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First discovery of Perkinsus beihaiensis in Mediterranean mussels (Mytilus galloprovincialis) in Tokyo Bay, Japan
Journal of Invertebrate Pathology 166 (2019) 107226
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First discovery of Perkinsus beihaiensis in Mediterranean mussels (Mytilus galloprovincialis) in Tokyo Bay, Japan
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Naoki Itoha, , Yoshiki Komatsua, Kazuki Maedaa, Shotaro Hiraseb, Tomoyoshi Yoshinagaa a
Laboratory of Fish Diseases, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1, Yayoi, Bunkyo, Tokyo 113-8657, Japan Fisheries Laboratory, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 2971-4, Bentenjima, Maisaka, Nishi-ku, Hamamatsu, Shizuoka 4310214, Japan
b
A R T I C LE I N FO
A B S T R A C T
Keywords: Perkinsus beihaiensis Mediterranean mussel Mytilus galloprovincialis Epidemiology
During analyses of the invasive Mediterranean mussel Mytilus galloprovincialis for pathologies in Tokyo Bay, infection by the protozoan parasite Perkinsus beihaiensis was found through histological examination, Ray’s Fluid Thioglycollate Medium assays, and molecular analyses. Specific PCR assays for each Perkinsus species also revealed the presence of an indigenous congeneric species, Perkinsus olseni, but P. beihaiensis was dominant in M. galloprovincialis. Sequences of the ribosomal internal transcribed spacer region I of P. beihaiensis found in Japan were genetically more similar to those found in South American countries (Panama and Brazil) than in Asian countries (China and India). Though Mediterranean mussels have become widespread in Japanese waters since their invasion in the 1930s, epidemiological surveys show that mussels collected outside Tokyo Bay are free of any Perkinsus infections. Based on these results, it was strongly suggested that P. beihaiensis invaded Tokyo Bay by transportation of bivalves originating from South America but has not yet spread to other parts of Japan. The possibility is not ruled out, however, that the parasite is indigenous in Japan but the environment in Tokyo Bay favors its transmission to Mediterranean mussels.
1. Introduction Various infectious diseases have emerged with the development of bivalve aquaculture and are now commonly accepted as serious obstacles for the further development of the industry. Currently, seven important infectious diseases affecting mollusks are listed as notifiable diseases by the World Organisation for Animal Health (OIE, 2018). In Japan, Akoya oyster disease, the abnormal enlargement of the ovary in the Pacific oyster, and perkinsosis in the Manila clam are serious infectious diseases seen during bivalve production (Morizane et al., 2001; Matsusato and Masumura, 1981; Shimokawa et al., 2010). Recently, infection by ostreid herpesvirus 1 (OsHV-1) and Francisella halioticida were added as novel health threats for juvenile Pacific oysters and the Yesso scallop, respectively (Nagai and Nakamori, 2018; Kawahara et al., 2018), and the gravity of these infectious diseases and the problems they may cause are gradually gaining recognition in Japan. It is known that introduction of exotic bivalves has often caused introduction and spread of pathogenic agents of bivalves (Reunault, 1996; Goedknegt et al., 2016). Haplosporidium nelsoni invaded North America by introduction of the Pacific oyster from Asian countries and
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caused detrimental impacts on the production of the eastern oyster Crassostrea virginica (Burreson and Ford, 2004). Also, Batista et al. (2015) suggested that introduction of the Pacific oyster from Asia resulted in the introduction of OsHV-1 to Europe. Thus field surveys for infectious agents in exotic bivalves are highly important for early biosecurity measures, such as zoning or compartmentalization. In 2016, we conducted a field survey targeting infectious agents of the Mediterranean mussel Mytilus galloprovincialis in Tokyo Bay. Since this alien bivalve is considered a nuisance species and is not a major aquaculture species in Japan, our survey was not concerned with health impacts on the mussel itself. Rather, this species is known to be infected with diseases such as marteiliosis and disseminated neoplasia (Berthe et al., 2004; Ciocan and Sunila, 2005). We were concerned that these diseases could be introduced and spread by the mussels, and have a negative influence on the production of edible bivalve species in Japan. During the survey, we discovered the presence of the protozoan parasite Perkinsus beihaiensis, which had never been reported in this mussel species, or in Japan. Genus Perkinsus is a protistan parasite group containing pathogens that are highly deleterious for mollusk production; for example, P. marinus in the eastern oyster, P. olseni in Manila clam, grooved clam and Australian abalones, and P. qugwadi in
Corresponding author. E-mail address: [email protected] (N. Itoh).
https://doi.org/10.1016/j.jip.2019.107226 Received 6 May 2019; Received in revised form 27 July 2019; Accepted 29 July 2019 Available online 29 July 2019 0022-2011/ © 2019 Elsevier Inc. All rights reserved.
Journal of Invertebrate Pathology 166 (2019) 107226
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Fig 1. A map of sampling sites in this study. Sampling sites in Tokyo Bay are shown in the left-above inlet. Detailed information for each sampling site is provided in Table 1.
supplemented with antibiotics. Individuals heavier than 1 g were cut at median lines, and halves of the bodies were used for the assay. After incubation at 25 °C for 7 days in the dark, the tissues were collected by centrifugation and treated with 2 M NaOH at 60 °C until the tissues were lysed. Prezoosporangia were then collected by centrifugation, washed twice with distilled water, and counted in 96-well plates after staining with Lugol’s iodine. The mean intensity of infection was calculated as “log10 (number of prezoosporangia/wet tissue weight + 1)”. For the molecular identification of Perkinsus sp. in Mediterranean mussels, five individuals (mean shell height ± SD: 38.9 ± 2.7 mm) collected on the same day as above were used for the RFTM assay. After being incubated at 25 °C for 7 days, the tissues were placed in a Petri dish, stained with Lugol’s iodine, and observed under a dissecting microscope CK2 (Olympus, Tokyo, Japan). Tissues containing prezoosporangia were recovered from two individuals and stored in 70% ethanol until DNA extraction. DNA was extracted using a solution of 10% Chelex (Bio-Rad, Hercules, CA, USA) resin as described by de Faveri et al. (2009). PCR targeting the internal transcribed spacer (ITS) region for Perkinsus spp. was conducted using the genus-specific primers PerkITS-85 and PerkITS-750 developed by Casas et al. (2002). The final volume of the PCR reaction was 20 µL containing 1x Takara HS Ex Taq Buffer (Takara Bio Inc., Shiga, Japan), 0.2 mM dNTPs, 12 pmol of each PCR primer, 0.1 µL of Takara Ex Taq Hot Start version (Takara Bio Inc., Shiga, Japan) and
Yesso scallops (Villalba et al., 2004). In order to assess the potential for spillover of this parasite to other bivalve species in Japan, we examined the current geographic distribution. During the epidemiological surveys, we also conducted phylogenetic analyses to estimate the relationships with P. beihaiensis strains found outside Japan.
