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

Frontiers in Zoology Integrated taxonomy: traditional approach and DNA barcoding for the identification of filarioid worms and related parasites (Nematoda) Emanuele Ferri 1 2 Michela Barbuto 1 Odile Bain 0 Andrea Galimberti 1 Shigehiko Uni 5 Ricardo Guerrero 4 Hubert Fert 3 Claudio Bandi 2 Coralie Martin 0 Maurizio Casiraghi 1 0 Parasitologie Comparee et Modeles experimentaux USM 307, Museum National d'Histoire Naturelle , 75231 Paris Cedex 05 , France 1 Dipartimento di Biotecnologie e Bioscienze, ZooPlantLab, Universita degli Studi di Milano Bicocca , P.zza della Scienza 2, 20126 Milano , Italy 2 Dipartimento di Patologia Animale, Igiene e Sanita Pubblica Veterinaria, Sezione di Patologia Animale e Parassitologia, Universita degli Studi di Milano , via Celoria 10, 20133 Milano , Italy 3 JE 2533 USC AFSSA Vecpar UFR de Pharmacie , 51 rue Cognacq-Jay, 51096 Reims , France 4 Instituto de Zoologia Tropical, Faculdad de Ciencias, Universidad Central de Venezuela , PO Box 47058, 1041 Caracas , Venezuela 5 Department of Medical Zoology, Osaka City University Medical School , 1-4-3 Asahimachi, Abeno-ku, Osaka 545-8585 Japan 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. - Background The identification of living species is one of the major goals of modern biology. Species can be delimited only in relation to other species, it is trivial, but many discriminators and species concepts can be used for this purpose. Molecular data have become widely used to aid rapid assessment of species diversity, and the DNA barcoding initiative [1] is one prominent line of research within this field, coordinated by the Consortium for the Barcode of Life (CBoL, http://barcoding.si.edu). DNA barcoding involves rapid sequencing of one or a few genes from several representatives of a species, as well as comparisons of these sequences within and between species. The method has revealed examples of cryptic species diversity in various taxa [2,3]. DNA barcoding aims at the development of a universal, standardized and economical tool, but the fear is that to gain a sufficient accuracy the sampling should be massive, and, accordingly, the PCR and sequencing efforts expensive. Finding a balance between standardization, low costs and accuracy is difficult, and researchers have to take key decisions on the level of accuracy they want to get and the costs they can reasonably sustain. A major strength of DNA barcoding is that it allows correlating any life stages of a living organism, or also a small part of it, to a single molecular entity (Molecular Operational Taxonomic Unit, MOTU; sensu Floyd et al. [4] and does not necessarily require taxonomy skilled personnel, at least in the step of the molecular data generation, to be used in the analysis. Nevertheless, the power and pitfalls of the DNA barcoding approach have not yet been fully evaluated. In particular, the proper methods to analyse DNA barcoding data are still under study (e.g. see the progress at CBoL working groups, http://www.bolin fonet.org/casestudy/index.php/browse). coxI sequences are widely used for DNA barcoding of metazoans, but several markers have been proposed as putative barcodes [5,6] and different authors underline the importance of a DNA barcoding approach based on multiple markers [7,8]. Ribosomal mitochondrial genes are often used as alternatives to coxI marker for different reasons: easy to amplify, good source of synapomorphies in loop regions and abundance of sequences in databases [8-10]. Predicted problems in DNA barcoding studies include: poor taxonomy (e.g. single species misidentified as two or more species and viceversa); insufficient sampling within a taxon, or insufficient sampling of taxa (see for instance [11] and consequent criticisms in [12-14]); polyphyletic or paraphyletic species [15]. This work focuses on an integrated approach at the identification of a group of nematodes, belonging to the order Spirurida, which includes the relevant superfamily Filarioidea. Several species of filarioid nematodes are agents of tropical diseases both for human and other animals of economical value. All the filarioids are transmitted through haematophagous vectors in which they span different juvenile stages [16-18]. The identification of these juvenile stages is a necessary condition for establishing the potential of transmission in endemic areas but it is difficult, due to the small size of the juvenile stages (about 1 mm) and paucity of characters. Identification of juvenile stages is also useful to detect any possible emergent zoonotic filarial disease at its beginning. Laboratories typically deals with fragments of parasitic nematodes recovered from host tissues, or with specimens representing a single developmental stage, and the diagnostic characters are often not present in these pieces of worms. The identification of filarioid and related nematodes via DNA barcoding is an ambitious and desirable goal for many reasons: 1) a fast identification engine, available not only for taxonomists, but validated by them, is useful for quicker diagnoses of filariasis; 2) filarioids cause diseases of high relevance in medical and veterinary fields throughout the world; 3) DNA barcoding can be useful for those cases of difficult or impossible i (...truncated)