Identification of glycine betaine as a host-derived molecule required for the vegetative proliferation of the protozoan parasite Perkinsus olseni.

Perkinsus olseni is an industrially significant protozoan parasite of Manila clam, Ruditapes philippinarum. So far, various media, based on Dulbecco's Modified Eagle Medium and Ham's F-12 nutrient mixture with supplementation of fetal bovine serum (FBS), have been developed to proliferate the parasitizing trophozoite stage of P. olseni. The present study showed that P. olseni did not proliferate in FBS-deficient Perkinsus broth medium (PBMΔF), but proliferated well in PBMΔF supplemented with tissue extract of host Manila clams, indicating that FBS and Manila clam tissue contained molecule(s) required for P. olseni proliferation. Preliminary characterization suggested that the host-derived molecule(s) was a heat-stable molecule(s) with a molecular weight of less than 3 kDa, and finally a single molecule required for the proliferation was purified by high-performance liquid chromatography processes. High-resolution electrospray ionization mass spectrometry and nuclear magnetic resonance analyses identified this molecule as glycine betaine (=trimethylglycine), and the requirement of this molecule for P. olsseni proliferation was confirmed by an assay using chemically synthesized, standard glycine betaine. Although glycine betaine was required for the proliferation of all examined Perkinsus species, supplementation of glycine betaine precursors, such as choline and betaine aldehyde, enhanced the proliferation of 4 Perkinsus species (P. marinus, P. chesapeaki, P. mediterraneus and P. honshuensis), but not of 2 others (P. olseni and P. beihaiensis). Thus, it was concluded that the ability to biosynthesise glycine betaine from its precursors varied among Perkinsus species, and that P. olseni and P. beihaiensis lack the ability required to biosynthesize glycine betaine for proliferation.

Effect of Biokos, a natural lipopeptide surfactant extracted from a bacterium of the Pseudomonas genus, on infection of Cryptocaryon irritans.

Cryptocaryoniasis (marine white spot disease), caused by Cryptocaryon irritans, is a major threat to marine fish cultures in tropical and subtropical waters, and a serious nuisance to hobbyists with saltwater fish tanks. With only classical treatment schedules such as copper salts or hyposaline baths being available, control of the disease remains a challenge. In this study, we investigated the effect of Biokos, a viscosin-like lipopeptide surfactant extracted from a bacterium of the Pseudomonas genus, on the external life stages of C. irritans, including theronts, protomonts and tomonts. The present study demonstrated that the compound has an antiparasitic effect on all tested external stages of the parasite. In particular, when Biokos was used at 48 mg/L, it was able to kill almost all theronts and protomonts within 1 h in in vitro experiments, and using the same concentration in an in vivo challenge experiment, the parasitic load was reduced by more than 95% compared to the control group with no Biokos. Additionally, cultured fish cells were able to proliferate, and fish showed no adverse signs at Biokos concentrations that were effective in killing the parasite. Thus, Biokos may be a promising way for preventing or reducing the burden of this parasitic disease in the future.

Supplementation with lipids enhances zoosporulation of Perkinsus species.

The protozoan parasite Perkinsus olseni is a major pathogen of marine mollusks. A large number of zoospores (the most infective stage) is required to experimentally examine the transmission and invasion processes of this parasite, but the conventional zoosporulation method in seawater is inefficient. A recent study found that P. olseni zoosporulation was highly enhanced in a nutrient-rich medium which was developed for trophozoite proliferation in Perkinsus. Here, we examined the effect of each medium component on P. olseni zoosporulation and found that lipids enhanced zoosporulation. Moreover, zoospores failed to develop into trophozoites in seawater supplemented with lipids, indicating that this zoosporulation method using seawater supplemented with lipids is ideal for preparing a large number of P. olseni zoospores for various biological assays. Lipid supplementation also significantly enhanced zoosporulation in P. honshuensis, P. chesapeaki, and P. marinus, although the enhancing effect of the lipid supplementation varied by species. Considering the limited availability of lipids in the sea water column, our findings suggest that Perkinsus zoosporulation likely occurs mainly in the vicinity of hosts (not in the sea water column), such as in the mantle cavity or on the body surface of mollusk hosts.

