PSEUDOKLOSSIA SEMILUNA N . SP. (APICOMPLEXA: AGGREGATIDAE):

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PSEUDOKLOSSIA SEMILUNA N . SP. (APICOMPLEXA: AGGREGATIDAE):

A COCCIDIAN PARASITE OF THE KIDNEY OF BLUE MISSELS, SPECIES OF MYTILUS, FROM BRITISH COLUMBIA, CANADA

DESSER S.S.*, BOWER S.M.** & HONG H.*

S u m m a r y :

Three of 9 1 mussels, taken from Pacific coastal waters in Nanaimo, British Columbia, were infected with a new species of coccidian parasite. Gamogonic and sporogonic development were observed in renal tubular epithelial cells. Mature macrogametocytes were crescent-shaped. Oocysts sporulated within the host. Mature oocysts were spherical, mean 2 3 . 9 μm (range 22-25 μm| with approximately 2 4 ellipsoidal sporocysts (approximately 6 x 3 μm), each of which contained two sporozoites. Ultrastructural features of immature and mature macrogametocytes are described. Although found in all five populations of mussels from various locations in British Columbia, prevalence of infection was usually less than 1 6 % , intensity of infection was usually light (less than 5 0 coccidia per histological section of kidney tissue), and evidence of associated pathology was not observed.

KEY W O R D S : bivalve, coccidia, macrogametocyte, oocyst, sporocyst.

RÉSUMÉ : PSEUDOKLOSSIA SEMILUNA N. SP. (APICOMPLEXA: A G G R E G A T I D A E ) : COCCIDIE RÉNALE D E S MOULES BLEUES, ESPÈCES D E MYTILUS, EN C O L O M B I E BRITANNIQUE ( C A N A D A )

Sur un groupe de 91 moules récollées sur la côte Pacifique, à Nanaimo (Colombie Britannique), trois étaient infectées par une nouvelle espèce de coccidie. La gamogonie et la sporogonie s'effectuent dans les cellules épithéliales des tubules rénaux. Les macrogamétocytes mûrs sont en forme de croissant. Les oocystes sporulent dans l'hôte. Les oocystes mûrs sont sphériques, mesurent 23,9 μm (22-25 μm) de diamètre et ont 24 sporocystes

ellipsoïdaux, d'environ 6 x 3 mm, contenant chacun deux sporozoïtes. Description et interprétation des caractéristiques ultrastructurales des macrogamétocytes mûrs et immatures.

L'infection existe dans les cinq populations de moules prospectées en Colombie britannique, mais la prévalence est habituellement inférieure à 16%. La plupart du temps, l'infection est légère (moins de 50 coccidies par coupe histologique du rein) et n'entraîne pas de lésions notables.

MOTS CLÉS : moule, coccidie, macrogamétocytes, oocystes, sporocystes.

INTRODUCTION

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e w s p e c i e s o f c o c c i d i a n parasites have b e e n d e s c r i b e d from bivalves, and their t a x o n o m y and life c y c l e s are not well understood. Since Leger's ( 1 8 9 7 ) first description o f Hyaklossia pelseneeri from the kidneys o f Donax sp. and Tellina sp. from coastal F r a n c e , almost a d o z e n similar parasites have b e e n d e s c r i b e d , mainly from the kidneys o f E u r o p e a n and North American marine molluscs ( r e v i e w e d by D e s s e r & B o w e r , 1 9 9 7 ) . O n the basis o f multisporocystic o o c y s t s o b s e r v e d in the tissues o f their mol- luscan hosts, s e v e n o f these parasites w e r e designated s p e c i e s o f Pseudoklossia. B e c a u s e the majority o f t h e s e s p e c i e s e x h i b i t e d m e r o g o n i c d e v e l o p m e n t , D e s s e r & B o w e r ( 1 9 9 7 ) transferred four o f t h e m from

* Department OF Zoology, University of Toronto, Toronto, Ontario, Canada M5S 3G5.

** Department of Fisheries and Oceans, Pacific Biological Station, Nanaimo, British Columbia V9R 5K6.

Correspondence: Sherwin S. Desser.

Tel: (416) 978-6956 - Fax: (416) 978-8532.

