In vitro propagation of two Perkinsus spp. parasites from Japanese Manila clams Venerupis...

In Vitro Propagation of Two Perkinsus spp. Parasites from Japanese ManilaClams Venerupis philippinarum and Description of Perkinsus honshuensis n. sp.

CHRISTOPHER F. DUNGANa and KIMBERLY S. REECEb

aMaryland Department of Natural Resources, Cooperative Oxford Laboratory, 904 S. Morris St., Oxford, Maryland 21654, andbVirginia Institute of Marine Science, College of William and Mary, PO Box 1346, Gloucester Point, Virginia 23062

ABSTRACT. Perkinsus species are destructive parasites of commercial Manila clams, Venerupis philippinarum, in Japan, Korea, andSpain. However, in vitro parasite cultures from this important host clam are not available. Tissues of Manila clams collected during April2002 in Gokasho Bay, Japan harbored Perkinsus sp. parasites at a 97% prevalence (28/29) of moderate- and high-intensity infections.Perkinsus sp. cells in tissue samples were enlarged in alternative Ray’s fluid thioglycollate medium, before propagation in DME:Ham’s F-12 Perkinsus sp. culture medium. Enlarged parasite hypnospores zoosporulated at high frequencies to release motile zoospores, whichgave rise to continuous schizogonic cell lines that also zoosporulated continuously at low frequencies. Four Perkinsus sp. in vitro isolatescomprising two distinct morphotypes were cryopreserved, cloned, and archived for public distribution. For three isolates of one morpho-type, nucleotide sequences of the ribosomal DNA internal transcribed spacer region, of the large subunit rRNA gene, and of actin genes,were consistent with those reported for P. olseni. Similar sequences from one morphologically unique isolate differed from those of alldescribed Perkinsus species. These results show that at least two Perkinsus spp. infect Japanese Manila clams, and that one represents anew species, Perkinsus honshuensis n. sp.

Key Words. Asian clam disease, Perkinsus olseni, protists, Ruditapes philippinarum, Tapes philippinarum, Tapes semidecussatus.

PERKINSUS species are destructive parasites of the Manilaclam, Venerupis philippinarum, which has a current world-

wide distribution as an important commercial aquaculture species.Perkinsus sp. infections were associated with recent major de-clines in commercial clam production on both the south and westcoasts of Japan (Hamaguchi et al. 1998; Maeno, Yoshinaga, andNakajima 1999), and with mass mortalities of both husbanded andwild Manila clams on the Korean peninsula south and west coasts(Choi and Park 1997; Park and Choi 2001). In addition, mortali-ties from Perkinsus sp. infections also compromise Manila clamproduction on the Iberian Atlantic and Mediterranean coasts(Rodriguez and Navas 1995; Sagrista, Durfort, and Azevedo1996).

Diverse characteristics consistently confirm the taxonomic af-finities of some parasites in Asian Manila clams to the genus Per-kinsus. Perkinsus sp. from Asian Manila clams enlarge in Ray’sfluid thioglycollate medium (RFTM) (Ray 1963), RFTM-enlargedparasite hypnospores stain blue-black with Lugol’s iodine (Ham-aguchi et al. 1998), and enlarged Perkinsus sp. hypnospores alsozoosporulate upon transfer to sterile seawater (Ahn and Kim 2001;Maeno et al. 1999). Histologically, Perkinsus sp. in Japanese Ma-nila clams show the characteristic signet ring and schizont morph-ologies originally described for Perkinsus marinus by Mackin(1961). Histozoic parasite cell types in Japanese Manila clams arelabeled by anti-Perkinsus spp. antibodies (Maeno et al. 1999), andsimilar parasites in Korean Manila clams are labeled by in situhybridization (ISH) with a genus Perkinsus-specific DNA probethat targets both the small subunit ribosomal RNA (SSU rRNA)gene and the SSU rRNA product (Elston et al. 2004).

In an effort to identify the specific identity of Perkinsus sp. inJapanese Manila clams, DNAs extracted from RFTM-enlargedparasite hypnospores from infected clam tissues were PCR-amplified and sequenced at the internal transcribed spacer (ITS)region of the rRNA gene complex to reveal close homology withITS-region sequences reported for both P. atlanticus and P. olseni(Hamaguchi et al. 1998). Although their results clearly differen-tiated the sequenced Japanese Manila clam parasite from P. mari-nus, Hamaguchi et al. (1998) declined to propose its speciesalignment with either P. atlanticus or P. olseni, as reported nuc-

leotide sequences did not differentiate those parasites from eachother (Robledo, Coss, and Vasta 2000).

Synonymy of P. olseni and P. atlanticus, with the former namein priority, was subsequently demonstrated (Murrell et al. 2002),based on nucleotide sequences at both ITS and non-transcribedspacer (NTS) regions of rRNA gene complexes; so the Perkinsussp. previously reported from Japanese Manila clams is probably P.olseni by extension. Recent analyses of ITS and NTS sequencesfrom several apparent short-term in vitro Perkinsus sp. isolatesfrom Korean Manila clams also identify those as P. olseni (Parket al. 2005). However, the current lack of both an in vitro isolate ofa P. olseni holotype from an Australian abalone type host (Lesterand Davis 1981), and of archived in vitro isolates of Perkinsus sp.from Manila clams, deny the optimal source of genomic DNA forcomprehensive sequencing at multiple genomic loci for rigorousresolution of parasite taxonomic affinities.

To eliminate part of that deficiency, we report here axenic invitro propagation, cloning, and deposit for public distribution ofseveral Perkinsus spp. isolate cultures from Japanese Manilaclams, V. philippinarum. Details of parasite in vitro cell cytolo-gy and cell proliferation cycles are reported, as are results ofphylogenetic analyses using nucleotide sequences of LSU rRNAgenes, rDNA ITS regions, and actin genes. Parasite taxonomicaffinities within the genus Perkinsus were inferred from thesecollective characteristics to confirm that both P. olseni and a pre-viously undescribed Perkinsus sp. are represented among our iso-late cultures. The new clam parasite is described here as Perkinsushonshuensis n. sp.

MATERIALS AND METHODS

Manila clams. During April 2002, 50 Venerupis philippina-rum clams were collected at a commercial clam bed in GokashoBay, Mie Prefecture, Japan, from waters of 18 1C and 33 ppt sa-linity. Refrigerated and humidified clams were shipped to theCooperative Oxford Laboratory, where they were quarantined inre-circulating aquaria at 23 1C and 30 ppt until sacrificed.

