Hurdles in the evolutionary epidemiology of Angiostrongylus cantonensis: Pseudogenes, incongruence between taxonomy and DNA sequence variants, and cryptic lineages

Abstract

Angiostrongylus cantonensis, the rat lungworm, is a zoonotic pathogen that is one of the leading causes of eosinophilic meningitis worldwide. This parasite is regarded as an emerging pathogen with a global range expansion out of southeastern Asia post‐WWII. To date, molecular systematic/phylogeographic studies on A. cantonensis have mainly used two mitochondrial (mtDNA) markers, cytochrome c oxidase 1 (CO1) and cytochrome b (CYTB), where the focus has largely been descriptive in terms of reporting local patterns of haplotype variants. In order to look for more global evolutionary patterns, we herein provide a collective phylogenetic assessment using the six available whole mtDNA genome samples that have been tagged as A. cantonensis, A. malaysiensis, or A. mackerrasae along with all other GenBank CO1 and CYTB partial sequences that carry these species identifiers. The results reveal three important complications that researchers will need to be aware of, or will need to resolve, prior to conducting future molecular evolutionary studies on A. cantonensis. These three problems are (i) incongruence between taxonomic identifications and mtDNA variants (haplotypes or whole mtDNA genome samples), (ii) the presence of a CYTB mtDNA pseudogene, and (iii) the need to verify A. mackerrasae as a species along with other possible cryptic lineages, of which there is suggestive evidence (i.e., A. cantonensis could be a species complex). We provided a discussion of how these complications are hurdles to our understanding of the global epidemiology of angiostrongyliasis. We call for future studies to be more explicit in morphological traits used for identifications (e.g., provide measurements). Moreover, it will be necessary to repeat prior morphological and life‐history studies while simultaneously using sequence data in order to assess possible associations between critical epidemiological data (e.g., biogeography, virulence/pathology, host species use) and specific lineages.

Low diversity of Angiostrongylus cantonensis complete mitochondrial DNA sequences from Australia, Hawaii, French Polynesia and the Canary Islands revealed using whole genome next-generation sequencing

“…In Japan, Tokiwa et al [33] suggested colonization of the area by multiple genetic lineages spreading from the south to the north of Japan. The majority of phylogeographic studies have local character and are based on partial sequences [36]. Data on genetic and phenotypic diversity of A. cantonensis within invaded regions remain scarce [10, 37–40].…”

The population genetics of parasitic nematodes of wild animals

“…The population genetics of Angiostrongylus cantonensis and A. malaysiensis , parasites of rodents of the family Muridae [ 58 ], has been extensively studied, with genetic structure detected on both small and large geographical scales [ 59 – 69 ]. However, interpretation of these findings is made difficult by the recent discovery of at least two sympatric cryptic species within A. cantonensis , one of which may be conspecific with individuals identified as A. malaysiensis [ 70 , 71 ]. It is now not clear whether the population genetic structure detected previously really represents the geographical distribution of genetic variants in a single species, or rather results from the accidental sampling of multiple reproductively isolated species.…”

SYBR-Green-Based Quantitative Real-Time PCR for Discriminating Between Closely Related Angiostrongylus Cantonensis and A. Malaysisensis

“…In 2016, Rodpai et al revealed that A. cantonensis is widely distributed in Thailand, Lao PDR, Cambodia, and Myanmar, and its distribution broadly overlapped that of A. malaysiensis [23]. Dusitsittipon et al discussed the incongruence between the morphological and molecular identi cations and found that most of the misidenti ed specimens were from Thailand [17,24].…”

“…A. cantonensis and A. malaysiensis can be distinguished using the mitochondrial cytochrome b (Cytb) gene as a genetic marker because of the robust phylogenetic relationship between the closely related species [17,24]. This gene sequence shows su ciently high genetic variation between species, and species-speci c primers can be used to detect and discriminate between the two species with high sensitivity and speci city.…”

“…In this study, therefore, we designed species-speci c primers to Cytb partial gene sequences, which have su ciently high genetic variation between species to prevent cross-ampli cation [17,24]. We aimed to develop a highly sensitive and speci c method capable of quantitively detecting the third-stage larvae of A. cantonensis and A. malaysiensis in their intermediate host, Achatina fulica, and to determine the e cacy of the method for the detection of Angiostrongylus genomic material within the CSF of infected patients.…”