E-mail <wired@zoo.toronto.edu>.

the family Aggregatidae Labbe, 1 8 9 9 to the family Eimeriidae Minchen, 1903- T h e s e four s p e c i e s w e r e m o v e d to the n e w g e n u s , Margolisiella, w h i c h was e s t a b l i s h e d t o a c c o m m o d a t e a n e w s p e c i e s , M.

kabatai, a parasite in the kidneys o f native littleneck clams, Protothaca staminea, from British Columbia, Canada. T h e two remaining n a m e d s p e c i e s o f renal c o c c i d i a n s , m e r o n t s o f w h i c h w e r e not o b s e r v e d , w e r e retained in the g e n u s Pseudoklossia ( s e e D e s s e r

& B o w e r , 1 9 9 7 ) .

U n i d e n t i f i e d s p e c i e s o f Pseudoklossia h a v e b e e n reported in the kidney cells o f b l u e mussels, Mytilus edulis, from the east coast o f the United States (Farley, 1 9 8 8 ) and from M. edulis and Mytilus galloprovincialis in Galicia, Spain ( B o w e r & Figueras, 1 9 8 9 ; R o b l e d o et al., 1 9 9 4 ) . B o w e r ( 1 9 9 2 ) & B o w e r et al. ( 1 9 9 4 ) briefly described an unidentified c o c c i d i a n parasite with crescent-shaped g a m e t o c y t e s and multisporocystic oocysts in the cytoplasm o f renal epithelial cells o f the blue mussel, Mytilus sp. from British C o l u m b i a . In this study, w e describe and illustrate a n e w species o f Pseu- doklossia from the kidneys o f b l u e mussels b e l o n g i n g to the Mytilus edulis/galloprovincialis/trossulus s p e c i e s c o m p l e x from British Columbia.

Article available athttp://www.parasite-journal.orgorhttp://dx.doi.org/10.1051/parasite/1998051017

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DESSER S.S., BOWER S.M.& HONG H.

MATERIALS AND METHODS

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uring November 1995, the kidneys o f 91 Myti- lus sp. from Pacific coastal waters in Nanaimo, British Columbia, were examined for coccidian parasites. T h e mussels w e r e maintained in 5 0 L fibre- glass tanks supplied with flow-through ambient sea water at 8° C, and w e r e dissected, e x a m i n e d and pro

cessed within two w e e k s o f their collection. Each mussel was s h u c k e d and the kidneys w e r e e x c i s e d . Renal tissue was pressed b e t w e e n a glass slide and coverslip, and e x a m i n e d for parasites with a c o m p o u n d micro

s c o p e . Infected renal tissue w a s fixed in Davidson's solution and p r o c e s s e d for routine histological e x a m i nation. Sections, 5 μm in thickness, w e r e stained with Harris m o d i f i e d h a e m a t o x y l i n and 0 . 5 % a l c o h o l i c eosin. T e n fresh and ten fixed, sporulated oocysts, and ten fresh c r e s c e n t - s h a p e d g a m e t o c y t e s w e r e measured with an ocular micrometer. Various stages o f fresh and fixed parasites w e r e p h o t o g r a p h e d with a Zeiss p h o - t o m i c r o s c o p e e q u i p p e d with differential interference contrast ( D I C ) optics using Kodak T e c h n i c a l Pan film.

For electron m i c r o s c o p y , p i e c e s o f infected kidney w e r e fixed in cacodylate buffered 2 . 5 % glutaraldehyde, p o s t f i x e d in c a c o d y l a t e b u f f e r e d 2 . 0 % o s m i u m tetroxide, dehydrated in ethanol, and infiltrated and e m b e d d e d in Spurr's resin ( D e s s e r et ai, 1 9 8 3 ) . Ultra- thin s e c t i o n s w e r e e x a m i n e d using a Hitachi H 7 0 0 0 transmission electron m i c r o s c o p e .