Clam tissue samples. Thirty live, and several moribund,Manila clams (mean shell length 41.4 � 3.5 mm SD) were proc-essed to acquire culture inocula for Perkinsus sp. isolates, andhistological samples. Measured clams were aseptically opened,and shucked from their shells with a sterile scalpel onto a single-use sheet of virgin paraffin film. A sterile blade was used to excisea transverse tissue section through the visceral mass, from which

Corresponding Author: C. Dungan, Cooperative Oxford Laboratory,904 S. Morris St., Oxford, MD 21654, USA—Telephone number: (410)226-5193; FAX number: (410) 226-5925; e-mail: [email protected]

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J. Eukaryot. Microbiol., 53(5), 2006 pp. 316–326r 2006 The Author(s)Journal compilation r 2006 by the International Society of ProtistologistsDOI: 10.1111/j.1550-7408.2006.00120.x

duplicate gill and visceral mass tissue sub-samples were asepti-cally excised for inoculation into 2 ml of alternative Ray’s fluidthioglycollate medium (ARFTM) (Nickens et al. 2002) containedin separate wells of a sterile, 24-well culture plate. Duplicate tis-sue sub-samples were also directly inoculated into 850 mOsm/kg(29 ppt) DME:Ham’s F-12 Perkinsus sp. culture medium contain-ing 3% (v/v) fetal bovine serum (FBS) (DME/F12-3) (Burreson,Reece, and Dungan 2005) in separate plates. Both ARFTM andDME/F12-3 media were supplemented with penicillin (200 U/ml),streptomycin (200 mg/ml), gentamicin (200 mg/ml), and nystatin(50 U/ml).

Additional transverse visceral mass tissue samples were asep-tically excised, and histological samples were fixed for 48 hfixation in 10 vol of Davidson’s solution. Paraffin-infiltrated histo-logical tissues were embedded such that transverse histologicalsections from anterior tissue surfaces were obtained when blockswere sectioned at 5–6 mm thickness. Sections were stained withMayer’s hematoxylin and eosin for microscopic analyses.

Histological immunoassays. For comparison with results ofprevious investigations (Maeno et al. 1999), histological fluores-cence immunoassays for detection of Perkinsus spp. parasiteswere performed as described (Dungan and Roberson 1993), usingprotein A-purified rabbit polyclonal antibodies to P. marinus thatlabel all known Perkinsus species (Blackbourn, Bower, andMeyer 1998; Bushek, Dungan, and Lewitus 2002). De-waxed, re-hydrated, and blocked sections were incubated for 1 h in a 15 mg/ml solution of rabbit anti-P. marinus IgG primary antibodies,washed three times, and then incubated for 1 h in a 3 mg/ml solu-tion of affinity-purified and FITC-congugated goat anti-rabbit IgGsecondary antibodies. Immunostained sections were counter-stained with 0.05% (w/v) Evan’s blue. Positive control sampleswere sections of Crassostrea virginica oyster tissues infected withP. marinus. Negative controls included replicate test sections thatwere only de-waxed, blocked, and counterstained, to test forautofluorescence of sample components. Replicate sections onwhich normal rabbit IgG was substituted for the specific primaryantibody during immunostaining, controlled for non-specific an-tibody binding.

In situ DNA probe hybridization (ISH) assays. Histologicalsections from selected Manila clams were tested for hybridizationby a digoxigenin-conjugated, genus Perkinsus-specific SSUrRNA gene DNA probe as previously described (Elston et al.2004), except that an eosin counterstain, ethanol dehydration, andPermounts mounting medium were used following precipitationof the 5-bromo-4-chloro-3-indolyl-phosphate/4-nitro blue tetra-zolium chloride (BCIP/NBT) chromophore by alkaline phosphatase-conjugated, anti-digoxigenin antibodies. A combined positivehybridization control and probe specificity negative control wasa section from a Chesapeake Bay Crassostrea virginica oysterco-infected with both P. marinus and Haplosporidium nelsoni.Negative controls included duplicate histological sections of alltested samples, which received hybridization buffer without probeduring hybridization incubations.

In vitro isolate inocula. Alternative Ray’s fluid thioglycollatemedium assays were incubated at 27 1C for 48 h to induce en-largement of hypnospore cells of Perkinsus sp., and clam tissuebiopsies were observed unstained in culture plate wells for en-larged, refractile parasite hypnospores, using an inverted micro-scope equipped with Hoffman modulation contrast (HMC) optics.Relative densities of Perkinsus sp. hypnospore cells in individualtissue biopsies were categorized as absent (0), or ranging fromlight (1) to heavy (5) (modified from Choi et al. 1989), and themean sample intensity of infections by Perkinsus sp. (weightedprevalence of historic authors) was calculated for the sample asthe sum of individual clam categorical infection intensities divid-ed by the number of clams in the sample. Sample infection prev-

alence was calculated as the percentage of infected clams in thesample. Five gill tissue samples and two visceral mass tissuesamples with the highest enlarged parasite cell densities, and lowor absent apparent levels of other microbial contaminants, wereselected as in vitro isolate inocula for Perkinsus sp. cultures

In vitro pathogen propagation. Selected ARFTM-incubatedtissues with heavy Perkinsus sp. hypnospore densities were asep-tically transferred to culture plate wells containing 2 ml of anti-microbial-supplemented DME/F12-3 culture medium. Infectedtissues were disrupted and suspended in culture medium by gen-tle trituration with a sterile pipette. Resulting suspensions wereserially diluted at 0.5 ml/well into three additional wells contain-ing 2 ml of culture medium. Inoculated culture plates were cov-ered, incubated at 27 1C in an air atmosphere, and observed dailyfor proliferation of Perkinsus sp. isolates.

Alternative Ray’s fluid thioglycollate medium-enlarged Per-kinsus sp. hypnospores from clam tissues vigorously zoosporu-lated after transfer to the DME/F12-3 culture medium. Oncezoospore release and motility decreased, apparent proliferationby schizogony of vegetative isolates was confirmed at 10–14 dpost-inoculation into DME/F12-3 culture medium. Proliferatingparasite cell populations were sub-cultured for six sequential pas-sages through culture plate wells in which antimicrobials weresequentially reduced or eliminated from media. Axenic isolatecultures were subsequently expanded in culture flasks and cryo-preserved (Dungan and Hamilton 1995). At cryopreservation, iso-lates were also cloned by limiting dilution in 96-well cultureplates (Dungan et al. 2002). Monoclonal cultures proliferatedwithin 10 d post-inoculation, and one monoclonal cell line fromeach isolate was expanded and cryopreserved.

In vitro cell cycles and morphometrics. Selected represent-atives of two apparently different in vitro isolate morphotypes(Mie-3g, Mie-5mg) were simultaneously propagated in continu-ous exponential growth for 10 d before microscopic measure-ments of in vitro cell types were made to estimate ranges andmeans for diameters of trophozoites (merozoites), subdividingschizonts (meronts), and zoosporangia. Relative proportions ofthose cell types in exponential-phase in vitro populations werealso estimated, as were their qualitative adherence characteristics.Apparent degradation of phenol red medium pH indicator wastested by comparing the pH of the colorless medium conditionedfor 48 h by in vitro isolates, to the pH of uninoculated culturemedium.

Alternative Ray’s fluid thioglycollate medium-induced enlarge-ment of in vitro propagated Perkinsus sp. isolate cells and zoo-sporulation capabilities of ARFTM-enlarged isolate hypnosporeswere tested by transferring DME/F12-3 medium-propagated cellsof both morphotype-isolates to ARFTM for 48 h. Resulting meanhypnospore diameters were calculated from microscopic celldiameter measurements, and ARFTM-enlarged hypnospores weretested for staining with 33% (v/v) Lugol’s iodine. Aliquots ofARFTM-enlarged isolate hypnospores were returned to the DME/F12-3 culture medium for 36 h, before microscopic estimation ofzoosporulation frequencies and zoosporangia diameters amonghypnospores of both morphotype isolates.

