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

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.


Introduction
Global movements of aquatic animals have facilitated the emergence of infectious diseases and have caused great losses in aquaculture and aquatic wildlife populations [1]. These movements often involve unintentional introductions that result in the establishment and spread of incidental "hitchhiking" species [2,3]. For instance, several pathogens are known to have crept into new geographic areas and infected new hosts, resulting in emerging infectious diseases [1]. This is the case of Aphanomyces astaci Schikora 1903 (Oomycota), the pathogen responsible for the crayfish plague disease that caused the decimation and near extinction of the native European crayfish populations [4,5]. This organism chronically infects its natural hosts, North a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 main objective of this study is to determine whether these mortality events were caused by the crayfish plague pathogen A. astaci.

Ethical statement
All experimental procedures and animal manipulations, as well as field sampling, were performed according to the Japanese, EU and Spanish legislation. All analyses were carried out according to the regulations of Spanish Ministry MINECO. No additional permits were required for the laboratory studies, since the ethics approval in the Spanish law is not required for working with arthropod invertebrates. Moreover, this study was carried out in strict accordance with the recommendations and the protocols established in previous studies.

Crayfish sampling
A total of 15 dead specimens of C. japonicus originating from two mass mortality events were analyzed. The first mass mortality event occurred in Minami-ku, a ward south of the city of Sapporo, during October 2014. The second event occurred in Ishikari River during September 2015. Two C. japonicus individuals from the first event and four from the second were collected and preserved in ethanol 95% for further analyses (Fig 1, Table 1). Additionally, nine C. japonicus specimens from locations either nearby or far from the second mass mortality location were collected and analyzed (Table 1). All specimens were analyzed at the Laboratory of Molecular Systematics at the Real Jardín Botánico-CSIC, Madrid, Spain.
To test the prevalence of the pathogen A. astaci in introduced North American species, P. clarkii specimens from a population inhabiting Yasuharu, a vicinity with known C. japonicus populations (Fig 1) and P. leniusculus specimens from an established population in Shikaribetsu Lake in a central region of eastern Hokkaido Island (Fig 1) were collected during October 2015 for further analysis (Table 1).

Macroscopic and microscopic examination
All analyzed crayfish were examined macroscopically to check for the presence of melanized areas and microscopically for the presence of hyphae in the soft cuticle, both of which are indicators of A. astaci infection. For microscopic examination, the subabdominal cuticle was removed and observed using an Olympus CKX41SF inverted microscope (Olympus Optical, Tokyo, Japan). Light micrographs of the colonizing hyphae were captured using a QImaging Micropublisher 5.0 digital camera (QImaging, Burnaby, BC, Canada). Digital image analysis was performed using the software Syncroscopy-Automontage (Microbiology International Inc., Frederick, MD) as described by Diéguez-Uribeondo et al. 2003 [39].

Molecular analyses
Genomic isolation, PCR amplification and sequencing. Subabdominal soft cuticle samples were rehydrated from ethanol into TE buffer (TRIS 10 mM/ EDTA 1 mM, pH 8). Each sample was rinsed three times for 1 hour with TE prior to an overnight wash. Samples were transferred into individual 2 ml Eppendorf tubes, frozen at -80˚C and then lyophilized in a freeze dryer VirTis BenchTop K for 24 hours ( -50˚C; 20 mTorr). The samples were then mechanical ruptured using a TissueLyser (QIAGEN, Venlo, The Netherlands). Genomic DNA was isolated with the E.Z.N.A. 1 Insect DNA Kit (Omega Bio-Tek, Norcross, Georgia, USA). The extracted DNA and A. astaci diagnostic primers 42 [40] and 640 [41] (which amplify the ITS1 and ITS2 surrounding the 5.8S rDNA, and anchored in ITS1 and ITS2 regions, respectively) were used for a single round of PCR according to the assay described by Oidtmann et al. 2006 [40]. As a positive control, DNA extracted from a pure culture of the A. astaci strain AP03 [42], was used; distilled Milli-Q water was used as a negative control. Amplified products were analyzed by electrophoresis in 1% agarose TAE gels stained with SBYR 1 Safe (Thermo Fisher Scientific, Waltham, MA, USA). Both strands of PCR amplified products were sequenced using an automated sequencer (Applied Biosystems 3730xl DNA, Macrogen, The Netherlands). Each sequence strand was assembled and edited with Geneious 1 10.0.2 [43]. BLAST searches were performed to verify the identities of the obtained sequences.
Phylogenetic and haplotype analyses. Specimens of C. japonicus, P. clarkii and P. leniusculus that tested positive for A. astaci based on diagnostic primers 42 [40] and 640 [41] were further analyzed to characterize the phylogenetic relationships and haplotypes of A. astaci present in the crayfish cuticles. Mitochondrial rnnS and rnnL sequences were obtained as described by Makkonen et al. [21]. Briefly, mitochondrial ribosomal rnnS and rnnL primers pairs (AphSSUF/AphSSUR and AphLSUF/AphLSUR, respectively) [21] were used for the pathogen characterization. The aforementioned positive and negative controls were also included. Amplified products were analyzed and sequenced as described above. However, in this case, amplified products were first purified using a QIAquick PCR Purification Kit (QIAGEN).
Sequences were assembled and edited using the program Geneious 1 10.0.2 [43] and two phylogenetic approximations, Bayesian Interference (BI) and Maximum Likelihood (ML), were employed to reconstruct phylogenetic relationships as described by Makkonen et al. [21]. The following haplotype sequences from GenBank were used as references in the approximations: accession numbers MF973121-MF973149 for rnnS and MF975950-MF975978 for rnnL. Aphanomyces frigidophilus was used as outgroup. We analyzed rnnS and rnnL independently, and a concatenated rnnS and rnnL dataset with the same parameters.