2. Materials and methods 2.1. Initial survey for Mediterranean mussels in Tokyo Bay In March 2016, 51 Mediterranean mussels (mean shell height ± SD: 32.6 ± 5.3 mm) were collected from the Koitogawa fishery port in Chiba, Japan (Site 1 in Fig. 1 and Table 1). After shucking the shell, the tissues were sliced to include the gills, mantles, digestive gland, and gonads and fixed in Davidson’s solution for a few days. Fixed tissues were processed using routine histological protocols for paraffin embedding, and tissue sections were cut at 5 µm thickness and stained with Mayer’s hematoxylin and eosin stain. For detection of Perkinsus infection, ten Mediterranean mussels (mean shell height ± SD: 40.3 ± 3.5 mm) were collected from the same sampling site as mentioned above in April 2016 and used for whole body burden assays (Choi et al., 1989; Almeida et al., 1999). After shucking the shell, the soft bodies were weighed and whole bodies were inoculated in Ray’s Fluid Thioglycolate Medium (RFTM) 2
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Table 1 Detailed information for sampling sites and dates, species, shell lengths of sampled bivalves, and prevalence and infection intensity of Perkinsus spp. estimated by RFTM assay. Location numbers correspond to numbers in the sampling map of Fig. 1. Mean infection intensity of Perkinsus spp. shown was calculated as log (number of prezoosporangium/wet tissue weight (g) + 1). Location
Sites
Dates
Species
n
Shell height (mean ± SD mm)
+ve by RFTM assay
Infection intensity (mean ± SD)
1 2 3 4 5 6 7 8 9 10 11
Koitogawa, Chiba Ichikawa, Chiba Kanazawa, Kanagawa Kurihama, Kanagawa Jogashima, Kanagawa Yoichi, Hokkaido Hakodate, Hokkaido Mustu Bay, Aomori Noheji, Aomori Obuchinuma, Aomori Shimanokoshi, Iwate
June 7, 2016 June 22, 2016 June 22, 2016 June 7, 2016 June 7, 2016 May 11, 2018 May 8, 2018 Oct 2017 Oct 4, 2017 Oct 2, 2017 Oct 5, 2017
12 13 14 15 16 17 18
Ogatsu Bay, Miyagi Katase-Enoshima, Kanagawa Shimizu, Shizuoka Bentenjima, Shizuoka Hikamasima, Aichi Oshima, Wakayama Shirahama, Wakayama
May 29, 2017 Nov 19, 2017 Nov 13. 2017 Sept 16, 2016 April 2017 Nov 1, 2017 Oct 5, 2017
19
Ushimado, Okayama
Oct 4, 2016
20 21 22
Nagasu, Kumamoto Obama, Fukui Noto, Ishikawa
May 31, 2016 Apr 2, 2018 Mar 15, 2018
M. galloprovincialis M. galloprovincialis M. galloprovincialis M. galloprovincialis M. galloprovincialis M. galloprovincialis M. galloprovincialis M. galloprovincialis M. galloprovincialis M. galloprovincialis Mytilus sp. Septifer virgatus M. galloprovincialis M. galloprovincialis M. galloprovincialis M. galloprovincialis M. galloprovincialis Mytilus sp. M. galloprovincialis Perna viridis M. galloprovincialis P. viridis M. galloprovincialis M. galloprovincialis M. galloprovincialis
30 30 30 30 30 60 30 15 30 15 27 15 28 29 29 30 30 13 24 30 22 10 10 30 30
56.5 28.1 59.4 65.6 40.4 54.9 47.9 95.1 38.9 49.6 49.2 22.0 61.5 26.0 37.7 32.8 71.9 27.9 27.4 65.0 36.0 28.2 34.9 46.6 65.4
30 13 20 23 23 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
4.91 0.92 1.15 1.28 0.65 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
6.1 4.2 7.7 8.1 4.6 3.8 4.9 12.5 5.3 12.6 13.8 5.6 2.4 6.3 4.7 3.6 7.1 13.3 12.8 6.0 18.9 8.0 3.4 5.0 7.0
± ± ± ± ±
0.97 0.29 0.91 0.99 0.36
Perkinsus spp. was examined using the whole body burden assay as described above. Additionally, Mediterranean mussels (n = 562) and related mussel species (Perna viridis (n = 40), Septifer virgatus (n = 15), and an unidentified Mytilus sp. (n = 40) were collected from 17 sampling sites in Japanese waters (Sites 6–22 in Fig. 1 and Table 1). They were also subjected to the RFTM assay as described above.
1.0 µL of the extracted template DNA. The thermal cycler program consisted of an initial denaturation at 95 °C for 4 min; 40 cycles of 95 °C for 1 min, 55 °C for 1 min, and 65 °C for 3 min; and a final extension at 65 °C for 5 min. The PCR products were visualized on a 1.5% agarose gel stained with SYBR Safe DNA gel stain (Thermo Fisher Scientific, Waltham, MA, USA), and the bands of approximately 700 bp were extracted using the FastGene Gel/PCR Extraction Kit (Nippon Genetics, Tokyo, Japan). The extracted PCR products were sent to an external sequencing facility (Eurofins Genomics, Tokyo, Japan) and sequenced using the original primers used for each PCR. The obtained sequences were compared by BLAST search against the NCBI GenBank database (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_ TYPE=BlastSearch&LINK_LOC=blasthome). Since the Perkinsus species found in our mussels were identified to be P. beihaiensis based on the BLAST searches, in situ hybridization was conducted for confirmation as described in Moss et al. (2008).
2.3. Species identification of Perkinsus in Tokyo Bay In order to identify the Perkinsus species found in Tokyo Bay, DNA sequences of prezoosporangia produced in RFTM were analyzed (Audemard et al., 2008). In brief, 10 mussels were collected from Jogashima and Koitogawa (Sites 1 and 5 in Fig. 1 and Table 1) in August 2017. Gill leaflets were excised and incubated in RFTM at 25 °C for 7 days. After staining with Lugol’s iodine, tissues were placed in a plastic dish and observed under the microscope. Tissues containing prezoosporangia were recovered, stored in 70% ethanol, and then underwent DNA extraction using the buffer system of QIAamp DNA Mini Kit (QIAGEN, Venlo, Netherlands) and EconoSpin IIA column (GeneDesign Inc., Osaka, Japan). Extracted DNA was applied to PCR assays using probes specifically developed for P. marinus (Audemard et al., 2004), P. olseni (Umeda and
2.2. Epidemiological surveys for Perkinsus infection in Japanese waters In June and July 2016, 30 Mediterranean mussels were sampled from five sites in Tokyo Bay: Koitogawa, Ichikawa, Kanesawa, Kurihama, and Jogashima (Sites 1–5 in Fig. 1 and Table 1). Tissues of mussels were incubated in RFTM, and the intensity of infection by Table 2 List of PCR primers specific to each Perkinsus species used in this study. Primer name
Sequences
Target species
References
PerkITS-85 PerkITS-750 PmarITS-70F PmarITS600R PerkOF PerkOR PchesITS-F PchesITS-R PerkHF PerkHR PerkITS-430R
5′-CCG CTT TGT TTG GAT CCC-3′ 5′-ACA TCA GGC CTT CTA ATG ATG-3′ 5′-CTT TTG YTW GAG WGT TGC GAG ATG-3′ 5′-CGA GTT TGC GAG TAC CTC KAG AG-3′ 5′-CTT AAC GGG CCG TGT TA-3′ 5′-CAT AAC GAA CTA TCT CCG AAG-3′ 5′-AAA CCA GCG GTC TCT TCT TCG G-3′ 5′-CGG AAT CAA CCA CAA CAC AGT CG-3′ 5′-CTG CCT GGC AGG TGA T-3′ 5′-CGA ATT GGC TCA ATA AAT TG-3′ 5′-TCT GAG GGG CTA CAA TCA T-3′
Perkinsus spp. (except for P. qugwadi) Perkinsus spp. (except for P. qugwadi) P. marinus P. marinus P. olseni P. olseni P. chesapeaki P. chesapeaki P. honshuensis P. honshuensis P. beihaiensis
Casas et al. (2002) Casas et al. (2002) Audemard et al. (2004) Audemard et al. (2004) Umeda and Yoshinaga (2012) Umeda and Yoshinaga (2012) Burreson et al. (2005) Burreson et al. (2005) Umeda (2012) Umeda (2012) Moss et al. (2008)
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individuals. Similar cells were also often observed in the mantle and gill lamellae (Fig. 2B). The RFTM assay detected Perkinsus infection in all of the ten Mediterranean mussels collected from the same site, and the mean intensity of infection was 3.25 ± 1.14 (range: 1.70–5.31). DNA samples extracted from the prezoosporangia of another five mussels were positive according to the Perkinsus genus-specific PCR, and the ITS sequences (648 bp) obtained from two individuals were 99% identical to those of P. beihaiensis collected in Panama (e.g. KU172665 and KU172673). Those newly obtained sequences in Japan were deposited in GenBank under the accession numbers LC437316 and LC437317. In situ hybridization using oligonucleotide probes developed for P. beihaiensis (Moss et al., 2006) showed positive cells (Fig. 2C) in the connective tissues of Mediterranean mussels, confirming that this mussel species in Tokyo Bay is infected with P. beihaiensis.