In order to confirm the lack o f merogonic development in mussels from British Columbia, archived histological sections (stained with Harris modified haematoxylin and 0.5 % a l c o h o l i c e o s i n ) that c o n t a i n e d s e c t i o n s through the kidneys o f 4 7 3 mussels w e r e e x a m i n e d for the p r e s e n c e o f c o c c i d i a . T h e sections w e r e derived from 9 5 mussels that w e r e preserved immediately after c o l l e c t i o n from D e p a r t u r e B a y o n August 1 9 8 5 to O c t o b e r 1 9 8 6 ( 6 8 ) , S o o k e Harbour in O c t o b e r 1982 ( s e v e n ) , B o o k e r L a g o o n (five) and Indian Arm (five) in August 1987, and B e c h e r B a y in August 1 9 8 9 ( t e n ) . T h e remaining 3 7 8 mussels w e r e obtained from Depar

ture B a y in early N o v e m b e r 1 9 8 6 and held in labora

tory tanks supplied with flow through ambient sea water for 21 to 175 days b e f o r e b e i n g preserved.

RESULTS

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imeriorin parasites w e r e found in the kidneys o f three o f the 91 mussels ( 3 - 3 % ) e x a m i n e d . T h e parasites, which consisted mainly o f g a m e tocytes in various stages o f d e v e l o p m e n t and o c c a sionally, unsporulated and sporulated oocysts, w e r e o b s e r v e d in b o t h the renal tubular epithelium and the lumen o f the tubules.

Squash preparations o f fresh infected kidney contained large crescent-shaped gametocytes (Fig- 1), which w e r e readily distinguished from the surrounding host cells.

Developing macrogametocytes w e r e s e e n in the lumen o f infected tubules in histological s e c t i o n s (Fig. 2 ) . G a m e t o c y t e s w e r e spherical to ellipsoidal, d e p e n d i n g o n the plane o f section. T h e largest macrogametocytes w e r e c r e s c e n t - s h a p e d and fresh s p e c i m e n s m e a s u r e d 3 0 . 8 x 2 0 . 8 urn ( 3 0 - 3 2 x 18-26 urn). Microgametes w e r e o b s e r v e d budding from the peripheral cytoplasm o f a spherical m i c r o g a m e t o c y t e (Fig. 3 ) , w h i c h m e a s u r e d about 2 0 urn.

U n s p o a i l a t e d o o c y s t s w e r e spherical in s h a p e and w e r e surrounded b y a characteristic wall o f u n e v e n thickness (Figs. 4 and 5 ) . Striatums w e r e apparent in the t h i c k e n e d portion o f the oocyst wall o f fresh s p e c i m e n s e x a m i n e d by DIC m i c r o s c o p y (Fig. 7 ) . Sporu

lated oocysts contained about 24 closely p a c k e d ellip

soidal sporocysts, which measured about 6 x 3 |im, e a c h containing two sporozoites (Figs. 6 and 7 ) . Fresh s p o rulated oocysts measured 2 3 . 9 μm ( 2 2 - 2 5 μm) w h e r e a s fixed s p e c i m e n s measured 19.7 μm (19-21 μm).

Electron microscopy revealed that y o u n g m a c r o g a m e tocytes w e r e generally spherical to ovoid with a d e n s e , irregular boundary layer. T h e nucleus was large and vesicular with a prominent nucleolus (Fig. 8 ) . T h e cytoplasm o f immature m a c r o g a m e t o c y t e s contained abundant lipid inclusions and amylopectin (Fig. 9 ) . An extensive network o f cisternae o f granular endoplasmic- reticulum (ER) occurred in the peripheral cytoplasm which also contained numerous mitochondria and Golgi apparatus, and s o m e dense-walled spherical b o d i e s . D e e p invaginations were observed in several maturing macrogametocytes, several o f which appeared to b e folded sharply upon themselves (Fig. 10). T h e cytoplasmic components o f mature macrogametocytes differed consi

derably from those o f earlier stages (Figs. 10 and 11). Lipid inclusions and amylopectin w e r e less abundant and the ER cisternae, prevalent in immature gametocytes, w e r e n o longer evident. T h e cytoplasm c o n t a i n e d many vesicular bodies, two types o f which w e r e distinctive, and will b e referred to as T y p e s I and II. T y p e I vesicles w e r e spherical and had a loosely granular matrix often containing a m o r p h o u s d e n s e inclusions (Fig. 1 1 ) . Small, slender projections lined the inner sur

face o f the limiting m e m b r a n e s and e x t e n d e d a short distance into the vesicular matrix. W h e n s e c t i o n e d near their e d g e , the matrix o f T y p e I vesicles a p p e a r e d to b e filled with uniformly arranged d e n s e punctate b o d i e s (Fig. 11). O t h e r vesicles o f similar size and a p p e a r a n c e , but without the slender projections, w e r e presumably earlier stages o f development o f the T y p e I vesicles.