PCR assays, DNA sequencing, and parsimony analy-ses. Large subunit rRNA genes, ITS regions of the rRNA genecomplex, and actin gene fragments were amplified by PCR fromgenomic DNAs extracted from cells of all four monoclonal iso-lates of our Manila clam Perkinsus spp. PCR amplicons from ITSregions, and from LSU rRNA and actin gene amplifications ofMie-5mg and Mie-3g culture strain DNAs, were cloned andsequenced for comparison with nucleotide sequences publishedfor other Perkinsus spp. infecting marine molluscs.

For DNA isolation, Perkinsus sp. cell populations werepropagated in vitro and harvested at a density of �107 cells/ml.

317DUNGAN & REECE—PERKINSUS SPP. FROM JAPANESE MANILA CLAMS

Three- to 5-ml aliquots of each cell suspension were washed twicewith 1 ml of 12 ppt sterile artificial seawater (SASW) to removegrowth medium, and were re-suspended in lysis buffer for extrac-tion using the DNeasy Tissue Kit (Qiagen, Carlsbad, CA) follow-ing the manufacturer’s protocol for tissue samples.

For each primer set, DNA from each isolate was used in two orthree separate amplification reactions. Five to 20 ng of isolateculture DNA were used in a 25 ml PCR. The primers and ampli-fication parameters were as described previously (Burreson et al.2005). For each locus, amplicons (3–10) from multiple replicateamplifications of DNAs from isolates Mie-5mg and Mie-3g werecloned for sequencing, and amplicons from multiple replicateamplifications of the ITS region were cloned from all four ofthe isolate cultures. Amplicons were cloned using a TOPO TACloning Kit for Sequencings (Invitrogen, Carlsbad, CA) follow-ing the manufacturer’s protocols. Insert sequences were deter-mined either as previously described, using a LI-COR Model 4200automated DNA sequencer (Burreson et al. 2005), or were pro-cessed for sequence analysis on a Prism 3100 Genetic Analyzer(Applied Biosystems, Foster City, CA) as described below.

DNA clones with appropriate-size inserts were identified by aPCR-based screening method in which template DNA was ex-tracted from transformed bacterial colonies by picking cells fromagar plates with a sterile wooden toothpick, and lysing pickedcells in 10ml of sterile water in 200-ml plastic strip tubes. Inoc-ulated water samples were heated for 4 min at 94 1C to lyse cells,and 0.5ml of these preparations were used in PCR. The 25ml re-actions contained the following reagents: 20 mM Tris-HCl (pH8.4), 50 mM KCl, 1.5 mM MgCl2, 0.2 mM of each dNTP, 1 mM ofeach primer, 0.25 U/ml Taq polymerase, and 0.2 mg/ml BSA.Thermocycling conditions for this reaction were as follow: initialdenaturation at 94 1C for 2 min, followed by 30 cycles of 94 1C for30 s, 54 1C for 30 s, and 72 1C for 1 min, followed by a final elon-gation at 72 1C for 5 min. Following amplification using the M13primer pairs, 3 ml of PCR products were electrophoresed on a 2%agarose gel, stained with ethidium bromide, and visualized withUV light.

Before sequencing, PCR products from clones containing thecorrect insert size were treated with shrimp alkaline phosphatase(SAP) and exonuclease I (Exo I) (Amersham Biosciences, Piscat-away, NJ), in order to degrade nucleotides and single-strandedDNA (primers) remaining after PCR. Five microliters of the M13PCR product were combined with 0.5 U of SAP and 5.0 U of ExoI, and incubated at 37 1C for 30 min, 80 1C for 15 min, and 15 1Cfor 5 s. Clean PCR products from plasmid inserts were sequencedbi-directionally using the Big Dye Terminator kit (Applied Bio-systems, Norwalk, CT) with M13 sequencing primers, and using5 ml reactions with 0.125 times the concentration of Big Dye re-agent specified in the manufacturer’s protocols. Each 5-ml reactioncontained 0.0625 ml of Big Dye, 0.96875 ml of 5 � buffer,1.6 pmol of each primer, and 10 ng of clean PCR product.Thermocycling parameters were as follow: 25 cycles of 96 1Cfor 1 min, 96 1C for 10 s, 50 1C for 5 s, 60 1C for 4 min, followedby a final incubation at 4 1C until the sequencing reaction productswere precipitated using the ethanol/sodium acetate precipitationmethod (ABI User Bulletin, April 11, 2002). Precipitated se-quencing reaction products were re-suspended in 20ml of Hi-Diformamide (Applied Biosystems) and 10 ml of each were elect-rophoretically separated on an ABI 3100 Prism Genetic Analyzer.

Sequences were imported into MacVector 8.2 Sequence Anal-ysis Software (Accelrys Inc., San Diego, CA) for trimming vectorsequences and CLUSTAL-W alignments (Thompson, Higgins,and Gibson 1994). Phylogenetic analyses were performed usingthe PAUP� 4.0b10 (Swofford 2002) software. Genetic distanceand parsimony analyses were performed on the ITS region, LSUrRNA gene, and actin gene sequences of Perkinsus sp. isolates

from Manila clams that were aligned with sequences deposited inGenBank from both described and undescribed Perkinsus species.Internal transcribed spacer region sequences were aligned withgap penalties of eight for insertions and three for extensions, inboth pairwise and multiple alignment phases. The LSU rRNAgene and actin gene fragments were aligned using the default pa-rameters of 10 for insertions and five for extensions.

GenBank sequences included in the analyses for the ITS regionwere as follows: Perkinsus qugwadi AF151528; P. marinusU07700, AF150987, AY295180, AY295184, AY295186,AY295188, AY295189, AY295194, AY295197, AY295199; P.olseni (5 P. atlanticus) U07697, U07698, U07699, U07701,AY435092, AF140295, AF369967, AF369969, AF441209,AF441211, AF441213, AF473840, AF509333, AF522321;P. mediterraneus AY487834–AY487842; P. chesapeaki (5 P.andrewsi) AF091541, AF102171, AY876302, AY876304,AY876305, AY876306, AY876307, AY876311, AY876314,AY876316, AY876318; Perkinsus sp. AF252288, AF440464,AF440465, AF440467, AF440468, AF440471.

GenBank sequences included in analyses of the LSU rRNAgene were as follows: Prorocentrum micans X16108 (outgrouptaxon); P. marinus AY876319, AY876320, AY876322,AY876325, AY876328, AY876329; P. olseni AF509333,AY876330, AY876331, AY876332; P. chesapeaki AY876336,AY876337; Perkinsus sp. AY876338, AY876340, AY876341,AY876342, AY876343, AY876344, AY876345, AY876347,AY876348, AY876349.

GenBank sequences included in analyses of the actin geneswere as follows: Amphidinium carterae U84289, Prorocentrumminimum U84290 (outgroup taxa); Type 1 P. marinus, U84287,U84288, AY876350; Type 1 P. olseni AY876352, AY876355;Type 2 P. olseni AY876351, AY876353, AY876354; Type 1 P.chesapeaki AY876359, AY876360, AY876361; Type 2 P. chesa-peaki AY876358, AY876362; Type 1 Perkinsus sp. AY876368,AY876371, AY876372, AY876373, AY876374; Type 2 Perkin-sus sp. AY876364, AY876369.

