Paralogues of nuclear ribosomal genes conceal phylogenetic signals within the invasive Asian fish tapeworm lineage: evidence from next generation sequencing data

Highlights

• Complete mitochondrial genomes and nuclear rRNA operons of Schyzocotyle acheilognathi were characterised and annotated.

• Genetic variation and phylogenetic relationships of 8 globally distributed Asian fish tapeworm populations were assessed.

• Nuclear rRNA operon paralogues were detected in all populations, having serious consequences for previous genetic studies.

• A broadly applicable sequencing approach using the Illumina MiSeq platform was successfully utilised.

Abstract

Complete mitochondrial genomes and nuclear rRNA operons of eight geographically distinct isolates of the Asian fish tapeworm Schyzocotyle acheilognathi (syn. Bothriocephalus acheilognathi), representing the parasite’s global diversity spanning four continents, were fully characterised using an Illumina sequencing platform. This cestode species represents an extreme example of a highly invasive, globally distributed pathogen of veterinary importance with exceptionally low host specificity unseen elsewhere within the parasitic flatworms. In addition to eight specimens of S. acheilognathi, we fully characterised its closest known relative and the only congeneric species, Schyzocotyle nayarensis, from cyprinids in the Indian subcontinent. Since previous nucleotide sequence data on the Asian fish tapeworm were restricted to a single molecular locus of questionable phylogenetic utility—the nuclear rRNA genes-separating internal transcribed spacers—the mitogenomic data presented here offer a unique opportunity to gain the first detailed insights into both the intraspecific phylogenetic relationships and population genetic structure of the parasite, providing key baseline information for future research in the field. Additionally, we identify a previously unnoticed source of error and demonstrate the limited utility of the nuclear rRNA sequences, including the internal transcribed spacers that has likely misled most of the previous molecular phylogenetic and population genetic estimates on the Asian fish tapeworm.

Introduction

Schyzocotyle acheilognathi (Yamaguti, 1934), commonly known as the Asian fish tapeworm (AFT), represents a significant, commercially important fish pathogen with a global impact (Scholz et al., 2012). Belonging to an evolutionarily derived lineage of bothriocephalidean cestodes restricted to freshwater fish (Brabec et al., 2015a), S. acheilognathi is perhaps best known amongst the 134 currently recognised species of the order Bothriocephalidea Kuchta, Scholz, Brabec & Bray, 2008 and among cestodes generally. Its notoriety and familiarity are underpinned by its exceptionally low host specificity, its ability to colonise a broad variety of freshwater fish taxa, its status as an invasive species, and its human-assisted history of dissemination throughout all continents of the globe except Antarctica (see Choudhury and Cole, 2012, Scholz et al., 2012 for reviews). It has also been recorded as a potential human parasite (Yera et al., 2013). Adult worms inhabit intestines of a wide range of freshwater fish hosts (rarely also amphibians, reptiles and birds) where they may cause notable harm (Scholz et al., 2012).

Schyzocotyle acheilognathi was originally described in Japan by Yamaguti (1934) from Lake Ogura as Bothriocephalus acheilognathi, but historical evidence suggests that the pathogen’s distribution range may have been first limited to the Amur River in mainland eastern Asia (Choudhury and Cole, 2012), although opinions exist suggesting the parasite’s origin in Africa (Scholz et al., 2012). Since then, AFT has been described under at least 23 different names and most recently transferred to the genus Schyzocotyle Akhmerov, 1960 by Brabec et al. (2015a). In each case, eastern Asia is well documented as the primary focus from where AFT spread throughout the globe (see http://www.cabi.org/isc/datasheet/91669 and references therein for up-to-date information on AFT distribution). AFT was originally a parasite of cyprinid fish and while it remains predominantly confined to cyprinids in Eurasia and Africa (Retief et al., 2007), it managed to colonise a range of non-cyprinids mainly in North American and Australian regions (Dove and Fletcher, 2000, Salgado-Maldonado and Pineda-López, 2003, Méndez et al., 2010, Scholz et al., 2012). Currently it has been documented from more than 200 species of freshwater fish belonging to 10 orders and 19 families (Scholz et al., 2012), even though the parasite might not be able to reach maturity in some of the hosts (Dove and Fletcher, 2000). In the cases of some predatory fish, amphibians, reptiles and birds harbouring adults of S. acheilognathi, the hosts might in fact represent postcyclic hosts in which the adult parasites manage to re-establish after being preyed on together with their definitive host (Hansen et al., 2007, Scholz et al., 2012).

Despite the relative abundance of published surveys of the life history, geographical distribution and host ranges of AFT, studies utilising molecular data remain surprisingly scarce and largely limited to non-coding internal transcribed spacer sequences (ITS1, ITS2) separating the nuclear rRNA genes. Liao and Lun (1998) and Feng and Liao (2000) were the first to use molecular data to study genetic polymorphism of AFT, using a random amplified polymorphic DNA (RAPD) approach in both cases. However, datasets from their studies were uncomfortably small, with inferred results insufficiently convincing to support their conclusions that AFT from Opsariichthys bidens Günther should be considered a separate species, and that AFT parasitising grass and common carp, Ctenopharyngodon idella (Valenciennes) and Cyprinus carpio Linnaeus, in China represents two unrelated groups, possibly separate species. In order to obtain further insights into the intraspecific diversity of AFT, Luo et al., 2002, Luo et al., 2003 utilised information from ITS1 and ITS2 sequences and microsatellite loci situated within this region, respectively, on a set of S. acheilognathi specimens predominantly from China. Conclusions from both studies should be treated with caution. Luo et al. (2002) interpreted their results of phylogenetic analysis incorrectly, and in fact failed to find any convincing associations among the ITS genotypes. Luo et al. (2003) ignored the fact that they had genotyped eight physically tightly linked microsatellite loci of a multi-copy region that might display intragenomic variation, a feature leading to the violation of Mendelian inheritance assumptions (Selkoe and Toonen, 2006).

Bean et al. (2007) used ITS together with partial ssrDNA sequences to evaluate genetic distances of North American AFT representatives. Chaudhary et al. (2015) and Salgado-Maldonado et al. (2015) compared ITS and rDNA sequences, respectively, to test identity of their isolates to those characterised previously. lsrDNA sequences of S. acheilognathi were utilised as part of a taxonomic re-evaluation of bothriocephalidean cestodes from African fishes (Kuchta et al., 2012). This study revealed that AFT is closely related to the clade comprising all African bothriocephalids. The first published mitochondrial (mt) sequence of S. acheilognathi was a partial fragment of cytochrome c oxidase subunit 1 (cox1) obtained from the only documented clinical case (Yera et al., 2013). Recently, a multi-gene phylogenetic study of cestodes of the order Bothriocephalidea (Brabec et al., 2015a) confirmed that African bothriocephalids form a derived monophyletic clade and that AFT together with its congeneric species, Schyzocotyle nayarensis (Malhotra, 1983), form its sister lineage. The authors also confirmed that the genus Bothriocephalus Rudolphi, 1808 represents an artificial taxonomic entity (originally revealed by Škeříková et al. (2004)) and subsequently transferred B. acheilognathi Yamaguti, 1934 to Schyzocotyle as S. acheilognathi. The present study aims to evaluate the utility of the traditionally exploited genomic loci, the nuclear rRNA operon and mt genome sequences, to study diversification of the AFT and gain first insights into the phylogeography of this invasive species.