T h e cytoplasm o f mature m a c r o g a m e t o c y t e s c o n t a i n e d l o o s e a g g r e g a t e s o f e l e c t r o n - d e n s e material w h i c h

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PSEUDOKLOSSIA SEMILUNA N. SP. PARASITE OF BLUE MUSSELS

Figures 1-7. - Photomicrographs of the endogenous stages of Psendoklossia semiluna.

Fig. 1 : Differential interference contrast (DIC) photomicrograph of a fresh crescent-shaped macrogametocyte X 1,310. Figs. 2, 3, 5, 6 : His

tological sections of renal tissue containing various stages of development (stained with haematoxylin and eosin). Fig. 2 : Several macrogametocytes in the tubular lumen x 660. Fig. 3 : Microgametocyte with peripherally arranged, budding microgametes (arrow) x 1,330.

Fig.4 : DIC photomicrograph of a fresh unsporulated oocyst. Note the thickened portion (arrow) of the oocyst wallx 1,520. Fig. 5 : Llnspo- rulated oocyst (section) X 1,520. Fig. 6 : Sporulated oocyst with closely-spaced sporocysts (section) X 1,520. Fig. 7 : DIC photomicrograph of a sporulated oocyst. Note striations (arrows) in the thickened portion of the oocyst wall X 1,520.

w e r e s e e n free in t h e c y t o p l a s m and a l s o within vesicles (designated as Type II) (Fig. 11). Larger vesicles o f variable shape containing filamentous material w e r e also apparent.

T h e o o c y s t wall was c o m p o s e d o f t w o distinct layers in the thinner region, with an additional layer inter

p o s e d b e t w e e n them in the t h i c k e n e d region (Fig. 1 2 ) . T h e boundary o f the o o c y s t cytoplasm was lined by an u n e v e n layer o f d e n s e material, and was separated by a narrow s p a c e from the inner s m o o t h surface o f t h e d e n s e o o c y s t wall. T h e interposed, m e m b r a n e - b o u n d e d layer c o n t a i n e d agglomerations o f electron- d e n s e b o d i e s and small vesicular b o d i e s w h i c h lay in direct contact with the s m o o t h inner surface o f the d e n s e o o c y s t wall (Fig. 1 2 ) .

Fifty six o f the 4 7 3 mussels in archived histological sec

tions w e r e infected with Pseudoklossia semiluna. None o f the 1 6 8 4 P. semiluna o b s e r v e d in the 5 6 infected mussels w e r e undergoing m e r o g o n i c d e v e l o p m e n t . T h e p r e v a l e n c e o f infection was highest ( 3 3 % ) in the sample o f 18 mussels collected from Departure B a y o n 27 O c t o b e r 1 9 8 6 . In all other samples, the prevalence

o f infection was 16% o r less. T h e intensity o f infec

tion was also low, with only six o f the 5 6 infected mus

sels having m o r e than 5 0 P. semiluna present in o n e histological section through the kidney tissues. In all cases, the morphology o f the kidney was similar to that o f uninfected mussels ( e x c e p t for the infected epithe

lial cells w h i c h w e r e usually hypertrophied to a c c o m modate the relatively large parasite). Also, there was little to n o e v i d e n c e o f an accumulation o f haemocytes in r e s p o n s e to the infection.

TAXONOMIC SUMMARY

PSEUDOKLOSSIA SEMILUNA n. sp.

Suborder: Eimeriorina Leger, 1 9 1 1 . Family: Aggregatidae Labbe, 1 8 9 9 .

G a m o g o n i c and s p o r o g o n i c d e v e l o p m e n t in renal tissue; m e r o g o n y absent. Mature m a c r o g a m e t o c y t e s usually crescent-shaped, 3 0 . 8 x 2 0 . 8 (30-32 x 18-26) μm.