Establishment and characteristics of Perkinsus beihaiensis in vitro isolates from Mediterranean mussels Mytilus galloprovincialis.

Two monoclonal in vitro isolates of Perkinsus beihaiensis were established from tissues of infected Mediterranean mussels Mytilus galloprovincialis, using a lipid concentrate and yeast extract medium (LpcYM) to induce enlarged prezoospoangia from histozoic trophozoites, and Perkinsus broth medium (PBM) to support subsequent zoosporulation and schizogonic proliferation from those cells. Zoospores of the isolates developed into uninucleate signet-ring trophozoites followed by schizonts typical of Perkinsus spp., but an additional type of unknown botryoidal cell clusters appeared and increased in density. These botryoidal cell clusters had second-generation daughter schizonts within which further internal cells were produced. Although botryoidal cell clusters in primary cultures rarely proliferated in PBM, cells contained by botryoidal clusters enlarged as prezoosporangia in LpcYM, and a large number of proliferative stages (i.e. trophozoites and schizonts typical for Perkinsus spp.) were obtained from these prezoosporangia in PBM. At lower cell density, trophozoites transformed into the botryoidal cell clusters in PBM, and the number of cells increased little, while trophozoites rapidly increased by typical schizogony at higher density in PBM. Based on these observations, we determined an optimal cell density for continuous culture of P. beihaiensis isolates, confirmed their ability to proliferate and survive after cryopreservation, and describe an experimental protocol to culture this most recently described species in the genus Perkinsus.

Morphological and molecular characterization of Ceratomyxa binhthuanensis n. sp. (Myxosporea: Ceratomyxidae) from the gall bladder of blacktip grouper Epinephelus fasciatus (Perciformes: Serranidae) in the East Sea of Vietnam.

A new myxozoan species, Ceratomyxa binhthuanensis n. sp. (Myxosporea: Ceratomyxidae), was found in the gall bladder of blacktip grouper Epinephelus fasciatus (Perciformes: Serranidae) in the East Sea of Vietnam. Myxospores were observed floating free in the gall bladder of 3 out of 20 fish examined (15%). Mature myxospores were elongate and slightly crescent-shaped and measured 12.2 ± 1.3 (10.8-16.0) µm in thickness and 5.8 ± 0.6 (4.8-6.9) µm in length, with two smooth equal shell valves. The two polar capsules were spherical and equal in size, measuring 2.6 ± 0.3 (2.3-2.9) µm in diameter. The posterior angle was slightly concave, 153.7° ± 5.6° (148.9°-166.0°). Molecular analysis of SSU rDNA sequence showed that Ceratomyxa binhthuanensis n. sp. differs from other Ceratomyxa spp. available in GenBank. Phylogenetic analysis indicated that C. binhthuanensis n. sp. was closely related to three species, Ceratomyxa nolani, Ceratomyxa yokoyamai, and Ceratomyxa cutmorei, which also infect fish hosts of the genus Epinephelus.

Francisellosis of Yesso scallops Mizuhopecten yessoensis in Japan is caused by a novel type of Francisella halioticida.

Francisella halioticida, the causative agent of francisellosis of the giant abalone Haliotis gigantea, has also been isolated from Yesso scallops Mizuhopecten yessoensis, which presented with orange/pinkish lesions in the adductor muscle and experienced high mortality. However, it is not clear whether the F. halioticida isolated from the giant abalone and Yesso scallops are phenotypically and genetically identical to each other. The present study revealed that isolates from the giant abalone and Yesso scallops were phenotypically different, with slower growth in modified eugon broth and a lack of prolyl aminopeptidase and phenylalanine aminopeptidase in Yesso scallop isolates. Additionally, we found that 3 of 8 housekeeping genes were different between them. Based on these phenotypic and genetic differences, we propose that F. halioticida isolated from Yesso scallops in Japan be designated as the 'J-scallop type' to distinguish it from strains from abalone ('abalone type'). Whole-genome sequencing analysis of a strain belonging to the J-scallop type showed that the overall similarity between the J-scallop and abalone type strains was estimated to be 99.84%. In accordance with a lack of prolyl aminopeptidase activity, in general, all of the J-scallop type strains examined have a 1 bp deletion in the responsible gene encoding prolyl aminopeptidase. This deletion was confirmed in all F. halioticida in diseased Yesso scallops examined, suggesting that in Japan, francisellosis of Yesso scallops is caused by a novel type of F. halioticida and not by the abalone type.

