Phylogeographical studies of Ascaris spp. based on ribosomal and mitochondrial DNA sequences

Abstract

Background: The taxonomic distinctiveness of Ascaris lumbricoides and A. suum, two of the world's most significant nematodes, still represents a much-debated scientific issue. Previous studies have described two different scenarios in transmission patterns, explained by two hypotheses: (1) separated host-specific transmission cycles in highly endemic regions, (2) a single pool of infection shared by humans and pigs in non-endemic regions. Recently, A. suum has been suggested as an important cause of human ascariasis in endemic areas such as China, where cross-infections and hybridization have also been reported. The main aims of the present study were to investigate the molecular epidemiology of human and pig Ascaris from non-endemic regions and, with reference to existing data, to infer the phylogenetic and phylogeographic relationships among the samples.

Methodology: 151 Ascaris worms from pigs and humans were characterized using PCR-RFLP on nuclear ITS rDNA. Representative geographical sub-samples were also analysed by sequencing a portion of the mitochondrial cox1 gene, to infer the extent of variability at population level. Sequence data were compared to GenBank sequences from endemic and non-endemic regions.

Principal findings: No fixed differences between human and pig Ascaris were evident, with the exception of the Slovak population, which displays significant genetic differentiation. The RFLP analysis confirmed pig as a source of human infection in non-endemic regions and as a corridor for the promulgation of hybrid genotypes. Epidemiology and host-affiliation seem not to be relevant in shaping molecular variance. Phylogenetic and phylogeographical analyses described a complex scenario, involving multiple hosts, sporadic contact between forms and an ancestral taxon referable to A. suum.

Conclusions/significance: These results suggest the existence of homogenizing gene flow between the two taxa, which appear to be variants of a single polytypic species. This conclusion has implications on the systematics, transmission and control programs relating to ascariasis.

Figure 1. Molecular characterization of Ascaris by PCR-RFLP.

A representative gel displaying the RFLP profiles following the digestion of ITS amplicons with restriction endonuclease HaeIII. Genotype As: Ascaris suum banding pattern (lanes 1–3,5,7–9,11); genotype Al : A. lumbricoides banding pattern (lanes 4,10,12,13); genotype H: hybrid/heterozygote banding pattern (lane 6); R (right-hand lane): 100bp reference ladder.

Figure 2. Neighbor Joining tree of Ascaris samples.

Evolutionary history of the Ascaris sp. partial cox1 mitochondrial DNA sequences, obtained by Neighbor Joining analysis. The same topology was obtained for Bayesian analyses. Numbers at nodes branches represent posterior probability (BPP) values of Dataset 2, Dataset 1 and bootstrap values (A = Cluster A, including subclusters A1 and A2; B = Cluster B; C = Cluster C). The bar shows the mean number of base substitution per site. H and P in codes represent human and pig hosts, respectively. Colored dots indicate ITS RFLP genotypes (blue: A. lumbricoides, red: A. suum, green: hybrid genotype). Trend for host affiliation is indicated on the right of the tree, as defined by the proportional size of human and pig gray figures.

Figure 3. Parsimony network of Ascaris haplotypes.

Parsimony network of the haplotypes observed in the Dataset2, with 95% connection limit. Circles diameter is proportional to haplotypes frequency; circles at branches represent SNPs (A = Cluster A, including subclusters A1 and A2; B = Cluster B; C = Cluster C).