ZOOSPORULATION OF A NEW PERKINSUS SPECIES ISOLATED FROM THE GILLS OF THE SOFTSHELL CLAM MYA ARENARIA
MCLAUGHLIN S.M.*, TALL B.D.**, SHAHEEN A***,****, ELSAYED E.E.***,***** & FAISAL M.***
Summary:
A gill-associated Perkinsus sp. isolated from the softshell clam [Mya arenaria) is described as a new species, P. chesapeaki sp. nov.
Examination of the parasite in seawater cultures revealed life cycle stages and zoosporulation processes similar to those described for other species of the genus Perkinsus. Prezoosporangia developed thickened cell walls upon contraction of the cytoplasm and development of a distinctive clear area between the cell wall and the protoplast. Successive bipartition of the protoplast led to the formation of hundred's of zoospores within mature sporangia.
Zoospores were released into seawater through one or more discharge tubes. Ultrastructural studies revealed an oblong zoospore possessing two flagella that arose from a concave side located in the upper third of the zoospore body. The anterior flagellum possessed a unilateral array of hair-like structures. A large anterior vacuole and basolateral nucleus dominated the cytoplasm of the zoospore body. The presence of a rudimentary apical complex including on open-sided conoid, rhoptries, micronemes, and subpellicular microtubules were also discerned.
Differences in zoospore morphology, and sequence analyses of two genes previously reported, support the designation of the gill- associated Perkinsus from the softshell clam as a new species.
KEYWORDS :
Perkinsus spp., softshell clam [Mya arenaria), zoosporulation, zoospores, ultrastructure.Résumé :
ZOOSPORULATION D'UNE NOUVELLE ESPECE DE PERKINSUS ISOLÉE DES BRANCHIES DU MOLLUSQUE MYA ARENARIADescription de Perkinsus chesapeaki n. sp. associée aux branchies du mollusque bivalve Mya arenaria. L'examen du parasite dans les cultures en eau de mer révèle différentes phases du cycle biologique et un processus de zoosporulation semblable à celui déjà décrit chez d'autres espèces de Perkinsus. Les
prézoosporanges acquièrent des parois cellulaires épaissies à la suite de la rétraction du cytoplasme et de la formation d'une zone claire distincte entre paroi cellulaire et protoplasme. Les bipartitions successives du protoplasme aboutissent à la formation de
centaines de zoospores à l'intérieur de sporanges mûrs. Les zoospores sont déchargées dans l'eau de mer à travers un ou plusieurs tubes d'expulsion. L'étude ultrastructurale montre que les zoospores, de forme oblongue, possèdent deux flagelles insérés au niveau du tiers antérieur, sur le côté concave du corps. Le flagelle antérieur présente une zone unilatérale ornée de formations filiformes. Une grande vacuole antérieure et un noyau bosolatéral surplombent le cytoplasme du corps de la zoospore.
Un complexe apical rudimentaire avec conoïde ouvert latéralement, rhoptries, micronèmes et microtubules sous pelliculaires est discernable. Les particularités morphologiques de la zoospore et les analyses séquentielles de deux gènes publiés précédemment conduisent à considérer le Perkinsus associé à Mya arenaria comme espèce nouvelle.
MOTS CLÉS : Perkinsus spp., palourde à coquille lisse (Mya arenaria), zoosporulation, zoospores, ultrastructure.
INTRODUCTION
P
rotozoa o f the g e n u s Perkinsus c a u s e significant m o r t a l i t i e s o f b i v a l v e m o l l u s k s w o r l d w i d e . R e c e n t increases in Perkinsus spp. infections in t h e C h e s a p e a k e B a y h a v e b e e n a s s o c i a t e d w i t h d e c r e a s e d a b u n d a n c e s o f e c o l o g i c a l l y and e c o n o m i -* National Oceanic and Atmospheric Administration, National Ocean Service, Center for Coastal Environmental Health and Biomolecular Research, 904 S. Morris St., Oxford, MD 21654.
** Food and Drag Administration, Microbial Ecology Branch, 200 C St., S.W., Washington, DC 20204.
*** Virginia Institute of Marine Science, School of Marine Science, The College of William and Mary, Gloucester Point, VA 23062.