Neighbor joining analyses were carried out based on uncor-rected ‘‘P’’ values. Parsimony jackknife analyses were carried outusing a deletion value of 30% and 100 random additions of 1,000replicates.

RESULTS

ARFTM tissue assays. Enlarged, clustered, spherical, and ref-ractile hypnospore cells of Perkinsus sp. were present in mostARFTM-incubated clam tissue samples. Detection frequencies ofPerkinsus sp. from all ARFTM-assayed live clams from the Gok-asho Bay sample estimated that 97% (28/29) of sampled clamsharbored Perkinsus sp. infections, and that the mean infection in-tensity was moderately high at 3.3 on the 0–5 infection intensityranking scale. Among 20 Gokasho Bay clams analyzed byARFTM assays to identify and select optimum tissues as sourc-es of in vitro isolate inocula, 45% (9/20) showed enlarged parasitehypnospores in gill tissues, and 70% (14/20) showed hypnosporesin visceral mass tissues.

Histological assays. Perkinsus sp. lesions were observed his-tologically in 80% (24/30) of clams whose sections were exam-ined. Relatively large (2–12 mm) trophozoites of Perkinsus sp. andactively proliferating schizonts occurred abundantly in connectivetissue lesions, but were rare in epithelia. Eccentrically vacuolatedsignet ring trophozoites typically showed eccentric nuclei con-taining a prominent, often eosinophilic, nucleolus (Fig. 1). Le-sions were observed at similarly high frequencies (73%) in gilland digestive system connective tissues, and at decreasing relativefrequencies in gonad, mantle, kidney, and heart connective tis-sues. Occasional trophozoites were observed circulating in the

318 J. EUKARYOT. MICROBIOL., VOL. 53, NO. 5, SEPTEMBER– OCTOBER 2006

vasculature or within stomach epithelia. Typical lesions in all ex-amined clams were hemocyte-encapsulated granulomatous cystsin hemocyte-infiltrated connective tissues, containing severalapparent sibling parasite cells that were often enrobed in anamorphous eosinophilic matrix apparently secreted by V.philippinarum hemocytes.

Immunostained histological sections that were available fromthree of the four clams (Mie-3, Mie-4, Mie-13) whose tissue ino-cula yielded in vitro parasite isolates, all showed intensely labeledPerkinsus sp. trophozoite and schizont cells of 4–12mm diame-ters. Adjacent sections labeled by ISH with the genus Perkinsus-specific SSU rRNA gene probe all showed labeling of the sametypes of Perkinsus sp. cells detected by immunoassays (Fig. 2).Neither immunoassays nor ISH assays specifically differentiatedany systemically distributed mono-dispersed, small, eosinophilic,avacuolate trophozoites of Perkinsus sp. (Maeno et al. 1999).

In vitro isolates, cell cycles, and cytology. Four Perkinsus sp.in vitro isolates were propagated from ARFTM-enlarged hypno-spores in tissue inocula from different clams, upon transfer ofthose hypnospores to the DME/F12-3 Perkinsus sp. culture me-dium. Similar isolates also propagated from infected tissues inoc-ulated directly into the DME/F12-3 culture medium, but abundantciliate, flagellate, amoebae, and thraustochytrid contaminantsco-propagating in those cultures rendered the less-contaminatedARFTM hypnospores most expedient as axenic isolate cultureinocula (La Peyre and Faisal 1995).

Alternative Ray’s fluid thioglycollate medium-enlarged hypno-spores universally zoosporulated upon transfer to the DME/F12-3culture medium. Motility among free-swimming and zoo-sporangium-contained zoospores was transient (days), and wasfollowed by enlargement and schizogonic in vitro proliferation ofapparent formerly motile zoospores. Subsequent generations ofproliferating in vitro isolate cultures also included continuous andspontaneous zoosporulation by a minor subset of all isolate cellpopulations. In vitro trophozoites capable of proliferation byschizogony or zoosporulation bore a large vacuole typically con-taining a prominent refractile body (vacuoplast), a large eccentricnucleus containing a prominent nucleolus, and granular peripheralcytoplasm (Fig. 3). Four in vitro Perkinsus sp. isolates, and theirfour monoclonal derivatives, were cryopreserved, cloned, and ar-chived for public distribution by the American Type Culture Col-lection (ATCC, http://www.atcc.org) (Table 1).

Two different isolate morphotypes were represented among ourfour in vitro isolates. Isolate Mie-3g (ATCC PRA-176, PRA-177)uniquely represented a morphotype with smaller maximum diam-eters for trophozoites (20 mm) and schizonts (35 mm), and withrare continuous zoosporulation (Fig. 4). Isolate Mie-3g cells bothclustered together and adhered tenaciously to flask surfaces, byapparent agency of a visible, transparent extra-cellular polymer.Isolate Mie-3g cells also uniquely degraded the culture mediumpH indicator, phenol red, rendering media colorless over 48 h,without acidification. Three isolates (Mie-5mg, Mie-4g, Mie-13v)shared a morphotype characterized by larger maximum diametersof trophozoite (40 mm) and schizont (85 mm) cells, by regular low-frequency continuous zoosporulation, by cells that clustered to-gether but did not adhere to flask surfaces (Fig. 5), and by theirfailure to degrade phenol red. Isolates of both morphotypesshowed similar proportions of schizonts (19%–21%) in exponen-tial phase cultures (Table 2).

After propagation in DME/F12-3 culture medium, cells of allisolates were rapidly induced to form enlarged hypnospores dur-ing a 48-h incubation in ARFTM, after which hypnospore cellsstained blue-black with Lugol’s iodine. Relative and absolute en-largement in ARFTM of the small-cell morphotype representedby isolate Mie-3g greatly exceeded that of large-cell morphotypeisolate Mie-5mg cells. The resulting Mie-3g hypnospores andzoosporangia had mean diameters 1.9 and 1.4 times the respectivemean diameters of Mie-5mg hypnospores and zoosporangia(Table 2 and Fig. 6–8). Beginning approximately 36 h after theirreturn to either the DME/F12-3 propagation medium or to 30 pptsterile artificial seawater (SASW), ARFTM-enlarged hypnosporesof both morphotype-isolates zoosporulated at similarly highfrequencies (470%), producing and releasing abundant motilezoospores that were pyriform to ovoid with dimensions5–7mm � 2–3 mm (Fig. 9, 10).

PCR assays, DNA sequencing, and phylogenetic analy-ses. The expected 670–680 bp rRNA ITS region fragments were

Fig. 1–2. Trophozoites of Perkinsus sp. (Perkinsus honshuensis n. sp.)in gill connective tissue of Japanese Manila clam Mie-3. Scale bars 5 10mm. 1. Three hematoxylin & eosin-stained signet ring trophozoites witheccentric vacuoles, eccentric nuclei, and prominent nucleoli, encysted byclam hemocytes in a gill abscess lesion. 2. Cluster of five vacuolatedtrophozoites labeled (dark) by in situ hybridization with the genus Per-kinsus small subunit rRNA (SSU rRNA) gene DNA probe. CytoplasmicSSU rRNA and nuclear rDNA (o) both hybridize with the probe.