Yoshinaga, 2012), P. chesapeaki (Burreson et al., 2005), P. honshuensis (Umeda and Yoshinaga, 2012), or P. beihaiensis (Moss et al., 2008) (Table 2). The final volume of the PCR reactions was 20 µL containing 10 µL of KOD One PCR Master Mix -Blue- (Toyobo, Osaka, Japan), 30 pmol of each PCR primer, and 1.0 µL of the extracted DNA samples. The thermal cycler program for P. olseni, P. beihaiensis, and P. honshuensis consisted of 40 cycles of 98 °C for 10 sec, 50 °C for 5 sec, and 68 °C for 1 sec, while annealing temperatures of 53 °C and 56 °C were applied for P. marinus and P. chesapeaki, respectively. Before the assays, the specificity of the PCR protocols above was confirmed using positive control DNA of each Perkinsus species. PCR products with molecular weights similar to the positive controls were extracted and sequenced with the primers used for each PCR, as described above. 2.4. Haplotype analysis of Perkinsus beihaiensis based on the ribosomal ITS1 region
3.2. Epidemiological surveys of Perkinsus in Mediterranean mussels in Japanese waters
For the haplotype network analysis, ITS1 sequences of P. beihaiensis obtained from Japanese samples as above (n = 19) and 80 ITS1 sequences of P. beihaiensis with geographic information deposited in GenBank (supplementary data) were used. Using MEGA 7.0 software, 99 sequence data were aligned by CLUSTAL W (Thompson et al., 1994) at default settings. Two sequences (GenBank Accession No. MF595808 and MF595809) appeared to be genetically unrelated to the other sequences and were removed from further analyses. The haplotype network among 97 ITS1 sequences of P. beihaiensis was constructed with the TCS network method (Clement et al., 2000) using PopART (Leigh and Bryant, 2015). In this analysis, nucleotide sites containing ambiguous nucleotides were masked.
Perkinsus infection was detected in Mediterranean mussels collected from five sites in Tokyo Bay (Sites 1–5 in Fig. 1 and Table 1). The prevalence and mean intensity of infection were highest in Koitogawa (100% and 4.91, respectively) and lowest in Ichikawa (43.3% and 0.92, respectively) (Table 2). Meanwhile, Perkinsus infection was not detected in Mediterranean mussels nor in any related species collected from the other 17 sites outside Tokyo Bay (Table 2). 3.3. Identification of Perkinsus species in Mediterranean mussels
3. Results
The Mediterranean mussels collected from Jogashima (n = 10) and Koitogawa (n = 10) in August 2017 were all positive according to the RFTM assay. Following DNA extraction from tissues incubated in RFTM, PCR assays indicated infections by P. beihaiensis and P. olseni. These results were validated by sequencing the PCR products. In both sampling sites, P. beihaiensis was detected more often than P. olseni; 10 mussels were positive for P. beihaiensis and 5 were positive for P. olseni in Koitogawa, and 9 mussels were positive for P. beihaiensis and 1 was positive for P. olseni in Jogashima (Table 3). Nineteen sequences identified as P. beihaiensis were deposited in GenBank (LC437318 –
3.1. Initial detection of Perkinsus beihaiensis in Mediterranean mussels in Tokyo Bay Histological observations revealed that 24 of the 51 examined mussels had foci of hemocyte infiltration in the connective tissues around the digestive gland. Perkinsus-like cells with a large vacuole and eccentric nucleus were found in the foci of three individuals (Fig. 2A), although specific pathogen-like cells were not recognized in the other
Fig 2. Histological observations of Perkinsus cells in the Mediterranean mussel, Mytilus galloprovincialis. A. Perkinsus cells with vacuoles (arrows) are surrounded by hemocyte infiltration in the connective tissues around the digestive diverticula. Hematoxylin-Eosin Stain. B. A trophozoite of Perkinsus sp. (arrow) in the gill. Hematoxylin-Eosin Stain. C. Trophozoite cells of Perkinsus sp. were detected by in situ hybridization using specific oligonucleotide probes for P. beihaiensis.
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Table 3 Results of PCR assays specific for each Perkinsus species against Mediterranean mussels, Mytilus galloprovincialis, collected in Tokyo Bay, Japan.
Koitogawa Jogashima
P. beihaiensis
P. olseni
P. marinus
P. chesapeaki
P. mediterraneus
P. honshuensis
10/10 9/10
5/10 1/10
0/10 0/10
0/10 0/10
0/10 0/10
0/10 0/10
Fig 3. TCS network of 30 ITS1 haplotypes of Perkinsus beihaiensis estimated by PopART. Each line between haplotypes indicates a single nucleotide substitution. The size of each circle is proportional to the absolute haplotype frequency, Small black circles represent missing haplotypes and color shows localities where each haplotype was observed.
25
Brazil Panama
2 1
China Japan India Missing haplotype
LC437327 for sequences from Jogashima and LC437328 – LC437336 for samples from Koitogawa). Other Perkinsus species such as P. marinus, P. chesapeaki, and P. honshuensis were not detected in the Mediterranean mussels in this study (Table 3).