...» Faculty of Veterinary Medicine at Moshtohor, Benha, Egypt.
***** Faculty of Veterinary Medicine, Cairo University, Giza Egypt.
Correspondence: Mohamed Faisal.
Tel.: 1-804-684-7231 - Fax: 1-804-684-7186 - Email: faisal@vims.edu Disclaimer: Reference to trade names does not imply endorsement by NOAA.
cally important bivalve s p e c i e s , including the eastern oyster Crassostrea virginica (Burreson & Ragone Calvo, 1 9 9 6 ) and the softshell clam Mya arenaria (McLaughlin
& Faisal, in press). O u r previous studies have d e m o n s - trated the p r e s e n c e o f t w o distinct Perkinsus spp. in h e m o l y m p h and gill tissues o f infected softshell clams (McLaughlin & Faisal, 1998a; Kotob etal., 1999a, b ) . T h e softshell clam h e m o l y m p h isolate w a s identified as P. marinus b a s e d u p o n its m o r p h o l o g i c , p h e n o t y p i c a n d g e n e t i c c h a r a c t e r i s t i c s . T h e m o r p h o l o g y a n d s e q u e n c e s o f two g e n e s , small subunit ribosomal RNA and internal transcribed spacer regions, demarcated the gill-associated Perkinsus sp. isolate from other Per- kinsus s p e c i e s (McLaughlin & Faisal, 1998a; K o t o b et al, 1999a, b ) .
Levine ( 1 9 7 8 ) established the class Perkinsea based upon ultrastructural characteristics o f the zoospores o f the type species P. marinus, a deadly pathogen o f C. virginica, first described as Dermocystidium marinum Mackin,
Parasite, 2000, 7, 115-122
Mémoire 115
Article available athttp://www.parasite-journal.orgorhttp://dx.doi.org/10.1051/parasite/2000072115
MCLAUGHLIN S.M., TALL B.D., SHAHEEN A., ELSAYED E.E & FAISAL M.
O w e n , and Collier (Mackin et al, 1 9 5 0 ) . Characteris
tics o f the genus Perkinsus included the lack o f sexual reproduction and the p r e s e n c e o f biflagellated z o o s p o r e s possessing an anterior v a c u o l e , an array o f unilateral hair-like structures along the anterior fla- gellum, and a structure resembling the apical c o m p l e x characteristic o f a p i c o m p l e x a n protists. T h e rudimen
tary apical c o m p l e x o f P. marinus contains an i n c o m plete, or open-sided, c o n o i d instead o f the c o m p l e t e t r u n c a t e d c o n e p r e s e n t in a p i c o m p l e x a n s p e c i e s (Levine, 1 9 7 0 ; Perkins, 1 9 7 6 ) . Recently, h o w e v e r , the inclusion o f the class Perkinsea in the subphylum Api- c o m p l e x a has b e e n refuted b a s e d u p o n molecular evi
d e n c e o f a p h y l o g e n e t i c affinity with dinoflagellates and the p r e s e n c e o f an i n c o m p l e t e c o n o i d structure ( r e v i e w e d b y Siddall et al, 1 9 9 7 ) .