Fig. 3. Live trophozoite of the Mie-5mg Perkinsus sp. (Perkinsusolseni) in vitro isolate showing vacuole with refractile vacuoplast (o),granular cytoplasm, and eccentric nucleus with prominent nucleolus (�).Scale bar 5 10 mm, differential interference contrast.


amplified from all of our Mie clam Perkinsus sp. culture DNAs,and the amplified LSU rRNA and actin gene fragments were like-wise of the expected lengths of approximately 1,000 and 660 bp,respectively. Sequencing of the fragments confirmed that the tar-geted region was amplified in each case.

In many instances, identical ITS region sequences were foundin multiple DNA clones from each culture, and identical sequenc-es were found in DNA from the isolates Mie-4g, Mie-5mg, andMie-13v. Two ITS region DNA clones from Mie-4g, three fromMie-5mg, and four from Mie-13v isolates had sequences that wereidentical to each other, and to the GenBank ITS region sequencesU07697 (Goggin 1994) and AF473840 (Park et al. 2002) depos-ited as P. olseni. One of the Mie-13v DNA clones had a sequenceidentical to that of many other GenBank P. olseni sequences, in-cluding AF369969 (Casas et al. 2002) that was used in these anal-yses. One ITS region sequence from strain Mie-4g was unique, butshowed 4 99% similarity to other P. olseni sequences (Table 3).

All ITS region sequences from the Mie-3g isolate strain weredistinct from those determined for our other three Mie clam isolatestrains, and from all GenBank ITS region sequences deposited asPerkinsus spp. Two of seven ITS region sequences determinedfrom the Mie-3g isolate were identical, and the other variants alldemonstrated 4 99% similarity to each other.

The three LSU rRNA gene sequences from isolate Mie-5mgDNA were each unique, showed 4 99% similarity to each other,and each showed 4 99% similarity to the respective P. olsenisequences from GenBank (Table 4). Seven DNA clones with LSUrRNA gene fragments from Mie-3g isolate DNA were sequenced.Three of these sequences were identical to each other (one de-posited in GenBank), and the others showed 4 99% similarity toeach other, but were distinct from LSU rRNA gene sequencesfrom other Perkinsus spp. (Table 4).

The three Type 2 actin gene sequences from isolate Mie-5mgDNA were each unique, showed 4 99% similarity to each other,and each showed 4 99% similarity to the respective P. olsenisequences from GenBank. Type 1 actin gene sequences were notfound in the limited number of DNA clones from the Mie-5mg

Table 1. In vitro Perkinsus spp. isolated from specified Japanese Manila clam, Venerupis philippinarum, tissues, ATCC isolate accession numbers,GenBank DNA sequence accession numbers, and isolate taxonomic affinities inferred from the rDNA ITS region, and from LSU rRNA and actin genesequences.

Isolatecode

Tissueorigin

Clonality ATCCnumber

ITS rDNAGenBank sequences

LSU rRNAGenBank sequences

Actin GenBanksequences

Taxonomicaffinity

Mie-3g Gill Poly PRA-176 DQ516696–DQ516698 P. honshuensis n. sp.Mie-3g/H8 Gill Mono PRA-177 DQ516699–DQ516702 DQ516680–DQ516682,

DQ516684DQ516686–DQ516692 P. honshuensis n. sp.

Mie-4g Gill Poly PRA-178 P. olseniMie-4g/F6 Gill Mono PRA-179 DQ516703–DQ516705 P. olseniMie-5mg Mantle/gill Poly PRA-180 P. olseniMie-5mg/F8 Mantle/gill Mono PRA-181 DQ516706–DQ516708 DQ516679, DQ516683,

DQ516685DQ516693–DQ516695 P. olseni

Mie-13v Visceral Poly PRA-182 P. olseniMie-13v/H4 Visceral Mono PRA-183 DQ516709–DQ516715 P. olseni

ATCC, American Type Culture Collection; ITS, internal transcribed spacer; LSU, large subunit.

Fig. 4–5. Schizogonic in vitro cell cycles of different morphotypeisolates of Perkinsus spp. from different Japanese Manila clams. Hoffmanmodulation contrast images of live cells. Scale bars 5 20 mm. 4. IsolateMie-3g (Perkinsus honshuensis n. sp.) showing mature trophozoite (A),subdividing schizonts (B), and clusters of sibling daughter cells (C).5. Isolate Mie-5mg (P. olseni) showing mature trophozoites (A) and sub-dividing schizonts in progressive bipartite reductive divisions (B–D) thatproduce a mature schizont containing numerous 2mm daughter cells (E),which enlarge and release as clusters of sibling daughter cells (F).


isolate that were sequenced as part of this study. It is likely, how-ever, that our Japanese Manila clam Mie-5mg cultures also harborType 1 actin gene sequences that will be found by sequencingadditional DNA clones. Seven DNA clones with actin gene frag-ments were sequenced from Mie-3g isolate strain DNA. Four ofthe sequences (Type 1) showed 499% similarity to each other,but were only about 80% similar to the other three actin gene se-quences (Type 2), two of which were identical to each other andabout 96% similar to the third (Table 5).

In both distance and parsimony phylogenetic analyses, the nuc-leotide sequences of isolate DNAs from multiple genomic lociconsistently differentiated two genetic clades among sequencesfrom Perkinsus sp. DNAs of our Japanese Manila clam in vitroisolates. DNA sequences from our large-cell morphotype, whichincluded isolates Mie-4g, Mie-5mg, and Mie-13v, consistentlygrouped with P. olseni; but sequences from our Mie-3g isolatewere always found in distinct clades (Fig. 11–13). In all instances,

the sequences from GenBank listed above as Perkinsus sp. se-quences grouped with P. chesapeaki sequences. The overall top-ologies of the trees generated from both the distance andparsimony analyses were the same. In addition, the DNA se-quences from each described Perkinsus species formed distinctclades in both types of analyses that were performed with each ofthe three data sets (Fig. 11–13).

Internal transcribed spacer region sequences determined fromthe Mie-4g, Mie-5mg, and Mie-13v isolate DNAs all groupedwith GenBank deposited P. olseni ITS region sequences (Fig. 11)and showed pairwise genetic distances of less than 1.0% withthose sequences. Such variability is within the range of geneticdistances observed among the sequences previously determinedfor isolates of P. olseni (0%–1.2%) (Table 3). The ITS region se-quences from the Mie-3g isolate DNA formed a distinct clade,however, and demonstrated pairwise genetic distances (uncorrect-ed ‘‘p’’) with other Perkinsus species ITS region sequences

Fig. 6–8. Hypnospores and zoozsporangia of Perkinsus spp. enlarged in alternative Ray’s fluid thioglycollate medium (ARFTM) before propagationin DME:Ham’s F-12 Perkinsus sp. culture medium containing 3% (v/v) fetal bovine serum (DME/F12-3) culture medium. Hoffman modulation contrastimages of live cells. Scale bars 5 40 mm. 6. Pre-zoosporulation hypnospore of isolate Mie-3g (Perkinsus honshuensis n. sp.) with large eccentric nucleus(�) bulging into central vacuole. 7. Zoosporangium of isolate Mie-3g with extended germ tube (4). 8. Central hypnospore of isolate Mie-5mg (P. olseni)with eccentric nucleus (�), surrounded by zoosporangia with extended germ tubes (4).