So far, two species of Perkinsus, P. olseni and P. honshuensis, have been reported in Japanese waters (Hamaguchi et al., 1998; Dungan and Reece, 2006) and, thus, Perkinsus cells found in Mediterranean mussels in this study were initially expected to be one or both of these. However, DNA sequence analyses and subsequent in situ hybridization confirmed that the Perkinsus cells infecting the Mediterranean mussels were P. beihaiensis. This Perkinsus species was first described in China (Moss et al., 2008) and subsequently was reported in Brazil (Sabry et al., 2009), India (Sanil et al., 2012) and Panama (Pagenkopp Lohan et al., 2016). This is the first report from Japan. In addition, this species has so far been reported in 14 bivalve species (Pagenkopp Lohan et al., 2018); we showed that the Mediterranean mussel is also a host species for this parasite. PCR assays specific to each Perkinsus species revealed that Mediterranean mussels in Tokyo Bay were also infected with P. olseni, but the prevalence of P. beihaiensis was much higher than that of P. olseni, suggesting that P. beihaiensis is the dominant species in Mediterranean mussels in Tokyo Bay. This suggestion is also supported by our preliminary qPCR assays for P. olseni and P. beihaiensis in which the intensity of P. beihaiensis was much higher than P. olseni in Mediterranean mussels sampled in Tokyo Bay (Y. Komatsu, unpublished data). This mussel species seems to be highly susceptible to P. beihaiensis. In spite of susceptibility of Mediterranean mussels to parasitism by P. beihaiensis, Perkinsus infection has never been found in Mediterranean mussels outside Tokyo Bay in either previous (Hamaguchi et al., 2002) or current surveys. One of the possibilities to explain the absence of P. baihaiensis in the susceptible bivalves outside Tokyo Bay is that P. beihaiensis invaded Tokyo Bay from another marine area but has not yet spread to other parts of Japan. Mediterranean mussels invaded Japan around the 1930s, rapidly expanded their geographic distribution, and now can be found in most areas of Japan (Ishida et al., 2005). Therefore, if P. beihaiensis is an exotic species in Japan, this parasite invaded Tokyo Bay after the
3.4. Haplotype analysis of Perkinsus beihaiensis based on the ribosomal ITS1 region Based on the ITS1 region, 30 haplotypes were recognized in the P. beihaiensis used for this analysis. Two haplotypes from Brazil were relatively dissimilar genetically to the two major haplogroups. The other 28 haplotypes were divided into the two major haplogroups, which were separated by at least three nucleotide differences (Fig. 3). One of the haplogroups was composed of haplotypes found in Asian countries (China and India), while the other comprised haplotypes identified in the Americas (Brazil and Panama). Only two haplotypes were found in Japan, and both haplotypes were included in the haplogroups of Brazil and Panama (Fig. 3). 4. Discussion The initial surveys conducted in Tokyo Bay revealed the presence of Perkinsus beihaiensis in Mediterranean mussels, Mytilus galloprovincialis, for the first time in Japan. Subsequent epidemiological surveys in Japanese waters indicated that Mediterranean mussels in Tokyo Bay were frequently infected with this parasite; however, no Perkinsus infections were detected in individuals outside Tokyo Bay. PCR analyses specific to each Perkinsus species revealed the presence of both P. olseni and P. beihaiensis in Mediterranean mussels in Tokyo Bay, but P. beihaiensis appeared to be dominant in the mussels. Haplotype network analyses based on the ribosomal ITS1 region showed that the P. beihaiensis in Tokyo Bay is genetically more similar to P. beihaiensis found in South American countries than to that from in Asian countries. 5
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dispersion of Mediterranean mussels in Japanese waters, as otherwise it would have spread widely in Japanese waters along with the Mediterranean mussels. Following the hypothesis above, it is likely that P. beihaiensis in Japan originated from South America. We base this hypothesis on the finding that haplotypes found in Japan are included in the haplotypes from Panama and Brazil. Since only a few haplotypes have been detected in Japan through this study, introductions of P. beihaiensis from South America to Japan may have occurred only a few times. The present study did not provide any information regarding the invasion route of P. beihaiensis into Tokyo Bay. To our knowledge, Tokyo Bay does not have any aquaculture industry introducing bivalve seed from South American countries, and it is not likely that P. beihaiensis was introduced along with the introduction of bivalve seeds for aquaculture purposes. On the other hand, six international ports are located in Tokyo Bay, and maritime transportation in those ports often brings alien bivalve species into Tokyo Bay (Hiwatari et al., 2006). P. beihaiensis may have been introduced into Tokyo Bay with bivalves in ballast water or by attaching to a vessel surface, as suggested by Pagenkopp Lohan et al. (2018). We have not ruled out that P. baihaiensis is indigenous in Japan and that infection in Mediterranean mussels of Tokyo Bay is highly possibly due to characteristic environmental conditions of the bay. So far, surveys for infectious agents have been limited to commercially important mollusks species in Japan, and further surveys for non-commercial species outside Tokyo Bay may be required to determine if P. beihaiensis is indigenous or introduced. In the present study, we reported the presence of P. beihaiensis in Mediterranean mussel in Japan for the first time. The possibility was strongly suggested that P. beihaiensis invaded Tokyo Bay but has not yet spread to other parts of Japan. Host specificity of this parasite is relatively low, therefore, if the hypothesis above is correct, P. beihaiensis may be easily transmitted with the movement of various susceptible bivalve species and invade new areas and cause diseases problems in economically important bivalve species after expansion because parasitic organisms often show strong pathogenicity to new host species (host switching) after invading new locations. Additional field surveys for this parasite are urgently needed to identify geographic distribution. Also, in order to assess the potential risk of this parasite for Japanese fishery, the establishment of experimental infections for edible bivalve species is also needed.
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Hamaguchi, M., Suzuki, N., Usuki, H., Ishioka, H., 1998. Perkinsus protozoan infection in shortnecked clam Tapes (Ruditapes) philippinarum in Japan. Fish Pathol. 33, 473–480. Hiwatari, T., Shiotsuka, Y., Kohata, K., Watanabe, M., 2006. Exotic hard clam in Tokyo Bay identified as Mercenaria mercenaria by genetic analysis. Fish. Sci. 72, 578–584. Ishida, S., Iwasaki, K., Kuwahara, Y., 2005. Initial invasion history and process or range extension of Mytilus galloprovincialis – inferred from the specimens collected by T. Furukawa. Venus 64, 151–159. Kawahara, M., Kanamori, M., Meyer, G.R., Yoshinaga, T., Itoh, N., 2018. Francisella halioticida, identified as the most probable cause of adductor muscle lesions in Yesso scallops Patinopecten yessoensis cultured in southern Hokkaido, Japan. Fish Pathol. 53, 78–85. Leigh, J.W., Bryant, D., 2015. POPART: full-feature software for haplotype network construction. Meth. Ecol. Evol. 6, 1110–1116. Matsusato, T., Masumura, K., 1981. Abnormal enlargement of the ovary of oyster, Crassostrea gigas (Thurnberg) by an unidentified parasite. Fish Pathol. 15, 207–212. Morizane, T., Takimoto, S., Nishikawa, S., Matsuyama, N., Tyohno, K., et al., 2001. Mass mortalities of Japanese Pearl Oyster in Uwa Sea, Ehime in 1997–1999. Fish Pathol. 36, 207–216. Moss, J.A., Xiao, J., Dungan, C.F., Reece, K.S., 2008. Description of Perkinsus beihaiensis n. sp., a new Perkinsus sp. parasite in oysters of Southern China. J. Eukaryot. Microbiol. 55, 117–130. Nagai, T., Nakamori, M., 2018. Experimental infection of ostreid herpesvirus 1 (OsHV-1) JPType 1, a Japanese variant, in Pacific oyster Crassostrea gigas larvae spats. Fish Pathol. 53, 71–77. OIE, 2018. Aquatic Animal Health Code. OIE, Paris. http://www.oie.int/en/standard-setting/ aquatic-code/access-online/ (accessed 16 Nov 2018). Pagenkopp Lohan, K.M., Hill-Spanik, K.M., Torchin, M.E., Aguirre-Macedo, L., Fleischer, R.C., Ruiz, G.M., 2016. Richness and distribution of tropical oyster parasites in two oceans. Parasitology 143, 1119–1132. Pagenkopp Lohan, K.M., Hill-Spanik, K.M., Torchin, M.E., Aguirre-Macedo, L., et al., 2018. Phylogeography and connectivity of molluscan parasites: Perkinsus spp. in Panama and beyond. Int. J. Parasitol. 48, 135–144. Reunault, T., 1996. Appearance and spread of diseases among bivalve molluscs in the northern hemisphere in relation to international trade. Rev. Sci. Tech. Off. Int. Epiz. 15, 551–561. Sabry, R.C., Rosa, R.D., Magalhães, A.R., Barracco, M.A., Gesteira, T.C., da Silva, P.M., 2009. First report of Perkinsus sp. infecting mangrove oysters Crassostrea rhizophorae from the Brazilian coast. Dis. Aquat. Org. 88, 13–23. Sanil, N.K., Suja, G., Lijo, J., Vijayan, K.K., 2012. First report of Perkinsus beihaiensis in Crassostrea madrasensis from the Indian subcontinent. Dis. Aquat. Org. 98, 209–220. Shimokawa, J., Yoshinaga, T., Ogawa, K., 2010. Experimental evaluation of the pathogenicity of Perkinsus olseni in juvenile Manila clams, Ruditapes philippinarum. J. Invertebr. Pathol. 105, 347–351. Thompson, J.D., Higgins, D.G., Gibson, T.J., 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucl. Acids Res. 22, 4673–4680. Umeda, K., Yoshinaga, T., 2012. Development of real-time PCR assays for discrimination and quantification of two Perkinsus spp. in the Manila clam Ruditapes philippinarum. Dis. Aquat. Org. 99, 215–225. Umeda, K., 2012. Studies on Two Perkinsus Species Infecting the Manila Clam. PhD Dissertation. The University of Tokyo. Villalba, A., Reece, K.S., Ordas, M.C., Casas, S.M., Figueras, A., 2004. Prekinsosis in molluscs: a review. Aquat. Living Resour. 17, 411–432.