Presently, the g e n u s Perkinsus includes four s p e c i e s , n a m e l y P. marinus o f the eastern oyster (Mackin et al, 1950; Perkins, 1 9 7 6 ) , P. olseni o f the blacklip a b a l o n e Haliotis ruber (Lester & Davis, 1 9 8 1 ) , P. atlanticus o f the carpet shell clam Ruditapes decussatus (Azevedo, 1 9 8 9 ) , and P. qugwadi o f cultured J a p a n e s e scallops Patinopecten yessoensis ( B l a c k b o u r n et al, 1 9 9 8 ) . In t h e s e studies, description o f a n e w Perkinsus s p e c i e s w a s p r e m i s e d u p o n m o r p h o l o g y o f life cycle stages, ultrastructure o f zoospores, host affected, and host res
p o n s e to infection. Zoospore morphology, in particular, has b e e n thought important in differentiating a m o n g Perkinsus s p e c i e s . F o r e x a m p l e , P. atlanticus z o o s p o r e s are t a p e r e d b o t h anteriorly and posteriorly while P. marinus zoospores are rounded anteriorly and t a p e r e d posteriorly (Perkins, 1 9 7 6 , 1 9 9 6 ; A z e v e d o , 1 9 8 9 ) . In addition, z o o s p o r e s o f P. atlanticus are m o r e uniform in dimension and structure than other reported s p e c i e s ( A z e v e d o , 1 9 8 9 ) . Characteristics u n i q u e to P. quqwadi include the presence o f both vegetative and zoosporulation forms in host tissues, lack o f z o o s p o - rulation in seawater, and an inability to form h y p n o - spores in Ray's fluid thioglycollate assays ( B l a c k b o u r n et al, 1 9 9 8 ) . Differences in host s p e c i e s and size o f trophozoites provided the basis for the description o f P. olseni as a n e w s p e c i e s (Lester & Davis, 1 9 8 1 ) . Herein, w e report m o r p h o l o g i c characteristics o f the z o o s p o r e s and z o o s p o r a n g i a o f gill-associated Per
kinsus sp. o f the softshell clam.
Marine Enterprises Inc., Baltimore, M D ) p r e p a r e d at 12 ppt and maintained at 5 ° C .
ZOOSPORULATION IN ARTIFICIAL SEAWATER
P i e c e s o f gill (~ 6 m m ) from e a c h c l a m w e r e e x c i s e d and i n c u b a t e d for six days in tubes containing 6 ml o f Ray's fluid thioglycollate m e d i u m (RFTM, B e c t o n D i c kinson Microbiology Systems, Cockeysville, M D ) sup
p l e m e n t e d with 8 pi nyastatin (Sigma Chemical Co., St. Louis, M O ) as d e s c r i b e d b y Ray ( 1 9 5 2 ) . A small p i e c e o f incubated gill w a s m a c e r a t e d o n glass slides and stained with Lugol's iodine solution. H y p n o s p o r e s staining a b l u e - b l a c k c o l o r p a t h o g n o m o n i c to Per
kinsus sp. w e r e enumerated. T h e remaining gill tissues o f infected clams w e r e m a s h e d against the walls o f the tubes using cell scrapers o r sterile inoculating loops.
Following thorough maceration, 1 ml o f the m e d i u m was transferred to 25 c m2 flasks (Corning Glass W o r k s , Corning, N Y ) containing 5 ml J L - O D R P growth media p r e p a r e d as d e s c r i b e d b y LaPeyre & Faisal ( 1 9 9 6 ) using sterile t e c h n i q u e s . Cultures w e r e further incu
b a t e d for 24 h at 2 7 ° C, and transferred to artificial ste
rile s e a w a t e r ( 2 2 g / L ) in 25 c m2 tissue culture flasks.
Seawater cultures w e r e maintained at r o o m tempera
ture a n d the zoosporulation p r o c e s s w a s f o l l o w e d daily using an inverted m i c r o s c o p e .
ELECTRON MICROSCOPY
Zoosporulation preparations w e r e fixed in 2.5 % glu- taraldehyde, 0.1 M sodium cacodylate (pH 7 . 2 ) at r o o m temperature, post-fixed in 1 % osmium tetraoxide, and e m b e d d e d in E p o n a t e 8 1 2 . Ultra-thin sections w e r e stained with uranyl acetate and the sections e x a m i n e d with an e l e c t r o n m i c r o s c o p e .
Semi-thin sections w e r e stained with toluidine blue ( S i g m a ) and e x a m i n e d b y light m i c r o s c o p y . D i m e n sions o f the z o o s p o r e b o d y and flagella w e r e taken from the stained semi-thin sections.
Despite the disputed phyla classification (Siddall et al, 1 9 9 7 ) , terminology and classification criteria utilized in this report parallel previously published descriptions o f Perkinsus s p e c i e s for purposes o f simplicity and consistency.