Table 2. Morphometric and physical characteristics of two different Perkinsus spp. morphotype isolates propagated from Japanese Manila clams andmaintained in continuous exponential in vitro growth (DME/F12-3 medium), or enlarged in ARFTM before return to DME/F12-3 medium.

Morphotype isolates

DME/F12-3 medium continuous culture ARFTM to DME/F12-3 media

Trophozoitediameter

(mm)

Schizontdiameter

(mm)

Zoospor-angium

diameter(mm)

Schizont(%)

Zoospor-angium

(%)

Adherencec, cell;f, flask

Hypno-spore

diameter(mm)

Zoospor-angium

diameter(mm)

Zoospor-angium

(%)

Mie-3g(P. honshuensis n. sp.)

19 �1 c, f 470

Mean 10.2 20.3 78.3 49.4SD 3.7 5.4 15.3 8.9Range 3–20 10–35 35 35–120 30–70n 60 60 1 60 60

Mie-5mg (P. olseni) 21 o1 c 470Mean 10.0 23.5 26.3 42.1 35.6SD 4.6 18.7 4.8 13.4 6.8Range 5–40 10–85 20–30 20–80 25–60n 60 60 4 60 60

Diameter ranges and means for different cell types are listed, along with their occurrence frequencies and differentiating adherence characteristics.ARFTM, alternative Ray’s fluid thioglycollate medium; DME/F12-3, DME:Ham’s F-12 Perkinsus sp. culture medium containing 3% (v/v) fetal

bovine serum.


ranging from 3.5% to 12.9% (Table 3). This range of genetic dis-tances is similar to that observed between the ITS region sequenc-es differentiating currently accepted Perkinsus species (excludingP. qugwadi, which is more distant), which range from 4.4% to14.3%.

The LSU rRNA gene and actin gene DNA sequences from theMie-5mg isolate culture grouped in phylogenetic analyses with P.olseni sequences previously deposited in GenBank. Sequences forthese regions from the Mie-3g isolate DNA, however, formed adistinct clade within the genus Perkinsus sequences in both dis-tance (not shown) and parsimony analyses at each locus (Fig. 12,13). As with the ITS region sequences, the pairwise genetic dis-tances observed between the LSU rRNA (Table 4) and actin gene(Table 5) sequences from the Mie-3g isolate culture DNA andthose of the Perkinsus spp. were comparable to the distances ob-served between other Perkinsus species at those loci. The actinType 2 gene sequences amplified and sequenced from the Mie-5mg culture grouped with P. olseni actin Type 2 gene sequencesdeposited in GenBank. Sequences that grouped with actin Type 1and those that grouped with the Type 2 actin genes were found inthe Mie-3g isolate DNA, and these formed distinct clades withineach group (Fig. 13). Overall, there was only 70%–80% similaritybetween the actin Type 1 and Type 2 sequences from all Perkinsusspecies.

Parsimony jackknife support for the ITS region sequences wasvery high (100%) for a monophyletic P. olseni clade that includedthe Mie-5mg, Mie-4g, and Mie-13v isolate strains with previouslydetermined P. olseni ITS region sequences. High jackknife sup-port for monophyletic P. olseni clades with LSU rRNA and actingene fragments that included those sequences from the Mie-5mgisolate, were 99% and 100% respectively. Support for the mon-ophyletic clades of the ITS, LSU, and two actin gene type se-quences from the Mie-3g isolate were also very high (99%, 93%,100%, and 100%, respectively), and included only those sequenc-es determined from this unique isolate strain’s DNA.

In the parsimony jackknife analysis of the ITS region, the Mie-3g isolate clade was sister to P. mediterraneus with low support(54%); while in the LSU rRNA gene analysis, the Mie-3g isolateclade was sister to P. marinus with moderate support (73%).There are no LSU rRNA or actin gene sequences currently avail-able, however, for P. mediterraneus. Therefore, we do not know ifour Mie-3g isolate and P. mediterraneus would group together inanalyses with those data. The Type 2 actin gene sequence of theMie-3g isolate grouped with that of P. olseni Type 2 actin geneswith high support (99%). There was no resolution, however,among the Perkinsus species based on their available Type 1 ac-tin gene sequences (Fig. 13). At this time there is not enough ev-idence to suggest a sister relationship for the Mie-3g isolate withany described Perkinsus species. However, support for groupingthe Mie-3g isolate within the genus Perkinsus is very high with allthree data sets (90%–100%).

DISCUSSION

The high prevalence of variable-intensity Perkinsus sp. infec-tions documented by the present study in Japanese Manila clamsfrom the waters of Mie Prefecture is consistent with similar in-fection prevalences previously reported in samples of this clamspecies from both Hiroshima and Kumamoto prefectures (Ham-aguchi et al. 1998; Maeno et al. 1999). This suggests that suchinfections may be widespread and prevalent among Japanese Ma-nila clam populations. Our frequent detection of moderate- andhigh-intensity Perkinsus sp. infections in Japanese Manila clamsfrom temperate (18 1C) springtime waters is consistent with sim-ilar seasonal pathology reported for Manila clams in both Japan(Maeno et al. 1999) and Korea (Park and Choi 2001), suggestingthat Perkinsus spp. infecting these clams are well-adapted to path-ogenic proliferation at moderate water temperatures.

Defensive responses to Perkinsus sp. among the Japanese Ma-nila clams that we examined histologically, closely resembled thehemocyte encapsulation responses described in similarly infected

Fig. 9–10. Zoospores of Perkinsus spp. isolates from Japanese Manilaclams by differential interference contrast. Scale bars 5 5 mm. 9. Zoosporeof isolate Mie-3g (Perkinsus honshuensis n. sp.) showing biflagellate,pyriform profile, with inflated anterior end and recoiled anterior flagellum.10. Zoospore of isolate Mie-5mg (P. olseni) showing reniform profile,with anterior flagellum inserting at the ventral concavity.


V. philippinarum (5 Tapes semidecussatus) from the SpanishMediterranean coast, and are probably effected by the hem-ocyte-secreted proteins described for the same clam in Mediter-ranean waters (Montes, Durfort, and Garcia-Valero 1995;Sagrista, Durfort, and Azevedo 1995). Similar to apparent inef-fective defensive encapsulations of P. chesapeaki in ChesapeakeBay Mya arenaria clams (Dungan et al. 2002), encapsulated Per-kinsus sp. cells in granulomatous lesions of Japanese Manilaclams also frequently showed both normal morphology and evi-dence of proliferation.

Morphometric characteristics grouped our four in vitro isolatesfrom Japanese Manila clams into the same two clades to whichthey were consistently grouped by independent phylogenetic anal-yses of nucleotide sequences at three genomic loci. Our DNA se-quencing results consistently confirmed three of these isolates tobe strains of P. olseni, in agreement with the nucleotide sequencespreviously reported by Hamaguchi et al. (1998) from DNAs ofRFTM-enlarged Perkinsus sp. hypnospores harvested from infect-ed Japanese Manila clam tissues. In contrast, nucleotide sequenc-es at all three analyzed loci from one of our Japanese Manila clamisolates (Mie-3g/H8, ATCC PRA-177) consistently differentiatedthat isolate strain from any described Perkinsus species, and wereconsistent with the unique morphometric, metabolic, and otherphenotypic characteristics of that isolate strain that are describedhere.