A new myxosporean species, Henneguya lata n. sp. (Myxozoa: Myxobolidae), from the gills of yellowfin seabream Acanthopagrus latus (Perciformes: Sparidae) in the Gulf of Tonkin, Vietnam.

During a survey of myxosporean parasites of marine fish in the coastal region of Vietnam, a species of the genus Henneguya (Myxosporea: Bivalvulida) was found in the gill of yellowfin seabream Acanthopagrus latus (Perciformes: Sparidae). White and oval cysts, measuring 145-220 µm in diameter, were detected in the gill lamellae of 4 of 15 fish examined (26.7%). Mature myxospores were elongate, with smooth valves, two similar polar capsules, and having the following dimensions: spore body length 9.9 ± 0.5 (8.9-12.5) µm, body width 6.7 ± 0.3 (6.1-7.6) µm, thickness 5.1 ± 0.2 (4.8-5.4) µm, caudal appendage length 10.0 ± 1 (8.3-11.6) µm, and total myxospore length 19.3 ± 1.4 (16.5-21.5) µm. The polar capsules were ovoid, measuring 3.2 ± 0.2 (2.8-3.9) µm long and 1.9 ± 0.2 (1.5-2.3) µm wide. Each polar capsule has a polar filament with 4-5 coils. Histological analysis revealed plasmodia in the connective tissues of the gill lamellae, but inflammation and deformation of the gills were not observed. In the phylogenetic tree reconstructed from the small subunit ribosomal DNA (SSU rDNA), sequences of the Henneguya specimens found in this study form a distinct branch. Morphological characteristics and molecular data identified a new species, namely Henneguya lata n. sp.

The effects of environmental and nutritional conditions on the development of Perkinsus olseni prezoosporangia.

Perkinsus olseni is a widely distributed protozoan pathogen that infects a wide range of marine mollusks. Prezoosporulation of P. olseni trophozoites is easily observed in Ray's fluid thioglycollate medium, but in nature, trophozoites within host tissue should be able to develop into prezoosporangia without any additional artificial medium after the host dies. How this process might work in field conditions remains poorly understood, however, partly because of the lack of appropriate in vitro assays. In this study, we observed that trophozoites of P. olseni successfully developed into prezoosporangia when mixed with minced tissue of the Manila clam Ruditapes philippinarum and placed in seawater. We were thus able to establish a new method to examine the development of P. olseni to prezoosporangia under artificially simulated natural environmental conditions. Using this method, we found that low temperatures (5 °C, 15 °C) significantly suppressed prezoosporangia development. In addition, we found that prezoosporangia were developed in a wide range of salinities (10-50 practical salinity unit) and that P. olseni requires some nutrition factors from host tissue for prezoosporulation to occur. Because the transmission of P. olseni among a host population highly depends on the developmental process of prezoosporangia, which leads to production of the infective zoospore stage, these results will help further our understanding of the parasite's infection dynamics in nature.

A novel dimorphic microsporidian Ameson iseebi sp. nov. infecting muscle of the Japanese spiny lobster, Panulirus japonicus, in western Japan.

Japanese spiny lobsters (Panulirus japonicus) exhibiting white opaque abdominal muscle were found in Mie and Wakayama prefectures, in mid-Western Japan. Microscopically, two types of microsporidian spores, ovoid and rod-shaped, were observed infecting the muscle. Histologically, both types of spore were detected inside myofibers of the abdomen, appendages, and cardiac muscles and were often both observed in a single myofiber simultaneously. Transmission electron microscopy revealed that ovoid spores have villous projections on the surface, and that ovoid and rod-shaped spores have a polar filament with 12 coils and 6 to 8 coils respectively. Merogonic and sporogonic stages were observed around ovoid spores, but rarely around rod-shaped spores. The small subunit ribosomal DNA sequences obtained from both spore types were identical to each other, indicating that this microsporidian exhibits a clear spore dimorphism. Phylogenetic analysis based on the rDNA sequences indicates that this microsporidian is part of a clade consisting of the genera Ameson and Nadelspora, with the most closely related species being A. herrnkindi found in the Caribbean spiny lobster P. argus. Based on ultrastructural features, molecular phylogenetic data, host type and geographical differences among known species in these genera, the species found in whitened abdominal muscles of the Japanese spiny lobster is described as Ameson iseebi sp. nov.

