The genetic variation of Angiostrongylus cantonensis in the People’s Republic of China

Shan Lv, Yi Zhang, Peter Steinmann, Jürg Utzinger, Xiao-Nong Zhou

The People's Republic of China (P.R. China) is the presumptive home range of the rat lungworm Angiostrongylus cantonensis, a major aetiological agent of human eosinophilic meningitis. We present a study of the genetic variation of A. cantonensis in P.R. China. Our aim was to deepen the current knowledge pertaining to its origin and global spread from a molecular perspective. Adult A. cantonensis were collected in the frame of a national survey and identified based on morphological criteria. Polymerase chain reaction (PCR) was employed to amplify the target DNA sequences (cytochrome c oxidase subunit I (cox1), nicotinamide adenine dinucleotide dehydrogenase subunit 1 (nad1) and internal transcribed spacer (ITS)). The PCR product of cox1 was directly submitted to sequencing, while clone sequencing was used for nad1 and ITS. The identity of the samples was verified by comparing the sequences to those of accepted A. cantonensis specimens. The specific composition of substitutions in each gene was analysed, and the genotypes were compared based on the complete cox1, nad1 and ITS genes. We characterised the complete mitochondrial genes cox1 and nad1 of 130 specimens and obtained 357 nuclear sequences containing two complete ITS (ITS1 and ITS2) and 5.8S rRNA of the same samples. All specimens were genetically confirmed as A. cantonensis. Two major groups (i.e. I and II) were identified according to the phylogeny of cox1 sequences. Group I could be further categorised into six distinct clades. Almost half of the specimens (47.7%) belong to the clade Ia and 22.3% to the group II. The former was widely distributed across the study region. A variable number of repeat units in three microsatellites was observed, resulting in considerable length variation in ITS. Intragenomic variation of ITS sequences was found in a large proportion of the samples. Genotyping showed a striking difference between mitochondrial DNA and ITS. Our results demonstrate that A. cantonensis is the only rat lungworm species in P.R. China and shows high genetic diversity. Results of diversity and genotyping of A. cantonensis can be impacted by the sequencing strategy and biomarker. Although ITS may be a valuable marker for interspecific identification, it is not suitable for studying the intraspecific variation of A. cantonensis due to its high intragenomic variation and current challenges for direct sequencing.