In light of the genetic data that consistently differentiated ourMie-3g isolate strain from all described Perkinsus spp, and of itssimilarly distinct in vitro morphometric and phenotypic charac-teristics, we propose that this in vitro isolate represents a newparasite species from Japanese Manila clams. In reference to thegeographic origin of this new clam parasite, we designate isolateMie-3g/H8 strain (ATCC PRA-177) as the holotype representa-

tive of Perkinsus honshuensis n. sp., for which we provide thefollowing description.

Perkinsus honshuensis n. sp.

Diagnosis. Histologically, trophozoites in host tissues arespherical, 4–12mm in diameter, with a single, eccentric nucleusthat typically contains a prominent nucleolus, and a large, eccen-tric vacuole that occupies much of the cell volume. Lesions occurwith decreasing frequency among connective tissues of gills, go-nad, digestive tract, heart, mantle, and kidney, but epithelia are nottypically infected. Infected clam tissues are commonly infiltratedby granulocytic hemocytes, and groups of two to 10 parasitetrophozoites occurring within granulomatous cysts are variablyencapsulated in an amorphous, hemocyte-secreted, eosinophilicmatrix.

Mature in vitro propagated trophozoites are 10.2 � 3.7 mm(SD) mean diameter, and a schizont mean diameter of20.3 � 5.4mm (SD). Typical continuous culture zoosporangiumdiameter is 35.0mm, but ARFTM-induced zoosporangia are49.4 � 8.9mm (SD) diameter. In vitro zoosporulation is continu-ous at low (o1%) frequency, but may be induced at high (470%)frequency by transient, 48 h trophozoite incubation in ARFTMmedium. Zoospores assessed in killed whole mounts by DIC mi-croscopy are pyriform or ovoid with dimensions 5–7 mmlong � 2–3 mm wide.

DNA nucleotide sequences. In phylogenetic analyses, ITS re-gion sequences from the ribosomal RNA gene complex ofP. honshuensis will fall within a distinct monophyletic clade,separate from monophyletic clades of other Perkinsus species,including P. marinus, P. olseni, P. chesapeaki, and P. med-iterraneus.

Table 3. Range of sequence similarities and pairwise distances (uncorrected ‘p’ values) observed among rDNA ITS region sequences of currentlyaccepted Perkinsus spp. (except P. qugwadi) from GenBank, and those of the cultures Mie-4g, Mie-5mg, Mie-13v, and Mie-3g obtained in this study (bold).

Species P. marinus P. chesapeaki P. mediterraneus P. olseni Isolates Mie-4g,Mie-5mg, Mie-13v

(P. olseni)

Isolate Mie-3g(P. honshuensis n. sp.)

P. marinus 498.5% 86.7–88.2% 94.2–95.5% 93.9–95.3% 94.1–95.2% 93.4–95.0%P. chesapeaki 0.118–0.132 495.9% 86.1–87.5% 85.7–87.8% 86.0–87.8% 87.1–88.8%P. mediterraneus 0.045–0.058 0.125–0.139 498.9% 94.4–95.6% 94.4–95.6% 95.4–96.5%P. olseni 0.047–0.061 0.122–0.143 0.044–0.056 498.8% 99.0–100.0% 93.7–95.3%Isolates Mie-4g, Mie-5mg, Mie-13v

(P. olseni)0.048–0.059 0.122–0.140 0.044–0.056 0.000–0.010 499.0% 94.2–94.9%

Isolate Mie-3g (P. honshuensis n. sp.) 0.050–0.066 0.112–0.129 0.035–0.046 0.047–0.063 0.051–0.058 499.2%

The range (up to 100%) of observed within-species sequence similarity is given across the diagonal. Sequence similarity ranges between species andour isolate cultures from Mie clams are given above the diagonal, and ranges for raw distance values are given.

ITS, internal transcribed spacer.

Table 4. Range of sequence similarities and pairwise distances (uncorrected ‘p’ values) observed among LSU rRNA gene sequence fragments ofcurrently accepted Perkinsus spp. (except P. qugwadi or P. mediterraneus) from GenBank, and those of cultures Mie-5mg and Mie-3g obtained in thisstudy (bold).

Species P. marinus P. chesapeaki P. olseni Isolate Mie-5mg(P. olseni)


P. marinus 499.8% 96.0–97.4% 97.0–97.5% 97.0–97.3% 97.5–97.9%P. chesapeaki 0.036–0.040 499.1% 96.7–97.8% 96.6–97.6% 96.8–97.5%P. olseni 0.025–0.030 0.022–0.033 499.3% 99.3–100% 97.5–98.1%Isolate Mie-5mg (P. olseni) 0.027–0.030 0.024–0.034 0.000–0.007 499.4% 97.5–97.9%Isolate Mie-3g (P. honshuensis n. sp.) 0.021–0.025 0.025–0.032 0.019–0.025 0.021–0.025 499.5%

The range (up to 100%) of observed within-species sequence similarity is given across the diagonal. Sequence similarity ranges between species andour isolate cultures from Mie clams are given above the diagonal, and ranges for raw distance values are given.

LSU, large subunit.


Table 5. Range of sequence similarities and pairwise distances (uncorrected ‘P’ values) observed among actin type 1 and actin type 2 gene sequencefragments of currently accepted Perkinsus spp. (except P. qugwadi or P. mediterraneus) from GenBank, and those of the cultures Mie-5mg and Mie-3gobtained in this study (bold).

Species P. marinus P. chesapeaki P. olseni Isolate Mie-5mg(P. olseni)


P. marinus 97.5% 84.4–85.7% 86.8–88.1% NA 86.5–87.1%NA NA NA NA NA

P. chesapeaki 0.143–0.156 98.9% 84.9–85.4% NA 83.3–83.9%NA 491.1% 82.5–83.4% 82.8–83.6% 81.3–83.5%

P. olseni 0.119–0.132 0.146–0.151 100% NA 86.8–87.1%NA 0.166–0.175 499.4% 99.4–100% 84.2–83.9%

Isolate Mie-5mg (P. olseni) NA NA NA NA NANA 0.164–0.172 0.000–0.006 499.7% 84.6–86.0%

Isolate Mie-3g (P. honshuensis n. sp.) 0.129–0.135 0.161–0.167 0.129–0.132 NA 99.5%NA 0.165–0.187 0.141–0.158 0.140–0.154 495.3%

Within each cell, values for actin gene type 1 sequences are listed above those for actin gene type 2 sequences. The range (up to 100%) of observedwithin-species sequence similarity is given across the diagonal. Sequence similarity ranges between species and our isolate cultures from Mie clams aregiven above the diagonal, and ranges for raw distance values are given below. NA, not applicable due to lack of available sequences from that actin genetype (one for Mie-5mg, two for P. marinus).