Fresh sporulated oocysts spherical with u n e v e n wall, 2 2 . 9 μ m ( 2 2 - 2 5 (μm); fixed s p e c i m e n s 19.7 (μm ( 1 9 - 20 μm); with approximately 24 ellipsoidal sporocysts ( 6 x 3 μ m ) , e a c h containing two sporozoites.

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DESSER S.S., BOWER S.M. & HONG H.

Figures 8-11. - Electron micrographs of immature and mature macrogametocytes.

Fig. 8 : Immature gametocyte with a vesicular nucleus containing a prominent nucleolus x 1,600. Fig. 9 : Cytoplasmic components of an immature gametocyte include abundant cisternae of granular endoplasmic reticulum (Er), Golgi apparatus (Go), lipid inclusions (Li), amylopectin (Am), and mitochondria (Mi). Nu-nucleus x 9,450. Fig. 10 : Mature, sharply reflexed macrogametocytes possibly assuming the sphe

rical shape of an oocyst x 1,500. Fig. 11 : The cytoplasm of mature macrogametocytes contains many vesicular bodies. Regularly arranged fine projections line the inner margin of Type I vesicles ( V I ) . The punctate appearance of the projections lining the inner membrane of a Type I vesicle is apparent in a specimen sectioned through its edge ( V I ) . Dense granular inclusions occur within Type II vesicles (V2).

Vesicles (stars) similar to Type I but without the fine projections, and other vesicles (asterisks) containing flocculent material arc also seen.

Note the bilaminar appearance of an invaginated portion of the boundary layer (large arrowhead) x 23,800.

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PSEUDOKLOSSIA SEMILUNA N. SP. PARASITE OF BLUE MUSSELS

Fig. 12. - Transitional zone in oocyst wall from thinner to thickened region. In this region, the oocyst cytoplasm, which is lined by dense material (arrowheads), is retracted from the wall (Ow) and in the interposing, thickened area is a membrane delimited and composed of agglomerations of electron-dense bodies and small vesicular structures of relatively uniform size. This interposed layer lies in contact with the smooth inner surface of the electron-dense wall of oocyst, whose outer surface is irregular in appearance x 15,000

T y p e Host: Mytilus edulis/galloprovincialls/trossulus s p e c i e s c o m p l e x (Mollusca: Bivalvia: Mytilidae).

T y p e Locality: Departure B a y , adjacent to the Pacific Biological Station, Department o f Fisheries and O c e a n s , Nanaimo, British Columbia (geographical quadrilateral co-ordinates o f 4 9 ° N, 123° W ) .

Habitat: epithelium o f renal tubule.

Etymology: the specific n a m e refers to the often cres

cent or half-moon s h a p e o f mature macrogametocytes.

T y p e Material: histological s e c t i o n s containing various stages o f P. semiluma in Mytilus edulis have b e e n sub

mitted to the Canadian Museum o f Nature Parasite Col

lection, Ottawa, Ontario, Canada. Catalogue n u m b e r CMNPA1997-0050.

DISCUSSION

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é g e r & D u b o s c q ( 1 9 1 5 ) established the g e n u s Pseudoklossia to a c c o m m o d a t e the type s p e c i e s P. glomerata, which they observed in the kidney o f t w o s p e c i e s o f marine bivalves o f the g e n u s Tapes.

According to the criteria o f these authors, s p e c i e s o f Pseudoklossia h a v e z e r o to m a n y sporocysts, e a c h containing two sporozoites, d o not exhibit m e r o g o n y in the host in w h i c h s p o r o g o n y occurs, and possibly undergo h e t e r o x e n o u s d e v e l o p m e n t . T h e g e n u s Pseu

doklossia was included in the eimeriorin family Aggre- gatidae Labbé, 1 8 9 9 , several m e m b e r s o f w h i c h have h e t e r o x e n o u s life cycles with m e r o g o n i c d e v e l o p m e n t occurring in o n e host, and g a m o g o n i c and s p o r o g o n i c d e v e l o p m e n t in another.

Although m e r o g o n i c d e v e l o p m e n t was not s e e n in mussels infected with P. semiluna, asexual replication

may o c c u r in a s e c o n d host. It is also possible that oocysts are infective to mussels, and that sporozoites give rise directly to g a m e t o c y t e s in the renal tubular cells.