First discovery of Perkinsus beihaiensis in Mediterranean mussels (Mytilus galloprovincialis) in Tokyo Bay, Japan.

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.

Parallel studies confirm Francisella halioticida causes mortality in Yesso scallops Patinopecten yessoensis.

Francisella halioticida is a marine bacterium originally described as the causative agent of mass mortality among giant abalone Haliotis gigantea. Recent field studies in Canada and Japan have suggested that this bacterium is also the cause of adductor muscle lesions and high mortality of Yesso scallops Patinopecten yessoensis, although a causal relationship has not been established. In the present study, the pathogenicity of F. halioticida in Yesso scallops was assessed in both Canada and Japan using bacteria isolated from diseased Yesso scallops in each respective country. Independent laboratory experiments revealed that scallops challenged with F. halioticida via bath exposure resulted in high mortality and histological lesions characterized by massive haemocyte infiltration. The presence of F. halioticida was confirmed using PCR, and F. halioticida was re-isolated from a portion of dead and surviving specimens. These results fulfill Koch's classic criteria for establishing disease causation and provide conclusive evidence that F. halioticida causes adductor muscle lesions and high mortality in Yesso scallops.

New insights into the entrance of Perkinsus olseni in the Manila clam, Ruditapes philippinarum.

In order to understand interactions between Perkinsus olseni and its host mollusk species Manila clam Ruditapes philippinarum, this study focused on invasion processes of the parasite, particularly the mechanisms of zoospore transformation to trophozoites in its portal entry into the host. We exposed Manila clam to P. olseni zoospores, then periodically quantified parasite intensity in various host organs and tissues. We detected large numbers of parasite cells within gills and labial palps of the host clam from the early to the final stages, moderately within mantle and digestive organs but low numbers within hemolymph, foot and adductor muscles. Our results suggest that P. olseni first invades the gills and labial palps of the host clam with limited translocation throughout the host body via the host's circulatory system until 12 days post exposure to zoospores. P. olseni zoospores exposed to extracts of gills and labial palps transformed into trophozoites more efficiently than they did when exposed to other tissues; this transformation was not observed when zoospores were exposed to heated organ extracts. Our results suggest the involvement of a host molecule in the transformation of P. olseni zoospores, leading to initial infection primarily within gills and labial palps of the host clam.

Impact of Perkinsus olseni infection on a wild population of Manila clam Ruditapes philippinarum in Ariake Bay, Japan.

Many studies have addressed the production decline of Manila clam, Ruditapes philippinarum, in Japan, but infection of clams with Perkinsus olseni has received scarce attention. To evaluate the impact of P. olseni, infection levels and host density of a wild, unexploited clam population were monitored monthly or bimonthly on a tidal flat from June 2009 to January 2013. Real-time PCR analysis discriminating P. olseni and Perkinsus honshuensis detected only P. olseni in tested clams. The prevalence of P. olseni was 100% or nearly 100% in 7 cohorts throughout the study period, except in newly recruited clams. Infection intensity remained low and seldom reached 106 cells/g wet tissue in newly recruited clams until the month of September. Infection intensity reached 106 cells/g in autumn and remained high at 104-106 cells/g until each cohort of clams disappeared. Clam density began to decrease in the autumn when the infection intensities reached ca. 106 cells/g. Density was relatively stable in winter, increased in spring and decreased again in clams aged 1 year or older during summer and autumn in the following years. Survival of clams experimentally infected with P. olseni at ca. 106 cells/g and placed in a cage in the tidal flat for 1 or 2 months was significantly lower than survival of uninfected control clams. Our results suggested that heavy infection with P. olseni was a major cause of the clam density decrease, although other environmental and biological factors also may have contributed to the decline in density. In addition, uninfected clams were deployed in cages for 1-2 months from June 2010 to January 2013 and prevalence and infection intensity were monitored. Parasite transmission and infection progression increased in summer and autumn.

High genetic diversities between isolates of the fish parasite Cryptocaryon irritans (Ciliophora) suggest multiple cryptic species.