Macroscopic and microscopic examination
Macroscopic observations showed that all P. clarkii and P. leniusculus specimens exhibited characteristic melanized areas on the subabdominal cuticle, joints and chelae (Fig 2). Melanized patches or spots on the C. japonicus cuticles were not observed. However, microscopic September 2015 Location next to the second mass mortality Second mass mortality event detected CE15/36-10 d1 examination of the subabdominal soft cuticle of the C. japonicus samples revealed an abundance of non-melanized A. astaci hyphae (Fig 3). These hyphae had rounded tips and similar diameters, ca 10 μm, characteristics of an A. astaci infection. However, no melanized hyphae or micro-melanized spots were detected in any of the C. japonicus samples analyzed (Fig 3).

Molecular analyses
One C. japonicus from each of the two mortality event localities, one C. japonicus from the location proximate to the second outbreak, one P. leniusculus and five P. clarkii tested positive for A. astaci based on amplification of the ITS region with the diagnostic primers 42 [40] and 640 [41] (Table 1). BLAST analyses of the sequenced PCR products showed 100% similarity to strain SAP0877 Aphanomyces astaci (GenBank accession number KX555484), which originated from P. clarkii [44]. PCR amplification of the mitochondrial ribosomal rnnS and rnnL regions of the infected specimens produced 476 base pairs (bp) and 355 bp fragments, respectively (GenBank accession number for rnnS MG905008-MG905015 and for rnnL MG905000-MG905007). The BI and ML analyses of the rnnS (Fig 4A) and rnnL (Fig 4B) regions recovered congruent topologies and indicated the presence of a novel haplotype, d3. Analysis of the concatenated rnnS  and rnnL dataset supported a new clade comprised of the novel d3-haplotype, which corresponds to the D-haplogroup (Fig 4C). One of the C. japonicus specimen from the first crayfish plague outbreak and five of the P. clarkii specimens showed this haplotype (Table 1, Fig 4C). The presence of the d1-haplotype, grouped within the D-haplogroup, was supported for one of the C. japonicus specimen from the second crayfish plague outbreak (and one specimen from the proximate locality). The infected P. leniusculus specimen from Shikaribetsu Lake grouped within the b-haplotype in the B-haplogroup (Table 1, Fig 4C).
Observed haplotype diversity (Fig 5) is consistent with the phylogenetic analyses (Fig 4). The amplicons corresponding to the rnnS region registered three segregating sites, resulting in four different haplotypes (Fig 5A) (Table 2), whereas the amplicons from the rnnL region  registered eight segregating sites and five different haplotypes (Fig 5B) ( Table 2). The concatenated rnnS + rnnL dataset showed a total of 11 segregating sites, supporting the existence of six haplotypes (Fig 5C) (Table 2).