MATERIALS AND METHODS
CLAMS
F
ifty softshell clams w e r e c o l l e c t e d b y hydraulic escalator dredge o n S e p t e m b e r 7, 1 9 9 8 , from Swan Point in the Chester River, Maryland. T h e c l a m s w e r e a l l o w e d to p u r g e for 2 4 h in a 10-L container o f aerated, artificial seawater (Forty Fathoms,RESULTS
E
xamination o f seawater cultures revealed an active zoosporulation process. Large prezoosporangia d e v e l o p e d 24 hrs post-incubation and ranged in size from 2 0 to 135 p m ( m e a n = 69.3 u m ± 29.3) (Fig. 1 ) . Cell walls o f the p r e z o o s p o r a n g i a t h i c k e n e d u p o n contraction o f the cytoplasm with the formation o f a clear area b e t w e e n the cell wall and the proto-116
Mémoire
Parasite, 2000, 7, 115-122ZOOSPORULATION OF PERKINSUS CHESAPEAKI SP. NOV.
Figs 1-4. - Zoospoailation of Perkinsus chesapeaki sp. nov. 1: Prezoosporangia (Pz) and mature zoosporangia (Mz) containing several zoo
spores. 2-4: Discharge tube outgrowth formation (Dt) through which zoospores are released. (Semithin sections stained with toluidine blue.)
plast. Intermediate stages took place with s u c c e s s i v e bipartitions o f the protoplast. Cells within p r e z o o s p o rangia c h a n g e d from spheroidal to smaller rod-like forms. Mature zoosporangia which contained numerous b i f l a g e l l a t e d z o o s p o r e s w i t h p r o m i n e n t a n t e r i o r v a c u o l e s w e r e s e e n as early as 24 hours post-incuba
tion. Tubular outgrowths (Figs. 2 - 4 ) ranging from 2 . 5 - 20 um in length w e r e apparent 4 8 h post-incubation.
T h e beginning o f zoosporulation w a s r e c o g n i z e d b y the active m o v e m e n t o f z o o s p o r e s within the z o o - sporangia. Z o o s p o r e s s w a m out o f the z o o s p o r a n - gium through a discharge tube in an oscillatory m o v e m e n t , l e a v i n g b e h i n d e m p t y s p o r a n g i a ( F i g . 4 ) . O c c a s i o n a l l y , m o r e t h a n o n e d i s c h a r g e t u b e w a s o b s e r v e d in a single zoosporangium. T h e release o f z o o s p o r e s from different zoosporangia continued for 7-8 days. T h e z o o s p o r a n g i a s h o w e d n o synchroniza
tion in their d e v e l o p m e n t .
Mature zoosporangia had t h i c k e n e d cell walls c o m p o s e d o f distinctive inner and outer layers (Fig. 5 ) . L a m o s o m e s w e r e o b s e r v e d along the periphery o f the z o o s p o r a n g i u m cell wall (Fig. 5 ) . Lipid-like droplets w e r e also e m b e d d e d in the inner cell wall (Fig. 5 ) . T h e b o d i e s o f free zoospores w e r e usually o b l o n g with r o u n d e d anterior and posterior regions. T h e posterior region w a s narrower than the anterior in s o m e z o o -
P a r a s i t e , 2 0 0 0 , 7, 1 1 5 - 1 2 2
spores (Fig. 6 ) . Z o o s p o r e b o d y length ranged from 2.2 to 4.7 um ( m e a n = 3.73 ± 0 . 4 8 ) and the width from 1.4 to 2.5 pm ( m e a n = 2.41 ± 0 . 3 6 ) . Z o o s p o r e s w e r e bifla
gellated (Fig. 6 ) with o n e flagellum longer than the other (5.51 pm ± 1.19 x 3.0 um ± 0 . 6 5 ) . T h e flagella
Fig. 5. - Ultrastructural details of the peripheral zone of a zoospo
rangium showing the bilayered wall (outer wall = Ow; inner wall = Iw), a lamosome (Ls); and lipid-like bodies (Li).
Mémoire 117
M C L A U G H L I N S . M . , T A L L B . D . , S H A H E E N A . , E L S A Y E D E . E . & F A I S A L M.