T h e c r e s c e n t - s h a p e d g a m e t o c y t e s and a s y m m e t r i c configuration o f the oocyst wall o f P. semiluna appear to b e unique features a m o n g the s p e c i e s o f Pseudo

klossia described thus far. T h e sharply reflexed macro

g a m e t o c y t e s , s e e n b y e l e c t r o n m i c r o s c o p y , suggest that in the later stages o f their maturation, the cres

cent-shaped g a m e t o c y t e «round up» and assume a more typical spherical shape. Electron m i c r o s c o p y also provided s o m e information o n the synthesis o f mate

rial for the oocyst, and possibly the sporocyst walls.

T h e cytoplasm o f y o u n g m a c r o g a m e t o c y t e s contained considerable synthetic machinery, including numerous mitochondria, Golgi apparatus, abundant lipid and amylopectin inclusions, and an extensive network o f granular ER. T h e contents o f T y p e s I and II vesicles, s e e n in the cytoplasm o f mature m a c r o g a m e t o c y t e s , a p p e a r e d to contribute to the formation o f the oocyst wall. T h e vesicles w e r e probably analogous to the wall- forming bodies seen in macrogametocytes o f other c o c - cidian s p e c i e s ( H a m m o n d & Long, 1 9 7 3 ) . Unfortuna

tely, the m e c h a n i s m o f o o c y s t and s p o r o c y s t wall formation could not b e elucidated b e c a u s e o f the pau

city o f oocysts in the electron microscopic material and also the p o o r fixation o f the few s p e c i m e n s observed.

A low prevalence (1 % or less) o f Pseudoklossia sp. has b e e n reported in M. edulis and M. galloprovincialis from either sides o f the Atlantic O c e a n (Farley, 1 9 8 8

& R o b l e d o et al., 1994, respectively). T h e mussel host o f P. semiluna in the Pacific is similar to the mussel host o f Pseudoklossia sp. in the Atlantic. O n the Pacific

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DESSER S.S., BOWERS.M. & HONG H.

coast o f Canada the specific identity o f the blue mussels is confused. T h e t w o blue mussel sibling s p e c i e s , M. edulis a n d M. galloprovincialis, have b e e n intro- d u c e d to t h e Pacific coast o f North America a n d a r e morphologically similar to the native s p e c i e s Mytilus trossulus. In Departure B a y ( N a n a i m o ) , w h e r e most o f the mussels for the s p e c i e s description o f P. semiluna w e r e obtained, at least 5 % o f the mussels w e r e found to have a minimum o f o n e allele specific for either M.

edulis or M. galloprovincialis (Heath et al., 1 9 9 5 ) . T h e genetic identity o f e a c h host s p e c i m e n for P. semiluna is n o t k n o w n , a n d it is possible that this parasite o c c u r s in b l u e mussels other than M. trossulus. Thus, P. semiluna is described as infecting mussels o f the Mytilus edulis/galloprovincialis/trossulus s p e c i e s c o m - plex. It would b e o f value to e x a m i n e t h e Pseudo- klossia sp. that o c c u r s in Mytilus spp. in the Atlantic O c e a n t o a s c e r t a i n t h e t a x o n o m i c r e l a t i o n s h i p t o P. semiluna.

C o m p a r e d t o t h o s e s p e c i e s infecting vertebrate hosts, the c o c c i d i a n parasites o f invertebrates are poorly d o c u m e n t e d a n d understood. Elucidation o f the life cycles o f m o n o x e n o u s coccidia o f molluscs will b e dif- ficult, a n d the discovery o f alternate hosts o f hete- roxenous coccidian species o f molluscs especially chal- lenging.

ACKNOWLEDGEMENTS

W

e are grateful to J a n i c e B l a c k b o u r n a n d Gary M e y e r for their technical assistance, to Betty Kim, Kowthar Salim, T o d d Smith and Chongxie Xiao for reviewing the initial draft o f this p a p e r a n d t o the Natural S c i e n c e s a n d Engineering Research Council o f Canada ( R e s e a r c h Grant # 6 9 6 5 ) , the British Columbia Ministry o f the Environment, a n d the B.C. Ministry o f Agriculture and Fisheries, for finan- cial support.

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Reçu le 20 août 1997 Accepté le 21 octobre 1997

22 Mémoire Parasite, 1998, 5, 17-22