The ciliate protozoan Cryptocaryon irritans parasitizes marine fish and causes lethal white spot disease. Sporadic infections as well as large-scale outbreaks have been reported globally and the parasite's broad host range poses particular threat to the aquaculture and ornamental fish markets. In order to better understand C. irritans' population structure, we sequenced and compared mitochondrial cox-1, SSU rRNA, and ITS-1 sequences from 8 new isolates of C. irritans collected in China, Japan, and Taiwan. We detected two SSU rRNA haplotypes, which differ at three positions, separating the isolates into two main groups (I and II). Cox-1 sequences also support the division into two groups, and the cox-1 divergence between these two groups is unexpectedly high (9.28% for 1582 nucleotide positions). The divergence is much greater than that detected in Ichthyophthirius multifiliis, the ciliate protozoan causing freshwater white spot disease in fish, where intraspecies divergence on cox-1 sequence is only 1.95%. ITS-1 sequences derived from these eight isolates and from all other C. irritans isolates (deposited in the GenBank) not only support the two groups, but further suggest the presence of a third group with even greater sequence divergence. Finally, a small Ka/Ks ratio estimated from cox-1 sequences suggests that this gene in C. irritans remains under strong purifying selection. Taken together, the C. irritans species may consists of many subspecies and/or syngens. Further work is needed to determine if there is reproductive isolation between the groups we have defined.

Factors affecting sporoplasm release in Kudoa septempunctata.

The myxosporean parasite Kudoa septempunctata has been isolated from cultured olive flounder (Paralichthys olivaceus) and was recently identified as a cause of food poisoning in humans. Since the sporoplasm plays an important role in causing diarrhea by invading intestinal cells, the specific factors affecting the release of sporoplasm from spores should be determined. Thus, we investigated the effect of digestive and serum enzymes, fetal bovine serum (FBS), temperature, and the role of glucose in cell culture media on the release of sporoplasm. Sporoplasm release was observed in the groups treated with FBS and media containing glucose. In addition, 1,10-phenanthroline inhibited the release of sporoplasm in the FBS medium. These results indicate that K. septempunctata uses glucose for releasing its sporoplasm and that zinc or metalloprotease is related to the release mechanism. The present study provides important information for the development of agents to prevent sporoplasm release and the consequent food poisoning caused by K. septempunctata.

Effect of light exposure on circadian rhythm in theront excystment in Cryptocaryon irritans.

We examined the effects of light exposure on the theront excystment circadian rhythm in Cryptocaryon irritans using a newly invented apparatus, which enabled us to examine the excystment rhythms of theronts from tomonts with low labor. Using the apparatus, we examined the timings of theront excystment from tomonts exposed directly to light and from tomonts exposed to light-exposed seawater by counting the number of excysted theronts. We found that the theront excystment time changed only when tomonts were directly exposed to light, indicating that light reception is essential for circadian rhythm entrainment. When tomonts were exposed to light only once for 12 h, either on Day 1, Day 2, or Day 3 after leaving host and being encysted, the circadian rhythm was entrained according to the photoperiod given on tomonts. Tomonts exposed to a low light (1 lx) with 12L:12D photoperiod daily showed a circadian rhythm similar to that in tomonts exposed to an intense light (500 lx) under the same photoperiod. When tomonts were incubated at 22 °C, 25 °C, or 28 °C under the same photoperiod, almost the same circadian rhythm was developed, suggesting temperature has little effect on the circadian rhythm entrainment between the range, even though the date of excystment was delayed in lower temperatures. These results suggest the circadian rhythm of theront excystment can be entrained in tomonts on the seabed of inner bays where net-cage aquaculture is conducted, and be involved in the outbreaks of cryptocaryoniasis there.

Target Protein Expression on Tetrahymena thermophila Cell Surface Using the Signal Peptide and GPI Anchor Sequences of the Immobilization Antigen of Cryptocaryon irritans.

