoding and its applications using High Resolution Melting (Bar-HRM) ensure high universality and rapid response respectively, whereas High Throughput Sequencing techniques (HTS) provide a complete characterization of plant residues in complex matrices. The pros and cons of each approach have been evaluated with the final aim of proposing a general user’s guide to molecular identification directed to different stakeholder categories interested in the diagnostics of poisonous plants.