Acknowledgments Bivalves used in this study were kindly provided by Professors Kenji Ohkoshi (Toho University), Shotaro Izumi (Tokai University), Sho Shirakashi (Kindai University), Hiroaki Suetake (Fukui Prefectural University), Hiroyasu Kamei (Kanazawa University), Drs. Makoto Kanamori and Yohei Shimizu (Hokkaido Research Organization), Mrs. Fumiyasu Murayama (Okayama Prefectural Technology Center for Agriculture, Forestry, and Fisheries Research Institute), and Shozo Ogiso (Kanazawa University). This study was funded by JSPS KAKENHI (Grant Number JP16H04967 to Tomoyoshi Yoshinaga and Grant Number JP17H03858 to Naoki Itoh). Natalie Kim, PhD, from Edanz Group (www.edanzediting.com/ac) edited a draft of this manuscript. Appendix A. Supplementary material Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jip.2019.107226. References Almeida, M., Berthe, F., Thebault, A., Dinis, M.T., 1999. Whole clam culture as a quantitative diagnostic procedure of Perkinsus atlanticus (Apicomplexa, Perkinsea) in clams Ruditapes decussatus. Aquaculture 177, 325–332.
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First discovery of Perkinsus beihaiensis in Mediterranean mussels (Mytilus galloprovincialis) in Tokyo Bay, Japan
T
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Naoki Itoha, , Yoshiki Komatsua, Kazuki Maedaa, Shotaro Hiraseb, Tomoyoshi Yoshinagaa a
Laboratory of Fish Diseases, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1, Yayoi, Bunkyo, Tokyo 113-8657, Japan Fisheries Laboratory, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 2971-4, Bentenjima, Maisaka, Nishi-ku, Hamamatsu, Shizuoka 4310214, Japan
b
A R T I C LE I N FO
A B S T R A C T
Keywords: Perkinsus beihaiensis Mediterranean mussel Mytilus galloprovincialis Epidemiology
During analyses of the invasive Mediterranean mussel Mytilus galloprovincialis for pathologies in Tokyo Bay, infection by the protozoan parasite Perkinsus beihaiensis was found through histological examination, Ray’s Fluid Thioglycollate Medium assays, and molecular analyses. Specific PCR assays for each Perkinsus species also revealed the presence of an indigenous congeneric species, Perkinsus olseni, but P. beihaiensis was dominant in M. galloprovincialis. Sequences of the ribosomal internal transcribed spacer region I of P. beihaiensis found in Japan were genetically more similar to those found in South American countries (Panama and Brazil) than in Asian countries (China and India). Though Mediterranean mussels have become widespread in Japanese waters since their invasion in the 1930s, epidemiological surveys show that mussels collected outside Tokyo Bay are free of any Perkinsus infections. Based on these results, it was strongly suggested that P. beihaiensis invaded Tokyo Bay by transportation of bivalves originating from South America but has not yet spread to other parts of Japan. The possibility is not ruled out, however, that the parasite is indigenous in Japan but the environment in Tokyo Bay favors its transmission to Mediterranean mussels.
1. Introduction Various infectious diseases have emerged with the development of bivalve aquaculture and are now commonly accepted as serious obstacles for the further development of the industry. Currently, seven important infectious diseases affecting mollusks are listed as notifiable diseases by the World Organisation for Animal Health (OIE, 2018). In Japan, Akoya oyster disease, the abnormal enlargement of the ovary in the Pacific oyster, and perkinsosis in the Manila clam are serious infectious diseases seen during bivalve production (Morizane et al., 2001; Matsusato and Masumura, 1981; Shimokawa et al., 2010). Recently, infection by ostreid herpesvirus 1 (OsHV-1) and Francisella halioticida were added as novel health threats for juvenile Pacific oysters and the Yesso scallop, respectively (Nagai and Nakamori, 2018; Kawahara et al., 2018), and the gravity of these infectious diseases and the problems they may cause are gradually gaining recognition in Japan. It is known that introduction of exotic bivalves has often caused introduction and spread of pathogenic agents of bivalves (Reunault, 1996; Goedknegt et al., 2016). Haplosporidium nelsoni invaded North America by introduction of the Pacific oyster from Asian countries and
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caused detrimental impacts on the production of the eastern oyster Crassostrea virginica (Burreson and Ford, 2004). Also, Batista et al. (2015) suggested that introduction of the Pacific oyster from Asia resulted in the introduction of OsHV-1 to Europe. Thus field surveys for infectious agents in exotic bivalves are highly important for early biosecurity measures, such as zoning or compartmentalization. In 2016, we conducted a field survey targeting infectious agents of the Mediterranean mussel Mytilus galloprovincialis in Tokyo Bay. Since this alien bivalve is considered a nuisance species and is not a major aquaculture species in Japan, our survey was not concerned with health impacts on the mussel itself. Rather, this species is known to be infected with diseases such as marteiliosis and disseminated neoplasia (Berthe et al., 2004; Ciocan and Sunila, 2005). We were concerned that these diseases could be introduced and spread by the mussels, and have a negative influence on the production of edible bivalve species in Japan. During the survey, we discovered the presence of the protozoan parasite Perkinsus beihaiensis, which had never been reported in this mussel species, or in Japan. Genus Perkinsus is a protistan parasite group containing pathogens that are highly deleterious for mollusk production; for example, P. marinus in the eastern oyster, P. olseni in Manila clam, grooved clam and Australian abalones, and P. qugwadi in
Corresponding author. E-mail address: [email protected] (N. Itoh).
https://doi.org/10.1016/j.jip.2019.107226 Received 6 May 2019; Received in revised form 27 July 2019; Accepted 29 July 2019 Available online 29 July 2019 0022-2011/ © 2019 Elsevier Inc. All rights reserved.