Cryptocaryoniasis, caused by Cryptocaryon irritans, is a significant threat to marine fish cultures in tropical and subtropical waters. However, controlling this disease remains a challenge. Fish infected with C. irritans acquires immunity; however, C. irritans is difficult to culture in large quantities, obstructing vaccine development using parasite cells. In this study, we established a method for expressing an arbitrary protein on the surface of Tetrahymena thermophila, a culturable ciliate, to develop a mimetic C. irritans. Fusing the signal peptide (SP) and glycosylphosphatidylinositol (GPI) anchor sequences of the immobilization antigen, a surface protein of C. irritans, to the fluorescent protein, monomeric Azami-green 1 (mAG1) of the stony coral Galaxea fascicularis, allowed protein expression on the surface and cilia of transgenic Tetrahymena cells. This technique may help develop transgenic Tetrahymena displaying parasite antigens on their cell surface, potentially contributing to the development of vaccines using "mimetic parasites".

Effects of temperature on eggs and larvae of Anisakis simplex sensu stricto and Anisakis pegreffii (Nematoda: Anisakidae) and its possible role on their geographic distributions.

Effects of temperature on development of eggs, recently hatched larvae and L3 larvae of the marine parasitic nematodes Anisakis simplex sensu stricto (s.s.) and A. pegreffii were examined in vitro. The eggs of A. simplex s.s. hatched at 3-25 °C and those of A. pegreffii hatched at 3-27 °C. Days before hatching varied between 2 days at 25 °C and 35-36 days at 3 °C in A. simplex s.s. and between 2 and 3 days at 27 °C and 65 days at 3 °C in A. pegreffii. Hatching rates of A. simplex s.s. were maintained high at temperatures between 3 and 25 °C but decreased to 0% at 27 °C. In contrast, those of A. pegreffii were lowest particularly at 3 °C, but also at 27 °C. The mean 50% survivals of hatched larvae ranged from 5.3 days at 25 °C to 82.3 days at 9 °C in A. simplex s.s., while in A. pegreffii it ranged from 1.2 days at 27 °C to 77.2 days at 9 °C. L3 larvae of A. pegreffii exhibited higher survival rates and activity than those of A. simplex s.s., particularly at 20 and 25 °C. These results suggest that the early stages of A. simplex s.s. are more adapted to lower temperatures whereas those of A. pegreffii are more tolerant to warm environments, which may correspond to their distribution patterns in Japan and Europe.

Effects of temperature on Anisakis simplex sensu stricto L3 larvae experimentally challenged in rainbow trout (Oncorhynchus mykiss) and Mozambique tilapia (Oreochromis mossambicus).

Human anisakiasis is a foodborne disease that has been increasingly reported worldwide and is caused by the consumption of raw or undercooked seafood infected with zoonotic nematodes of the genus Anisakis Dujardin, 1845. One of the most frequently reported species, both in fish paratenic hosts as well as in human patients, is Anisakis simplex sensu stricto (s.s.), which is distributed within some of the globe's main fishing grounds in the North Pacific and Atlantic oceans. In order to clarify the influence of temperature on this parasite in vivo, third-stage larvae of A. simplex s.s. were surgically challenged in the body cavities of rainbow trouts (Oncorhynchus mykiss) and Mozambique tilapias (Oreochromis mossambicus). Larval survival and tissue migration were then analyzed after 6 and 12 weeks. The results showed that survival rates of larvae were lower at 27 and 33 °C than at 3, 9, 15 and 21 °C. Also, migration to the body muscle was observed to be highest at 9 °C. These results suggest that third-stage larvae of A. simplex s.s. are more adapted to lower temperatures in experimentally challenged fish, which may justify its distribution in cold northern waters.

Experimental challenges of wild Manila clams with Perkinsus species isolated from naturally infected wild Manila clams.

Manila clams, Ruditapes philippinarum, are widely harvested in the coastal waters in Japan. However, there have been significant decreases in the populations of Manila clams since the 1980s. It is thought that infection with the protozoan Perkinsus species has contributed to these decreases. A previous study demonstrated that high infection levels of a pure strain of Perkinsus olseni (ATCC PRA-181) were lethal to hatchery-raised small Manila clams, however, the pathogenicity of wild strain Perkinsus species to wild Manila clam is unclear. To address this, we challenged large (30-40 mm in shell length) and small (3-15 mm in shell length) wild Manila clams with Perkinsus species isolated from naturally infected wild Manila clams. We report high mortalities among the small clams, but not among the large ones. This is the first report to confirm the pathogenicity of wild isolate of Perkinsus species to wild Manila clams.