Mutation scanning for sequence variation in three mitochondrial DNA regions for members of theContracaecum osculatum (Nematoda: Ascaridoidea) complex

Abstract

Anisakid nematodes of seals from different geographical origins, previously identified by multilocus enzyme electrophoresis as Contracaecum osculatum A (CoA), C. osculatum B (CoB), C. osculatum C (CoC), C. osculatum D (CoD), C. osculatum E (CoE) and C. osculatum baicalensis (Cob), were characterised genetically using a mutation scanning approach, in order to define genetic markers for their specific identification and differentiation. Three mitochondrial DNA (mtDNA) regions, namely cytochrome c oxidase subunit I (COI), and the small and large subunits of rRNA (ssrRNA and IsrRNA, respectively) were amplified separately from individual nematodes by polymerase chain reaction (PCR), analysed by single-strand conformation polymorphism (SSCP), and samples displaying sequence variability were subjected to sequencing. Forty-six haplotypes were defined for 62-66 individuals (representing the six members of C. osculatum). All taxa except CoD and CoE could be identified, or delineated from one another, by nucleotide differences in the COI, ssrRNA and/or IsrRNA sequences. For all three mtDNA regions, 4 (10.5%), 7 (18.4%), 15 (39.5%) and 11 (28.9%) of 38 nucleotide positions were considered diagnostic (fixed) and could thus unequivocally delineate CoA, CoB, CoC and Cob. The lack of an unequivocal nucleotide difference in any of the three mtDNA sequences between CoD and CoE was in accordance with previous ribosomal DNA sequence data but inconsistent with multilocus enzyme electrophoretic data. Using all fixed nucleotide positions, CoA, CoD/E and CoB were genetically more similar to Cob than each was to CoC, similar to previous findings. In spite of not being able to distinguish among all six taxa of C. osculatum, the present study demonstrated clearly the usefulness and attributes of the mutation scanning approach for investigating population genetic structures of species of parasitic nematodes.

Molecular Phylogenetics and Diagnosis of Anisakis, Pseudoterranova, and Contracaecum From Northern Pacific Marine Mammals

“…These population-level allozyme studies have been instrumental in detecting evidence of genetic heterogeneity (noninterbreeding individuals within populations) among large samples of ascaridoids collected from paratenic or definitive hosts in nature (Paggi and Bullini, 1994;Bullini et al, 1997), and have led to the discovery and description of several new species. Such studies have facilitated the development of other molecular diagnostic tools for these species, in particular, those based on the polymerase chain reaction (PCR) including PCR-restriction fragment length polymorphism (PCR-RFLP) (Zhu, Gasser, Podolska, and Chilton, 1998;D'Amelio et al, 2000;Kijewska et al, 2002;Shih, 2004), single-strand conformational polymorphism (SSCP) of PCR products (Zhu, Gasser, Podolska, and Chilton, 1998;Zhu et al, 2000;Hu et al, 2001;Zhu et al, 2001Zhu et al, , 2002, and direct sequencing of PCR-amplified DNA (Zhu, Gasser, Podolska, and Chilton, 1998;Nadler et al, 2000;Zhu et al, 2000;Hu et al, 2001;Zhu et al, 2001Zhu et al, , 2002Mattiucci et al, 2003). These DNA-based diagnostic techniques are advantageous because individual alcohol-preserved adults and larvae can be identified; in contrast, allozyme techniques require enzymatic activities that are only preserved in frozen tissues.…”

“…consists of 2 sibling species, C. ogmorhini s. str. and Contracaecum margolisi (Mattiucci et al, 2003), with the latter species diagnosed genetically (Zhu et al, 2001;Mattiucci et al, 2003) from 1 locality (Vancouver Island, Canada) in Z. californianus (CSL). The third common genus of marine mammal ascaridoid, Pseudoterranova, is also a complex of at least 5 species (Paggi et al, 1991;Mattiucci et al, 1998;George-Nascimento and Urrutia, 2000;Paggi et al, 2000;Zhu et al, 2002) that can be diagnosed by allozyme markers (Paggi et al, 2000), nucleotide sequences (Zhu et al, 2002), and, for adult males, morphometric differences (Di Deco et al, 1994;Mattiucci et al, 1998;George-Nascimento and Urrutia, 2000;Paggi et al, 2000).…”

Forewarned is forearmed: harmonized approaches for early detection of potentially invasive pests and pathogens in sentinel plantings

“…The quality and accuracy of the sequences stored in the genetic databases might not always be satisfactory, especially considering that any user can access and add sequences (Hebert and Gregory 2005). In a recent survey of insects that colonized a sentinel plantation in China, DNA barcoding enabled to reliably identify only one quarter of sample insect species (Roques et al 2015) For nematodes, several genes are targeted for identification such as the mitochondrial cytochrome b locus (mtDNAcytb) (Mattiucci et al 2003), the gene encoding the mitochondrial cytochrome oxidase 2 (COX2) (Valentini et al 2006) and the mitochondrial cytochrome oxidase 1 (COXI) (Blouin, 2002), the ribosomal RNA of the small (ssrRNA) and large subunit (lsrRNA) (Hu et al 2001). Other nuclear genes were also selected such as the internal transcribed spacer 1 (ITS1) of rDNA to identify Strongylidae and Anisakidae (Roeber et al 2013).…”

Single‐Stranded Conformation Polymorphism Analysis

“…Utilizing the second internal transcribed spacer (ITS-2) of the ribosomal RNA gene by SSCP, 23 nematode species of the orders Ascaridida and Strongiylida could be identified (Gasser et al, 1997). Later, Trichinella isolates, hookworms, members of the Contracaecum osculatum complex, Benedeniinae, and members of the Pseudoterranova decipiens complex have been differentiated by SSCP (Gasser et al, 1998;Hu et al, 2001Hu et al, , 2002Zhu et al, 2002;Li et al, 2005).…”