Fig. 11. Parsimony jackknife tree resulting from analysis of the inter-nal transcribed spacer region sequences of Perkinsus spp., and rooted withPerkinsus qugwadi. Jackknife support values for each clade are givenwhen above 50%, and support for each genus clade is indicated in bold.The Perkinsus olseni and Perkinsus honshuensis n. sp. clades include newsequences determined for this study (bold GenBank accession numbers).Isolate sources for new sequences are listed in Table 1. aGroup 1 identicalsequences include U07697, DQ516703, DQ516704, and DQ516706–DQ516712; bGroup 2 identical sequences include AF369969 andDQ516715; cGroup 3 identical sequences include AF369967,DQ516713, and DQ516714.

Fig. 12. Parsimony jackknife tree resulting from analysis of the largesubunit rRNA gene sequences from Perkinsus spp. with Prorocentrummicans used as the outgroup taxon. Jackknife support values for each cladeare given when above 50% and support for each genus clade is indicated inbold. The Perkinsus olseni and Perkinsus honshuensis n. sp. clades includenew sequences determined for this study (bold GenBank accession num-bers). Isolate sources for new sequences are listed in Table 1.


Reference materials deposited. Viable cryopreserves ofmonoclonal holotype in vitro isolate Mie-3g/H8, and its unclonedhapantotype Mie-3g source strain, are deposited for public distri-bution by the American Type Culture Collection (www.atcc.org)as strains ATCC PRA-177 and ATCC PRA-176, respectively.

Nucleotide sequences for the ITS region and LSU rRNA geneof the ribosomal RNA gene complex, and for actin genes, are de-posited with GenBank (www.ncbi.nlm.nih.gov/Genbank) underthe accession numbers listed in Table 1.

Replicate hapantotype histological slides from the same clam(020430 RphilMie-3) that yielded the deposited holotype and ha-pantotype P. honshuensis in vitro isolate strains, were depositedwith the USDA National Parasite Collection (www.lpsi.barc.usda.gov/bnpcu) as USNPC 97122.

Type host. Manila clam, Venerupis (5 Tapes, 5 Ruditapes)philippinarum (5 semidecussatus).

Type locality. Gokasho Bay, Mie Prefecture, Honshu Island,Japan.

Etymology. The species name references the Japanese archi-pelago island of Honshu, from whose southern waters the clamthat yielded the in vitro parasite holotype strain was collected.

Higher classification. (Adl et al. 2005). Chromalveolata(super-group), Alveolata (first rank), Dinozoa (second rank),Perkinsidae (third rank).

Despite strong specific labeling of numerous 4–12mm, vacuo-lated trophozoites by both genus-specific in situ probes that we

used, neither our histological observations nor our ISH orimmunoassays assays specifically discriminated systemically dis-tributed mono-dispersed, small, eosinophilic, avacuolate, Perkin-sus sp. trophozoites like those described by Maeno et al. (1999). Itis possible that a small Perkinsus sp. cell-type was absent or rarein the limited material that we analyzed, or that such cells wereerroneously identified in the previous analyses of Maeno et al.(1999). Nevertheless, their report of diverse Perkinsus sp. cellmorphotypes in histological materials from individual clams isconsistent with our isolation of multiple parasite species fromdifferent individuals in the same Japanese Manila clam sample,since mixed-parasite infections in some individuals from suchclam populations are probable.

As Perkinsus honshuensis was represented by only one of fourin vitro isolates that we acquired from a single sample of JapaneseManila clams, that crude isolation frequency suggests that P.honshuensis infections are less common than P. olseni infectionsamong such clams. Although that inference is compatible withseveral reports of Perkinsus sp. that consistently show only P.olseni-like sequences in template DNAs extracted from infectedJapanese (Hamaguchi et al. 1998) and Korean (Park et al. 2005)Manila clams and their parasites, accurate delineation of the truegeographic range and prevalence of P. honshuensis infectionsamong Asian and European Manila clam populations, and poten-tial other molluscan hosts, will require a systematic survey of suchpotential host populations.

Perkinsus olseni is hypothesized to have been transferred toEuropean waters (Hine 2001) by possible inadvertent transfers ofinfected Asian V. philippinarum clams that were introduced asaquaculture stocks during the 1970s (Le Borgne 1996). If this in-ference is correct, P. honshuensis may also have been importedto Europe with Asian Manila clams, since current high-volumecommerce involving importation and common Japanese beach-freshening of live Korean Manila clams provides a simple anddirect mechanism for clam parasite transfers between Korean andJapanese waters.

Host-vectored dispersal mechanisms are neither documentednor hypothesized for inferred epidemiological links (Murrell et al.2002) between Perkinsus olseni infecting type-host abalone andother diverse Australian molluscs (Lester, Goggin, and Sewell1990), P. olseni infecting New Zealand Austrovenus stutchburyiclams (Hine and Diggles 2002), and P. olseni infecting Asia-Pacific clams. Alternative parasite dispersal mechanisms may alsoinclude both natural hydrographic and ballast water transfers,among others. Notwithstanding uncertainties about parasite dis-persal mechanisms, the broad native geographic range of P. olseniappears to currently include Pacific waters bathing Japan, easternAsia, New Zealand, and Australia, with a probable modern anthro-pogenic parasite range extension to European Atlantic and Med-iterranean waters.

The in vitro cell cycles and cytological morphologies of all invitro isolates described here are generally similar to those describedfor other Perkinsus spp. in vitro isolates; particularly those fromclams, which typically proliferate in vitro by simultaneous schizo-gony and zoosporulation (Burreson et al. 2005; Casas et al. 2002;Dungan et al. 2002). The rapid enlargement of in vitro-propagatedcells of each of our Japanese Manila clam Perkinsus spp. isolatesupon transfer to ARFTM, and the subsequent wholesale zoos-porulation of ARFTM-enlarged hypnospores upon their return toDME/F12-3 or 30 ppt SASW media, offer a convenient method forproduction of dense zoospore populations for experimental uses.

ACKNOWLEDGMENTS

Esteemed colleagues Dr. Tomoyoshi Yoshinaga and Dr. Ma-sami Hamaguchi generously provided infected Japanese Manila

Fig. 13. Parsimony jackknife tree resulting from analysis of actingenes sequences from Perkinsus spp., with Amphidinium carterae andProrocentrum minimum used as outgroup taxa. Jackknife support valuesfor each clade are given when above 50% and support for each genus cladeis indicated in bold. The Type-2 Perkinsus olseni clade and the two Per-kinsus honshuensis n. sp. clades (Types 1 and 2) include new sequencesdetermined for this study (bold GenBank accession numbers). Isolatesources for new sequences are listed in Table 1.


clams. Karen L. Hudson, Jessica A. Moss, and Rosalee M.Hamilton each made extensive, critical, and competent technicalcontributions to the generation of results reported here, includingDNA sequencing and sequence alignments, in situ immuno- andISH assays, and manipulating in vitro parasite isolates. Experttechnical contributions by Maryland DNR Oyster Disease Re-search Project histotechnician Judson Blazek are gratefully ac-knowledged. This manuscript is VIMS contribution #2755.

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Received: 02/16/06, 05/11/06; accepted: 05/12/06


In vitro propagation of two Perkinsus spp. parasites from Japanese Manila clams Venerupis...

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