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Fig 1. A map of sampling sites in this study. Sampling sites in Tokyo Bay are shown in the left-above inlet. Detailed information for each sampling site is provided in Table 1.
supplemented with antibiotics. Individuals heavier than 1 g were cut at median lines, and halves of the bodies were used for the assay. After incubation at 25 °C for 7 days in the dark, the tissues were collected by centrifugation and treated with 2 M NaOH at 60 °C until the tissues were lysed. Prezoosporangia were then collected by centrifugation, washed twice with distilled water, and counted in 96-well plates after staining with Lugol’s iodine. The mean intensity of infection was calculated as “log10 (number of prezoosporangia/wet tissue weight + 1)”. For the molecular identification of Perkinsus sp. in Mediterranean mussels, five individuals (mean shell height ± SD: 38.9 ± 2.7 mm) collected on the same day as above were used for the RFTM assay. After being incubated at 25 °C for 7 days, the tissues were placed in a Petri dish, stained with Lugol’s iodine, and observed under a dissecting microscope CK2 (Olympus, Tokyo, Japan). Tissues containing prezoosporangia were recovered from two individuals and stored in 70% ethanol until DNA extraction. DNA was extracted using a solution of 10% Chelex (Bio-Rad, Hercules, CA, USA) resin as described by de Faveri et al. (2009). PCR targeting the internal transcribed spacer (ITS) region for Perkinsus spp. was conducted using the genus-specific primers PerkITS-85 and PerkITS-750 developed by Casas et al. (2002). The final volume of the PCR reaction was 20 µL containing 1x Takara HS Ex Taq Buffer (Takara Bio Inc., Shiga, Japan), 0.2 mM dNTPs, 12 pmol of each PCR primer, 0.1 µL of Takara Ex Taq Hot Start version (Takara Bio Inc., Shiga, Japan) and
Yesso scallops (Villalba et al., 2004). In order to assess the potential for spillover of this parasite to other bivalve species in Japan, we examined the current geographic distribution. During the epidemiological surveys, we also conducted phylogenetic analyses to estimate the relationships with P. beihaiensis strains found outside Japan.
2. Materials and methods 2.1. Initial survey for Mediterranean mussels in Tokyo Bay In March 2016, 51 Mediterranean mussels (mean shell height ± SD: 32.6 ± 5.3 mm) were collected from the Koitogawa fishery port in Chiba, Japan (Site 1 in Fig. 1 and Table 1). After shucking the shell, the tissues were sliced to include the gills, mantles, digestive gland, and gonads and fixed in Davidson’s solution for a few days. Fixed tissues were processed using routine histological protocols for paraffin embedding, and tissue sections were cut at 5 µm thickness and stained with Mayer’s hematoxylin and eosin stain. For detection of Perkinsus infection, ten Mediterranean mussels (mean shell height ± SD: 40.3 ± 3.5 mm) were collected from the same sampling site as mentioned above in April 2016 and used for whole body burden assays (Choi et al., 1989; Almeida et al., 1999). After shucking the shell, the soft bodies were weighed and whole bodies were inoculated in Ray’s Fluid Thioglycolate Medium (RFTM) 2
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Table 1 Detailed information for sampling sites and dates, species, shell lengths of sampled bivalves, and prevalence and infection intensity of Perkinsus spp. estimated by RFTM assay. Location numbers correspond to numbers in the sampling map of Fig. 1. Mean infection intensity of Perkinsus spp. shown was calculated as log (number of prezoosporangium/wet tissue weight (g) + 1). Location
Sites
Dates
Species
n
Shell height (mean ± SD mm)
+ve by RFTM assay
Infection intensity (mean ± SD)
1 2 3 4 5 6 7 8 9 10 11
Koitogawa, Chiba Ichikawa, Chiba Kanazawa, Kanagawa Kurihama, Kanagawa Jogashima, Kanagawa Yoichi, Hokkaido Hakodate, Hokkaido Mustu Bay, Aomori Noheji, Aomori Obuchinuma, Aomori Shimanokoshi, Iwate
June 7, 2016 June 22, 2016 June 22, 2016 June 7, 2016 June 7, 2016 May 11, 2018 May 8, 2018 Oct 2017 Oct 4, 2017 Oct 2, 2017 Oct 5, 2017
12 13 14 15 16 17 18
Ogatsu Bay, Miyagi Katase-Enoshima, Kanagawa Shimizu, Shizuoka Bentenjima, Shizuoka Hikamasima, Aichi Oshima, Wakayama Shirahama, Wakayama
May 29, 2017 Nov 19, 2017 Nov 13. 2017 Sept 16, 2016 April 2017 Nov 1, 2017 Oct 5, 2017
19
Ushimado, Okayama
Oct 4, 2016
20 21 22
Nagasu, Kumamoto Obama, Fukui Noto, Ishikawa
May 31, 2016 Apr 2, 2018 Mar 15, 2018
M. galloprovincialis M. galloprovincialis M. galloprovincialis M. galloprovincialis M. galloprovincialis M. galloprovincialis M. galloprovincialis M. galloprovincialis M. galloprovincialis M. galloprovincialis Mytilus sp. Septifer virgatus M. galloprovincialis M. galloprovincialis M. galloprovincialis M. galloprovincialis M. galloprovincialis Mytilus sp. M. galloprovincialis Perna viridis M. galloprovincialis P. viridis M. galloprovincialis M. galloprovincialis M. galloprovincialis
30 30 30 30 30 60 30 15 30 15 27 15 28 29 29 30 30 13 24 30 22 10 10 30 30
56.5 28.1 59.4 65.6 40.4 54.9 47.9 95.1 38.9 49.6 49.2 22.0 61.5 26.0 37.7 32.8 71.9 27.9 27.4 65.0 36.0 28.2 34.9 46.6 65.4
30 13 20 23 23 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
4.91 0.92 1.15 1.28 0.65 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
6.1 4.2 7.7 8.1 4.6 3.8 4.9 12.5 5.3 12.6 13.8 5.6 2.4 6.3 4.7 3.6 7.1 13.3 12.8 6.0 18.9 8.0 3.4 5.0 7.0
± ± ± ± ±
0.97 0.29 0.91 0.99 0.36
Perkinsus spp. was examined using the whole body burden assay as described above. Additionally, Mediterranean mussels (n = 562) and related mussel species (Perna viridis (n = 40), Septifer virgatus (n = 15), and an unidentified Mytilus sp. (n = 40) were collected from 17 sampling sites in Japanese waters (Sites 6–22 in Fig. 1 and Table 1). They were also subjected to the RFTM assay as described above.
1.0 µL of the extracted template DNA. The thermal cycler program consisted of an initial denaturation at 95 °C for 4 min; 40 cycles of 95 °C for 1 min, 55 °C for 1 min, and 65 °C for 3 min; and a final extension at 65 °C for 5 min. The PCR products were visualized on a 1.5% agarose gel stained with SYBR Safe DNA gel stain (Thermo Fisher Scientific, Waltham, MA, USA), and the bands of approximately 700 bp were extracted using the FastGene Gel/PCR Extraction Kit (Nippon Genetics, Tokyo, Japan). The extracted PCR products were sent to an external sequencing facility (Eurofins Genomics, Tokyo, Japan) and sequenced using the original primers used for each PCR. The obtained sequences were compared by BLAST search against the NCBI GenBank database (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_ TYPE=BlastSearch&LINK_LOC=blasthome). Since the Perkinsus species found in our mussels were identified to be P. beihaiensis based on the BLAST searches, in situ hybridization was conducted for confirmation as described in Moss et al. (2008).
