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Crayfish plague in Japan: A real threat to the endemic Cambaroides japonicus.

ABSTRACT: Global introductions of aquatic species and their associated pathogens are threatening worldwide biodiversity. The introduction of two North American crayfish species, Procambarus clarkii and Pacifastacus leniusculus, into Japan in 1927 seems to have negatively affected native Japanese crayfish populations of Cambaroides japonicus. Several studies have shown the decline of these native populations due to competition, predation and habitat colonization by the two invasive North American crayfish species. Here, we identify an additional factor contributing to this decline. We report the first crayfish plague outbreaks in C. japonicus populations in Japan, which were diagnosed using both histological and molecular approaches (analyses of the internal transcribed spacer region). Subsequent analyses of the mitochondrial ribosomal rnnS and rnnL regions of diseased specimens indicate that these outbreaks originated from a P. clarkii population and identify a novel haplotype of Aphanomyces astaci, d3-haplotype, hosted by P. clarkii. Overall, our findings demonstrate the first two cases of crayfish plague in Japan, and the first case in a non-European native crayfish species, which originated from the red swamp crayfish P. clarkii. This finding is a matter of concern for the conservation of the native freshwater species of Japan and also highlights the risk of introducing crayfish carrier species into biogeographic regions harboring species susceptible to the crayfish plague.

SUBMITTER: Martin-Torrijos L

PROVIDER: S-EPMC5884544 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Crayfish plague in Japan: A real threat to the endemic Cambaroides japonicus.

Martín-Torrijos Laura L Kawai Tadashi T Makkonen Jenny J Jussila Japo J Kokko Harri H Diéguez-Uribeondo Javier J

PloS one 20180404 4

Global introductions of aquatic species and their associated pathogens are threatening worldwide biodiversity. The introduction of two North American crayfish species, Procambarus clarkii and Pacifastacus leniusculus, into Japan in 1927 seems to have negatively affected native Japanese crayfish populations of Cambaroides japonicus. Several studies have shown the decline of these native populations due to competition, predation and habitat colonization by the two invasive North American crayfish ...[more]

PMID: 29617418

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Project description:The oomycete Aphanomyces astaci is regarded as the causative agent of crayfish plague and represents an evident hazard for European crayfish species. Native crayfish populations infected with this pathogen suffer up to 100% mortality. The existence of multiple transmission paths necessitates the development of a reliable, robust and efficient test to detect the pathogen. Currently, A. astaci is diagnosed by a PCR-based assay that suffers from cross-reactivity to other species. We developed an alternative closed-tube assay for A. astaci, which achieves robustness through simultaneous amplification of multiple functionally constrained genes.Two novel constitutively expressed members of the glycosyl hydrolase (GH18) gene family of chitinases were isolated from the A. astaci strain Gb04. The primary amino acid sequence of these chitinase genes, termed CHI2 and CHI3, is composed of an N-terminal signal peptide directing the post-translational transport of the protein into the extracellular space, the catalytic GH18 domain, a proline-, serine-, and threonine-rich domain and a C-terminal cysteine-rich putative chitin-binding site. The A. astaci mycelium grown in a pepton-glucose medium showed significant temporal changes in steady-state CHI2 and CHI3 mRNA amounts indicating functional constraint. Their different temporal occurrence with maxima at 48 and 24 hours of incubation for CHI2 and CHI3, respectively, is in accordance with the multifunctionality of GH18 family members. To identify A. astaci-specific primer target sites in these novel genes, we determined the partial sequence homologs in the related oomycetes A. frigidophilus, A. invadans, A. helicoides, A. laevis, A. repetans, Achlya racemosa, Leptolegnia caudata, and Saprolegnia parasitica, as well as in the relevant fungi Fusarium solani and Trichosporon cutaneum. An A. astaci-specific primer pair targeting the novel genes CHI2 and CHI3 as well as CHI1 - a third GH18 family member - was multiplexed with primers targeting the 5.8S rRNA used as an endogenous control. A species was typed unambiguously as A. astaci if two peaks were concomitantly detected by melting curve analysis (MCA). For sensitive detection of the pathogen, but also for quantification of agent levels in susceptible crayfish and carrier crayfish, a TaqMan-probe based real-time PCR (qPCR) assay was developed. It targets the same chitinase genes and allows quantification down to 25 target sequences.The simultaneous qualitative detection of multiple sequences by qPCR/MCA represents a promising approach to detect species with elevated levels of genetic variation and/or limited available sequence information. The homogenous closed-tube format, reduced detection time, higher specificity, and the considerably reduced chance of false negative detection achieved by targeting multiple genes (CHI1, CHI2, CHI3, and the endogenous control) at least two of which are subject to high functional constraint, are the major advantages of this multiplex assay compared to other diagnostic methods. Sensitive quantification achieved with TaqMan qPCR facilitates to monitor infection status and pathogen distribution in different tissues and can help prevent disease transmission.

Dataset Information

Crayfish plague in Japan: A real threat to the endemic Cambaroides japonicus.

Publications

Crayfish plague in Japan: A real threat to the endemic Cambaroides japonicus.

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