Fig. 6. - Longitudinal section of a free zoospore of Perkinsus che- sapeaki sp. nor. showing nucleus (Nu), mitochondria (Mi), and vacuole (Va), flagella (F), and a transverse section of the axenome (FTSA). Note the unilateral array of hair-like structures on the ante
rior flagellum (FH).
originated from the upper third o f the b o d y and arose from a region that a p p e a r e d as a c o n c a v e impression.
T h e anterior flagellum possessed an array o f unilateral hair-like structures measuring 2-4 run in diameter (Figs. 6 and 9 ) . T h e basal b o d i e s o f the two flagella w e r e oriented at an acute angle and an electron-dense body was observed within the lumen of the kinetosomes (Fig. 7 ) . T h e longitudinal bundle o f microtubles forming the flagellar a x e n o m e was organized into an arrange
ment o f doublets with a nine plus two array (Fig. 8 ) . A large anterior vacuole was apparent in zoospores (Figs.
6, 10, 12, and 13). Cortical alveoli w e r e located exter
nally o n the anterior surface o f the zoospore (Fig. 10).
Zoospores had a single nucleus located laterally in the posterior portion o f the cells. T h e nucleus was large, o c c u p i e d nearly one-third o f the z o o s p o r e body, and possessed an uneven condensation o f chromatin (Figs. 6 and 10). Mitochondria either appeared as a longitudinal organelle (Fig. 6 ) or as multiple round to oval b o d i e s (Fig. 11). Lipid-like droplets w e r e occasionally observed posteriorly in the cytoplasm (Fig. 1 0 ) .
Longitudinal and transverse sections revealed structures resembling the apical c o m p l e x described by Perkins ( 1 9 7 6 ) including a c o n o i d , conoid-associated micro- n e m e s , r e c t i l i n e a r m i c r o n e m e s ( c a l l e d t h e a p i c a l ribbon, ribbon-associated vesicles, and toxicysts b y Siddall et al, 1 9 9 7 ) , rhoptries, and subpellicular micro
tubles (Figs. 10-13). T h e c o n o i d w a s open-sided and located anteriorly at the apical pole (Fig. 10). T h e rhop-
Figs 7-9. - L'ltrastructural details of the flagella in a free zoospore of Perkinsus chesapeaki sp. nov. 7: Kinetosome of both flagella (arrows) showing a cylindrical inclusion (I) and a cup-like structure (C). 8: Transverse section of axoneme (TSA) showing typical micro- tubular arrangement (9 + 2 array). 9: Hair-like structures (FH) located unilaterally along the anterior flagellum.
tries w e r e electron d e n s e and often vase-shaped with the narrower n e c k portion extending into the c o n o i d region (Fig. 1 0 ) .
Rectilinear m i c r o n e m e s e x t e n d e d vertically from the c o n o i d to the posterior region o f the cell in a straight line and e n d e d in a small bulb (Fig. 1 2 ) . T h e m o r e convoluted and curvilinear c o n o i d - a s s o c i a t e d micro
n e m e s e x t e n d e d posteriorly from the c o n o i d , w r a p p e d a r o u n d the n u c l e u s , and then e x t e n d e d anteriorly (Figs. 12 and 1 3 ) . Subpellicular microtubules w e r e also o b s e r v e d (Fig. 1 1 ) .
118 Mémoire Parasite, 2000, 7, 115-122
ZOOSPORULATION OF PERKINSUS CHESAPEAKI SP. NOV.
Fig. 10. - Longitudinal section of a mature zoospore showing vacuole (Va), alveoli (Alv), nucleus (Nu), mitochondrion (Mi), lipid- like bodies (Li), conoid (Co), and rhoptries (Rh).
Fig. 11. - Transmission electron micrograph of a free zoospore sho
wing subpellicular microtubules (arrows), several oval or round mito
chondria (Mi), and the kinetosome of a flagellum (F).