2.3. Species identification of Perkinsus in Tokyo Bay In order to identify the Perkinsus species found in Tokyo Bay, DNA sequences of prezoosporangia produced in RFTM were analyzed (Audemard et al., 2008). In brief, 10 mussels were collected from Jogashima and Koitogawa (Sites 1 and 5 in Fig. 1 and Table 1) in August 2017. Gill leaflets were excised and incubated in RFTM at 25 °C for 7 days. After staining with Lugol’s iodine, tissues were placed in a plastic dish and observed under the microscope. Tissues containing prezoosporangia were recovered, stored in 70% ethanol, and then underwent DNA extraction using the buffer system of QIAamp DNA Mini Kit (QIAGEN, Venlo, Netherlands) and EconoSpin IIA column (GeneDesign Inc., Osaka, Japan). Extracted DNA was applied to PCR assays using probes specifically developed for P. marinus (Audemard et al., 2004), P. olseni (Umeda and
2.2. Epidemiological surveys for Perkinsus infection in Japanese waters In June and July 2016, 30 Mediterranean mussels were sampled from five sites in Tokyo Bay: Koitogawa, Ichikawa, Kanesawa, Kurihama, and Jogashima (Sites 1–5 in Fig. 1 and Table 1). Tissues of mussels were incubated in RFTM, and the intensity of infection by Table 2 List of PCR primers specific to each Perkinsus species used in this study. Primer name
Sequences
Target species
References
PerkITS-85 PerkITS-750 PmarITS-70F PmarITS600R PerkOF PerkOR PchesITS-F PchesITS-R PerkHF PerkHR PerkITS-430R
5′-CCG CTT TGT TTG GAT CCC-3′ 5′-ACA TCA GGC CTT CTA ATG ATG-3′ 5′-CTT TTG YTW GAG WGT TGC GAG ATG-3′ 5′-CGA GTT TGC GAG TAC CTC KAG AG-3′ 5′-CTT AAC GGG CCG TGT TA-3′ 5′-CAT AAC GAA CTA TCT CCG AAG-3′ 5′-AAA CCA GCG GTC TCT TCT TCG G-3′ 5′-CGG AAT CAA CCA CAA CAC AGT CG-3′ 5′-CTG CCT GGC AGG TGA T-3′ 5′-CGA ATT GGC TCA ATA AAT TG-3′ 5′-TCT GAG GGG CTA CAA TCA T-3′
Perkinsus spp. (except for P. qugwadi) Perkinsus spp. (except for P. qugwadi) P. marinus P. marinus P. olseni P. olseni P. chesapeaki P. chesapeaki P. honshuensis P. honshuensis P. beihaiensis
Casas et al. (2002) Casas et al. (2002) Audemard et al. (2004) Audemard et al. (2004) Umeda and Yoshinaga (2012) Umeda and Yoshinaga (2012) Burreson et al. (2005) Burreson et al. (2005) Umeda (2012) Umeda (2012) Moss et al. (2008)
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individuals. Similar cells were also often observed in the mantle and gill lamellae (Fig. 2B). The RFTM assay detected Perkinsus infection in all of the ten Mediterranean mussels collected from the same site, and the mean intensity of infection was 3.25 ± 1.14 (range: 1.70–5.31). DNA samples extracted from the prezoosporangia of another five mussels were positive according to the Perkinsus genus-specific PCR, and the ITS sequences (648 bp) obtained from two individuals were 99% identical to those of P. beihaiensis collected in Panama (e.g. KU172665 and KU172673). Those newly obtained sequences in Japan were deposited in GenBank under the accession numbers LC437316 and LC437317. In situ hybridization using oligonucleotide probes developed for P. beihaiensis (Moss et al., 2006) showed positive cells (Fig. 2C) in the connective tissues of Mediterranean mussels, confirming that this mussel species in Tokyo Bay is infected with P. beihaiensis.
Yoshinaga, 2012), P. chesapeaki (Burreson et al., 2005), P. honshuensis (Umeda and Yoshinaga, 2012), or P. beihaiensis (Moss et al., 2008) (Table 2). The final volume of the PCR reactions was 20 µL containing 10 µL of KOD One PCR Master Mix -Blue- (Toyobo, Osaka, Japan), 30 pmol of each PCR primer, and 1.0 µL of the extracted DNA samples. The thermal cycler program for P. olseni, P. beihaiensis, and P. honshuensis consisted of 40 cycles of 98 °C for 10 sec, 50 °C for 5 sec, and 68 °C for 1 sec, while annealing temperatures of 53 °C and 56 °C were applied for P. marinus and P. chesapeaki, respectively. Before the assays, the specificity of the PCR protocols above was confirmed using positive control DNA of each Perkinsus species. PCR products with molecular weights similar to the positive controls were extracted and sequenced with the primers used for each PCR, as described above. 2.4. Haplotype analysis of Perkinsus beihaiensis based on the ribosomal ITS1 region
3.2. Epidemiological surveys of Perkinsus in Mediterranean mussels in Japanese waters
For the haplotype network analysis, ITS1 sequences of P. beihaiensis obtained from Japanese samples as above (n = 19) and 80 ITS1 sequences of P. beihaiensis with geographic information deposited in GenBank (supplementary data) were used. Using MEGA 7.0 software, 99 sequence data were aligned by CLUSTAL W (Thompson et al., 1994) at default settings. Two sequences (GenBank Accession No. MF595808 and MF595809) appeared to be genetically unrelated to the other sequences and were removed from further analyses. The haplotype network among 97 ITS1 sequences of P. beihaiensis was constructed with the TCS network method (Clement et al., 2000) using PopART (Leigh and Bryant, 2015). In this analysis, nucleotide sites containing ambiguous nucleotides were masked.
Perkinsus infection was detected in Mediterranean mussels collected from five sites in Tokyo Bay (Sites 1–5 in Fig. 1 and Table 1). The prevalence and mean intensity of infection were highest in Koitogawa (100% and 4.91, respectively) and lowest in Ichikawa (43.3% and 0.92, respectively) (Table 2). Meanwhile, Perkinsus infection was not detected in Mediterranean mussels nor in any related species collected from the other 17 sites outside Tokyo Bay (Table 2). 3.3. Identification of Perkinsus species in Mediterranean mussels
3. Results
The Mediterranean mussels collected from Jogashima (n = 10) and Koitogawa (n = 10) in August 2017 were all positive according to the RFTM assay. Following DNA extraction from tissues incubated in RFTM, PCR assays indicated infections by P. beihaiensis and P. olseni. These results were validated by sequencing the PCR products. In both sampling sites, P. beihaiensis was detected more often than P. olseni; 10 mussels were positive for P. beihaiensis and 5 were positive for P. olseni in Koitogawa, and 9 mussels were positive for P. beihaiensis and 1 was positive for P. olseni in Jogashima (Table 3). Nineteen sequences identified as P. beihaiensis were deposited in GenBank (LC437318 –
3.1. Initial detection of Perkinsus beihaiensis in Mediterranean mussels in Tokyo Bay Histological observations revealed that 24 of the 51 examined mussels had foci of hemocyte infiltration in the connective tissues around the digestive gland. Perkinsus-like cells with a large vacuole and eccentric nucleus were found in the foci of three individuals (Fig. 2A), although specific pathogen-like cells were not recognized in the other
Fig 2. Histological observations of Perkinsus cells in the Mediterranean mussel, Mytilus galloprovincialis. A. Perkinsus cells with vacuoles (arrows) are surrounded by hemocyte infiltration in the connective tissues around the digestive diverticula. Hematoxylin-Eosin Stain. B. A trophozoite of Perkinsus sp. (arrow) in the gill. Hematoxylin-Eosin Stain. C. Trophozoite cells of Perkinsus sp. were detected by in situ hybridization using specific oligonucleotide probes for P. beihaiensis.
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Table 3 Results of PCR assays specific for each Perkinsus species against Mediterranean mussels, Mytilus galloprovincialis, collected in Tokyo Bay, Japan.
