The Mitochondrial Genomes of the Zoonotic Canine Filarial Parasites Dirofilaria (Nochtiella) repens and Candidatus Dirofilaria (Nochtiella) Honkongensis Provide Evidence for Presence of Cryptic Species

Background Cutaneous dirofilariosis is a canine mosquito-borne zoonosis that can cause larva migrans disease in humans. Dirofilaria repens is considered an emerging pathogen occurring with high prevalence in Mediterranean areas and many parts of tropical Asia. In Hong Kong, a second species, Candidatus Dirofilaria hongkongensis, has been reported. The present study aimed to compare mitochondrial genomes from these parasites and to obtain population genetic information. Methods and Findings Complete mitochondrial genomes were obtained by PCR and Sanger sequencing or ILLU-MINA sequencing for four worms. Cytochrome oxidase subunit 1 sequences identified three as D. repens (all from Europe) and one as C. D. hongkongensis (from India). Mitochondrial genomes have the same organization as in other spirurid nematodes but a higher preference for thymine in the coding strand. Phylogenetic analysis was in contradiction to current taxonomy of the Onchocercidae but in agreement with a recent multi-locus p hylogenetic analysis using both mitochondrial and nuclear markers. D. repens and C. D. hongkongensis sequences clustered together and were the common sister group to Dirofilaria immitis. Analysis of a 2.5 kb mitochondrial genome fragment from macrofilaria or canine blood samples from Europe (42), Thailand (2), India (1) and Vietnam (1) revealed only small genetic differences in the D. repens samples including all European and the Vietnam sample. The Indian C. D. hongkongensis and the two Thai samples formed separate clusters and differences were comparatively large. Conclusion Genetic differences between Dirofilaria spp. causing cutaneous disease can be considerable whereas D. repens itself was genetically quite homogenous. C. D. hongkongensis was identified for the first time from the Indian subcontinent. The full mitochondrial genome sequence strengthens the hypothesis that it represents an independent species and the Thai samples might represent another cryptic species, Candidatus Dirofilaria sp. 'Thailand II', or a quite divergent population of C. D. hongkongensis

Fifth European Dirofilaria and Angiostrongylus Days (FiEDAD) 2016

Peer reviewe

Correction: The Mitochondrial Genomes of the Zoonotic Canine Filarial Parasites Dirofilaria (Nochtiella) repens and Candidatus Dirofilaria (Nochtiella) Honkongensis Provide Evidence for Presence of Cryptic Species.

[This corrects the article DOI: 10.1371/journal.pntd.0005028.]

Schematic map of the 13,672 bp <i>Dirofilaria repens</i> mitochondrial genome.

<p>On the outer circle, positions of restriction enzymes recognition sites (with positions in the genome) are indicated for enzymes that cut only once in the genome. The position of tRNA genes and the AT-rich control region (CR) are also shown on this circle. The next circle shows the position of the genes encoding proteins (gray) or rRNAs (blue). In the center, the positions on the map is provided in base pairs (bp). All genes, including the genes encoding rRNAs are orientated in clockwise direction. The map was drawn using the sequence with the accession no KX265047.</p

Annotated genes in the mitochondrial genome of <i>Dirofilaria repens</i>.

<p>Annotated genes in the mitochondrial genome of <i>Dirofilaria repens</i>.</p

Phylogenetic analysis of <i>Dirofilaria</i> repens like sequences from samples collected in Europe and Asia.

<p>Sequences of an approximately 2.5 kb mitochondrial DNA fragment including parts of the <i>ctc-3</i> and the <i>trnF</i> genes, the AT-rich control region, the complete protein coding genes for <i>ndfl4</i> and <i>nduo-1</i>, the ribosomal RNA gene <i>rrnS</i> and the genes for the tRNAs <i>trnA</i>, <i>trnL</i>^UUR, <i>trnN</i>, <i>trnM</i> and <i>trnK</i>. Sample origin in terms of countries are shown behind an in-house sample number/code. All sequences have been deposited in GenBank under the accession nos. KX265047—KX265093. In addition, samples of human origin are indicated, all other samples were from dogs. Numbers at the internal nodes represent results for the Shimodaira-Hasegawa and a Bayesian transformation of the likelihood ratio test before and after the slash, respectively. If only one number is shown, the results of the tests were identical. Small letters were used if the space was too limited to write the support values directly at the branch. The branches labelled with “<i>Dirofilaria repens</i>”, “<i>Candidatus</i> Dirofilaria (<i>C</i>. D.) hongkongensis” and <i>Dirofilaria</i> (<i>D</i>.) sp. ‘Thailand II’ contain only the respective (putative) species. The branch labeled with “<i>Candidatus</i> Dirofilaria (<i>C</i>. D.) hongkongensis like” clusters together this proposed species with sequences of <i>C</i>. D. sp. ‘Thailand II’ that might belong to the same species or represent an additional species. The yellow area indicates all sequences that belong to an apparently monophyletic group containing a large number of samples from Poland and Hungary. The green color labels a second apparently monophyletic group with most samples coming from South-Western Europe and Hungary.</p