Figs 12-13. - Longitudinal sections ot tree zoospores showing micro- nemes associated with conoid. 12: Rectilinear microneme (Rm) extending from the conoid to the nucleus (Nu) and terminating in bulb-like structure (arrow). Note the longitudinal and transverse sec
tions of the flagella (F). 13: Conoid-associated micronemes (Cm) extending posteriorly from the conoid, curving around the region of the nucleus (Nu), and then extending anteriorly.
Perkins US chesapeaki sp. nov.
Description
Uninucleated, transparent trophozoites ellipsoid to sphe
rical, containing an eccentric nucleus with a large, single nucleolus and e c c e n t r i c v a c u o l e . M o r p h o l o g y o f tro
p h o z o i t e s in vitro and within host tissues are detailed in McLaughlin & Faisal (1998a, b ) . Prezoosporangia cul
tured in RFTM and JL-ODRP media u n d e r g o s u c c e s sive binary fissions and p r o d u c e n u m e r o u s z o o s p o r e s . Z o o s p o r e s are u n i n u c l e a t e d , biflagellated, slightly o b l o n g (3.7 pm ± 0 . 4 8 x 2.41 pm ± 0 . 3 6 ) and p o s s e s s a large l a t e r o b a s a l n u c l e u s a n d a single, a n t e r i o r vacuole. Flagella are unequal in length (5.51 pm ± 1.19 and 3-0 pm ± 0 . 6 5 ) and arise from a c o n c a v e side located in the u p p e r third o f the z o o s p o r e body.
T a x o n o m i c summary
Type host: T r o p h o z o i t e s in softshell clam Mya are- naria (Mollusca, Bivalvia).
Type Locality: C h e s a p e a k e Bay, Maryland.
Prevalence: S e a s o n a l with p e a k prevalences occurring in late s u m m e r and fall.
Site of infection: Gills appear to b e the target tissue although a b s c e s s e s o c c u r in other organs in a d v a n c e d infections.
Etymology: Chesapeaki derived from C h e s a p e a k e B a y Specimens deposited: Stained slides o f infected softshell clam gills w e r e deposited in the Registry o f Tumors in L o w e r A n i m a l s , G e o r g e W a s h i n g t o n U n i v e r s i t y , W a s h i n g t o n DC ( A c c e s s i o n n u m b e r 7 1 2 1 ) . C l o n e d c u l t u r e s w e r e d e p o s i t e d in t h e l a b o r a t o r y o f Dr.
Parasite, 2 0 0 0 , 7, 1 1 5 - 1 2 2
M emoire 119
M C L A U G H L I N S . M . , T A L L B.D., S H A H E E N A . , E L S A Y E D E . E . & F A I S A L M .
M o h a m e d Faisal, Virginia Institute o f Marine S c i e n c e (VIMS), G l o u c e s t e r Point, Virginia. T h e SSU rRNA and ITS-5.8S g e n e sequences are accessible from G e n B a n k at retrieve@ncbi.nlm.nih.gov (Accession numbers AF042707 and A F 0 9 1 5 4 1 , respectively).
DISCUSSION
T he p r o c e s s e s o f prezoosporulation and z o o s p o - rulation o f Perkinsus chesapeaki sp. nov. in s e a - water w e r e identical to those reported during in vitro p r o p a g a t i o n o f the parasite in culture m e d i a (McLaughlin & Faisal, 1 9 9 8 a ) . Further, prezoosporula- tion and zoosporulation p r o c e s s e s o f P. chesapeaki sp.
nov. in s e a w a t e r w e r e similar to t h o s e reported for P. marinus and P. atlanticus (Perkins & Menzel, 1967;
A z e v e d o et al., 1 9 9 0 ; A u z o u x - B o r d e n a v e et al., 1 9 9 5 ; Ordas & Figueras, 1 9 9 8 ) . T h e life cycle o f P. chesa
peaki sp. nov. is characterized b y s c h i z o g e n y o f v e g e tative forms with multiple divisions o f mother cells into several daughter cells and successive bipartition o f the cytoplasm o f p r e z o o s p o r a n g i a into hundred's o f z o o spores. T h e softshell clam Perkinsus s p e c i e s appears to b e u n i q u e in its ability to u n d e r g o both s c h i z o g e n y and active zoosporulation in culture media (McLaughlin
& Faisal, 1 9 9 8 a ) .