Koitogawa Jogashima
P. beihaiensis
P. olseni
P. marinus
P. chesapeaki
P. mediterraneus
P. honshuensis
10/10 9/10
5/10 1/10
0/10 0/10
0/10 0/10
0/10 0/10
0/10 0/10
Fig 3. TCS network of 30 ITS1 haplotypes of Perkinsus beihaiensis estimated by PopART. Each line between haplotypes indicates a single nucleotide substitution. The size of each circle is proportional to the absolute haplotype frequency, Small black circles represent missing haplotypes and color shows localities where each haplotype was observed.
25
Brazil Panama
2 1
China Japan India Missing haplotype
LC437327 for sequences from Jogashima and LC437328 – LC437336 for samples from Koitogawa). Other Perkinsus species such as P. marinus, P. chesapeaki, and P. honshuensis were not detected in the Mediterranean mussels in this study (Table 3).
So far, two species of Perkinsus, P. olseni and P. honshuensis, have been reported in Japanese waters (Hamaguchi et al., 1998; Dungan and Reece, 2006) and, thus, Perkinsus cells found in Mediterranean mussels in this study were initially expected to be one or both of these. However, DNA sequence analyses and subsequent in situ hybridization confirmed that the Perkinsus cells infecting the Mediterranean mussels were P. beihaiensis. This Perkinsus species was first described in China (Moss et al., 2008) and subsequently was reported in Brazil (Sabry et al., 2009), India (Sanil et al., 2012) and Panama (Pagenkopp Lohan et al., 2016). This is the first report from Japan. In addition, this species has so far been reported in 14 bivalve species (Pagenkopp Lohan et al., 2018); we showed that the Mediterranean mussel is also a host species for this parasite. PCR assays specific to each Perkinsus species revealed that Mediterranean mussels in Tokyo Bay were also infected with P. olseni, but the prevalence of P. beihaiensis was much higher than that of P. olseni, suggesting that P. beihaiensis is the dominant species in Mediterranean mussels in Tokyo Bay. This suggestion is also supported by our preliminary qPCR assays for P. olseni and P. beihaiensis in which the intensity of P. beihaiensis was much higher than P. olseni in Mediterranean mussels sampled in Tokyo Bay (Y. Komatsu, unpublished data). This mussel species seems to be highly susceptible to P. beihaiensis. In spite of susceptibility of Mediterranean mussels to parasitism by P. beihaiensis, Perkinsus infection has never been found in Mediterranean mussels outside Tokyo Bay in either previous (Hamaguchi et al., 2002) or current surveys. One of the possibilities to explain the absence of P. baihaiensis in the susceptible bivalves outside Tokyo Bay is that P. beihaiensis invaded Tokyo Bay from another marine area but has not yet spread to other parts of Japan. Mediterranean mussels invaded Japan around the 1930s, rapidly expanded their geographic distribution, and now can be found in most areas of Japan (Ishida et al., 2005). Therefore, if P. beihaiensis is an exotic species in Japan, this parasite invaded Tokyo Bay after the
3.4. Haplotype analysis of Perkinsus beihaiensis based on the ribosomal ITS1 region Based on the ITS1 region, 30 haplotypes were recognized in the P. beihaiensis used for this analysis. Two haplotypes from Brazil were relatively dissimilar genetically to the two major haplogroups. The other 28 haplotypes were divided into the two major haplogroups, which were separated by at least three nucleotide differences (Fig. 3). One of the haplogroups was composed of haplotypes found in Asian countries (China and India), while the other comprised haplotypes identified in the Americas (Brazil and Panama). Only two haplotypes were found in Japan, and both haplotypes were included in the haplogroups of Brazil and Panama (Fig. 3). 4. Discussion The initial surveys conducted in Tokyo Bay revealed the presence of Perkinsus beihaiensis in Mediterranean mussels, Mytilus galloprovincialis, for the first time in Japan. Subsequent epidemiological surveys in Japanese waters indicated that Mediterranean mussels in Tokyo Bay were frequently infected with this parasite; however, no Perkinsus infections were detected in individuals outside Tokyo Bay. PCR analyses specific to each Perkinsus species revealed the presence of both P. olseni and P. beihaiensis in Mediterranean mussels in Tokyo Bay, but P. beihaiensis appeared to be dominant in the mussels. Haplotype network analyses based on the ribosomal ITS1 region showed that the P. beihaiensis in Tokyo Bay is genetically more similar to P. beihaiensis found in South American countries than to that from in Asian countries. 5
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dispersion of Mediterranean mussels in Japanese waters, as otherwise it would have spread widely in Japanese waters along with the Mediterranean mussels. Following the hypothesis above, it is likely that P. beihaiensis in Japan originated from South America. We base this hypothesis on the finding that haplotypes found in Japan are included in the haplotypes from Panama and Brazil. Since only a few haplotypes have been detected in Japan through this study, introductions of P. beihaiensis from South America to Japan may have occurred only a few times. The present study did not provide any information regarding the invasion route of P. beihaiensis into Tokyo Bay. To our knowledge, Tokyo Bay does not have any aquaculture industry introducing bivalve seed from South American countries, and it is not likely that P. beihaiensis was introduced along with the introduction of bivalve seeds for aquaculture purposes. On the other hand, six international ports are located in Tokyo Bay, and maritime transportation in those ports often brings alien bivalve species into Tokyo Bay (Hiwatari et al., 2006). P. beihaiensis may have been introduced into Tokyo Bay with bivalves in ballast water or by attaching to a vessel surface, as suggested by Pagenkopp Lohan et al. (2018). We have not ruled out that P. baihaiensis is indigenous in Japan and that infection in Mediterranean mussels of Tokyo Bay is highly possibly due to characteristic environmental conditions of the bay. So far, surveys for infectious agents have been limited to commercially important mollusks species in Japan, and further surveys for non-commercial species outside Tokyo Bay may be required to determine if P. beihaiensis is indigenous or introduced. In the present study, we reported the presence of P. beihaiensis in Mediterranean mussel in Japan for the first time. The possibility was strongly suggested that P. beihaiensis invaded Tokyo Bay but has not yet spread to other parts of Japan. Host specificity of this parasite is relatively low, therefore, if the hypothesis above is correct, P. beihaiensis may be easily transmitted with the movement of various susceptible bivalve species and invade new areas and cause diseases problems in economically important bivalve species after expansion because parasitic organisms often show strong pathogenicity to new host species (host switching) after invading new locations. Additional field surveys for this parasite are urgently needed to identify geographic distribution. Also, in order to assess the potential risk of this parasite for Japanese fishery, the establishment of experimental infections for edible bivalve species is also needed.
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Acknowledgments Bivalves used in this study were kindly provided by Professors Kenji Ohkoshi (Toho University), Shotaro Izumi (Tokai University), Sho Shirakashi (Kindai University), Hiroaki Suetake (Fukui Prefectural University), Hiroyasu Kamei (Kanazawa University), Drs. Makoto Kanamori and Yohei Shimizu (Hokkaido Research Organization), Mrs. Fumiyasu Murayama (Okayama Prefectural Technology Center for Agriculture, Forestry, and Fisheries Research Institute), and Shozo Ogiso (Kanazawa University). This study was funded by JSPS KAKENHI (Grant Number JP16H04967 to Tomoyoshi Yoshinaga and Grant Number JP17H03858 to Naoki Itoh). Natalie Kim, PhD, from Edanz Group (www.edanzediting.com/ac) edited a draft of this manuscript. Appendix A. Supplementary material Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jip.2019.107226. References Almeida, M., Berthe, F., Thebault, A., Dinis, M.T., 1999. Whole clam culture as a quantitative diagnostic procedure of Perkinsus atlanticus (Apicomplexa, Perkinsea) in clams Ruditapes decussatus. Aquaculture 177, 325–332.
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