Discussion
In this study, we report and describe the first cases of crayfish plague mass mortalities in Japan using histological and molecular approaches. These two cases also represent the first reported crayfish plague outbreaks in a native crayfish population outside of Europe and Asia minor. We found that these mass mortalities in C. japonicus populations originated from P. clarkii populations, based on the presence of the A. astaci d1-and d3-haplotypes. The d3-haplotype is a novel haplotype reported here for the first time. These two haplotypes belong to the D-haplogroup, which is associated with P. clarkii. Furthermore, we detected the novel d3-haplotype in P. clarkii specimens from Japan. Although the susceptibility of C. japonicus species to A. astaci was first demonstrated by Unestam in 1969 [4], no massive mortalities associated with A. astaci have been described until our study. We have shown that the pathogen A. astaci can cause mass mortalities among native Japanese crayfish populations as it has in native European crayfish populations [5]. Furthermore, histological analyses of C. japonicus tissues revealed abundant and non-melanized hyphae of A. astaci growing within the cuticle, similar to what has been observed in European species [45]. In contrast to the highly resistant North American crayfish species, P. clarkii and P. leniusculus [45,46], we did not observed signs of resistance against this pathogen, i.e., melanized hyphae or spots, in C. japonicus. The North American species are often chronically infected by the pathogen due to a strong immune response [47], which contains the pathogen but allows the dispersion of its infectious units, the biflagellate zoospores, which can then colonize new crayfish hosts, such as C. japonicus.
The crayfish plague outbreaks in Minami-ku and Ishikari River occurred in the vicinity of a P. clarkii population in Yasuharu (Fig 1). In this study, we also provide evidence, based on mtDNA rnnS and rnnL analyses of clinical samples, that both outbreaks are consequences of the transmission of the pathogen from P. clarkii. Our analyses indicated that the A. astaci haplotype present in C. japonicus from Minami-ku and P. clarkii from Yasuharu is the d3-haplotype. On the other hand, the specimens from the second mass mortality event in Ishikari River presented the d1-haplotype (of the D-haplogroup). This finding suggests that a different P. clarkii population infected these particular C. japonicas specimens. These results represent an additional concern in Japan, as the two haplotypes associated with the crayfish plague outbreaks here belong to a virulent D-haplogroup. The physiological properties of this haplogroup's strains allow them to grow, sporulate, and produce zoospores at higher temperatures than other strains [17]. Although the two P. clarkii associated haplotypes, d1-and d3-haplotypes, were found to be the cause of the mass mortalities, we also detected the presence of the b-haplotype in its natural carrier P. leniusculus from Shikaribetsu Lake in the central region of eastern Hokkaido Island. Therefore, two strains with different temperature preferences are now in Japan, which creates the potential for native C. japonicus to be infected by the pathogen at a wider temperature range. This is also very similar to the scenario in Southern Europe, where both B-and D-haplogroups (with their respective b-and d1-and d2-haplotypes) coexist and have driven the native European crayfish species Austropotamobius pallipes to a risk of extinction [10,13,48].
Numerous studies have warned about the risks concerning the North American crayfish carrying A. astaci [9,46,[49][50][51][52][53][54]; these risks were specifically discussed for Japan by Mrugala in 2016 [38]. Several studies carried out in Japan have indicated that aggressive interaction for shelter and predation by P. leniusculus is causing the decline of C. japonicus [30,37,55]. However, P. clarkii has not been implicated in its decline, until now. The risk posed by P. clarkii was probably overlooked as C. japonicus and P. clarkii, generally speaking, have different habitats due to their individual environmental requirements [32]. Thus, it should be taken into account that P. clarkii possesses great adaptability, making it a successful colonizer in the aquatic ecosystem of Japan [56], including in C. japonicus habitats.
Our results demonstrate that the pathogen A. astaci constitutes an actual threat to the endemic and endangered C. japonicus. Consequently, we urge authorities to rapidly develop and implement action plans, including strategies that aim to restore and manage native C. japonicus populations and to control and/or eradicate invasive crayfish species, especially P. clarkii and P. leniusculus. In Europe, the implementation of similar plans have allowed the conservation of the native European crayfish [57]. Moreover, preventing new introductions and translocations of North American crayfish species in Japan needs to be prioritized. The results presented in this study also pose as a warning of the potential risk of similar episodes of A. astaci spreading with alien crayfish to continents thus far free of the crayfish plague pathogen.