Examination o f the ultrastructure o f P. chesapeaki sp.
nov. in this study s h o w e d the zoospores to possess cha
racteristics typical o f other m e m b e r s o f the genus Per
kinsus. T h e zoospores are biflagellated with the ante
rior flagellum possessing unilateral hair-like extensions o f the flagellar m e m b r a n e . T h e p r e s e n c e o f an open- sided conoid-like structure and associated vesicles fur
ther support the classification o f the softshell clam para
site in the genus Perkinsus. Z o o s p o r e s o f P. marinus, P. atlanticus, and P. quqwadi possess similar morpho
logical features (Perkins 1976; Azevedo, 1989; Azevedo et al, 1990; B l a c k b o u r n et al, 1 9 9 8 ) . Detailed mor
phology o f P. olseni z o o s p o r e s has not b e e n reported;
however, zoosporulation studies produced biflagellated zoospores (Lester & Davis, 1 9 8 1 ) .
Differences observed in the ultrastructure o f P. chesapeaki sp. nov. zoospores compared with other described spe
cies include the larger size o f the nucleus, the presence o f a single, large anterior vacuole, and multiple mito
chondria o f varying shapes. Moreover, the zoospore body o f P. chesapeaki sp. nov. (3.7 pm ± 0.48 x 2.41 pm
± 0 . 3 6 ) differs in his dimension from P. marinus (4-6 x 2-3 p m ) , P. atlanticus (4.5 p m ± 0.6 x 2.9 pm ± 0.4 pm), and P. quqwadi ( 3 . 8 6 pm ± 0.31 x 2.47 pm ± 0.2 pm).
Interestingly, the dimensions o f the z o o s p o r e b o d y o f P. chesapeaki sp. nov. b e a r s o m e r e s e m b l a n c e to those o f a Perkinsus sp. reported in the Baltic clam Macoma balthica (Andrews, 1 9 5 4 ; Perkins, 1 9 6 8 ; Kleinschuster
et al, 1 9 9 4 ) . In P. chesapeaki sp. nov., the z o o s p o r e b o d y ranges in length from 2.2 to 4.7 p m and in width from 1.4 to 2.5 p m while the Baltic c l a m Perkinsus sp.
z o o s p o r e s m e a s u r e from 3 to 5 and 2 to 3 pm, res
pectively (Perkins, 1 9 6 8 ) . Similarly, the z o o s p o r e s o f the Perkinsus sp. o f Baltic clams and P. chesapeaki sp.
nov. o f softshell clams share a slightly o b l o n g mor
phology. O n the contrary, P. marinus z o o s p o r e s are r o u n d e d anteriorly and tapered posteriorly while z o o spores o f P. atlanticus are m o r e irregularly ellipsoidal and tapered at b o t h ends. Further, the z o o s p o r e s o f P. quqwadi are typically ovoid and have a tapered pos
terior.
Flagella o f P. chesapeaki z o o s p o r e s are inserted at an acute angle to o n e another and are located in the ante
rior third o f the z o o s p o r e s , a position similar to that o b s e r v e d in P. marinus (Perkins, 1969) and P. atlan
ticus (Azevedo, 1 9 8 9 ) . O n the o t h e r hand, flagellar b a s e s o f P. quqwadi are parallel in s o m e z o o s p o r e s and at right angles in others ( B l a c k b o u r n et al, 1 9 9 8 ) . B o t h flagella o f P. chesapeaki sp. nov. ( 5 . 5 1 p m ± 1.19 and 3-0 pm ± 0 . 6 5 ) appear to b e shorter than those des
cribed in other Perkinsus s p e c i e s including the M. bal
thica isolate (Perkins, 1968, 1969; Azevedo, 1989; Black
b o u r n et al, 1 9 9 8 ) b a s e d u p o n stained thin sections.
Anterior flagella m e a s u r e 1 0 - 1 8 pm in P. marinus, 12.7 ± 2.4 p m in P. atlanticus, 9-01 ± 1 . 2 pm in P. quq
wadi, and 13-17 pm in Perkinsus sp. o f M. balthica.
Posterior flagella measure 6-10 pm, 10.7 ± 3-2 pm, 7.95 ± 1.2 pm, and 7-9 pm, respectively. Electron- d e n s e bodies in the lumen o f the k i n e t o s o m e o f P. che
sapeaki sp. nov. w e r e observed in P. marinus (Perkins, 1969; 1 9 8 8 ; 1 9 9 6 ) and P. atlanticus (Azevedo, 1 9 8 9 ) but w e r e missing in P. quqwadi ( B l a c k b o u r n et al, 1 9 9 8 ) . Multiple round to oval mitochondria and single bar-shaped mitochondria w e r e o b s e r v e d with similar frequency in P. chesapeaki sp. nov. and occurred m o r e frequently than other d e s c r i b e d s p e c i e s . Further, most mitochondria o f P. marinus o c c u r r e d singly and w e r e often bar-shaped (Perkins, 1996) while P. quqwadi mitochondria w e r e lobulated ( B l a c k b o u r n et al, 1 9 9 8 ) . T h e cortical alveoli is l o c a t e d anteriorly in P. chesa
peaki sp. nov., P. marinus, and P. atlanticus while the entire surface o f P. quqwadi is surrounded by alveoli (Perkins, 1976; Azevedo, 1989; Blackbourn et al, 1998).
A n e w s p e c i e s o f Perkinsus isolated from softshell clams is p r o p o s e d b a s e d u p o n characteristics o f tro
phozoite m o r p h o l o g y , ultrastructure o f organelles in z o o s p o r e s , s h a p e and size o f z o o s p o r e cell body, and length o f flagella. This proposal is supported by mole
cular evidence ( K o t o b et al, 1999a, b ) . Further, the abi
lity o f P. quqwadi to develop at temperatures b e l o w 20° C ( B o w e r et al, 1998; B l a c k b o u r n et al, 1 9 9 8 ) was similarly o b s e r v e d in t h e softshell c l a m isolate b y McLaughlin & Faisal (in press). Prevalences o f Perkin-
1 2 0 Memoire Parasite, 2000, 7, 115-122
ZOOSPORULATION OF PERKINSUS CHESAPEAKI SP. NOV.
sus spp. infections in softshell clams collected from l o w salinity sites indicate the softshell clam isolate is also able to d e v e l o p at salinities b e l o w 12-15 ppt (McLaughlin &
Faisal, in press). Virus-like particles reported in the n u c l e u s a n d cy to plas ms o f P. marinus c o l l e c t e d from Virginia (Perkins, 1 9 6 9 ) w e r e lacking in P. chesapeaki sp. nov. In addition, P. marinus infections in oysters b e c o m e rapidly systemic (Mackin, 1 9 5 1 ) w h i l e t h e spread o f infections in clams by P. atlanticus and P. cbe
sapeaki sp. nov. a p p e a r to b e delayed b y initial e n c a p sulating r e s p o n s e s o f the hosts (Chagot et al., 1987;
McLaughlin & Faisal, 1 9 9 8 b ) .
A C K N O W L E D G E M E N T S
T
h e authors gratefully a c k n o w l e d g e Earl J . Lewis ( N O S , O x f o r d ) for his instruction in g r a p h i c s software a p p l i c a t i o n s . T h e r e s e a r c h w a s s u p p o r t e d b y a grant from t h e National O c e a n i c a n d A t m o s p h e r i c A d m i n i s t r a t i o n ( N O A A ) , Virginia S e a Grant C o l l e g e Program a n d t h e U.S.-Spain J o i n t C o m m i s s i o n o n Scientific a n d T e c h n o l o g i c a l C o o p e r a tion, Madrid, Spain. T h e v i e w s e x p r e s s e d h e r e i n a r e t h o s e o f t h e a u t h o r s a n d d o n o t n e c e s s a r i l y reflect t h e v i e w o f NOAA o r a n y o f its s u b - a g e n c i e s . Virg i n i a I n s t i t u t e o f M a r i n e S c i e n c e c o n t r i b u t i o n n u m b e r 2 2 9 7 .
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