U L T R A S T R U C T U R A L FEATURES OF T H E GAMETOGENIC A N D SPOROGONIC DEVELOPMENT OF HEPATOZOON SIPEDON (APICOMPLEXA: ADELEORINA)
The applicability of ultrastructural data in differentiating among Hepatozoon species
SMITH T.G.* & DESSER S.S.*
Summary :
Stages of gametogony and sporogony of the haemogregarine Hepatozoon sipedon, an apicomplexan parasite of the Northern water snake, Nerodia sipedon sipedon, were studied in the mosquito, Culex pipiens, by electron microscopy. Four days after mosquitoes fed on an infected snake, microgamonts and macrogamonts were observed in syzygy in parasitophorous vacuoles within fat body cells of the haemocoel. During
microgametogenesis, two biflagellated microgametes wete formed, one of which fertilized the macrogamete. After fertilization, zygotes increased rapidly in size, accumulating reserve material in the form of lipid inclusions. Beginning at 16 days post-feeding (PF), the nucleus of the immature oocyst underwent multiple divisions in the first stage of sporoblast formation. Small crystalloid bodies initially appeared in the cytoplasm of the dividing oocyst at 2 0 days PF.
At 2 4 days PF, the oocyst contained hundreds of sporoblasts, each of which matured as early as 2 8 days PF into thick-walled sporocysts containing eight sporozoites and a large residual body.
Sporozoites contained a large crystalloid body comprised of tightly-packed particles assembled in a paracrystalline array. The use of ultrastructural characters in the differentiation of Hepatozoon species is discussed in context with the current phylogenetic hypotheses of adeleorin taxa.
KEY WORDS : Culex pipiens, gametogony, haemogregarine, Hepatozoon, mosquito, sporogony, ultrastructure.
Résumé: CARACTÉRISTIQUES ULTRASTRUCTURALES DU DÉVELOPPEMENT GAMÉTOGÉNIQUE ET SPOROGONIQUE D'HEPATOZOON SIPEDON (APICOMPLEXA: ADELEORINA) : Applicabilité des caractéristiques ultrastructurales pour la séparation des espèces d'Hepatozoon Les stades de la gamétogonie et de la sporogonie de
l'hémogrégarine Hepatozoon sipedon, un parasite du phylum Apicomplexa qui infeste la Couleuvre d'eau (Nerodia sipedon sipedon), ont été étudiés dans le moustique Culex pipiens à l'aide de la microscopie électronique. Quatre jours après que le moustique se soit nourri sur une couleuvre, un microgamonte et un macrogamonte se sont associés (syzygyie) dans une vacuole parasitophore à l'intérieur de l'hémocoèle du moustique. La microgamétogénèse a produit deux microgamètes bi-flagellés, dont un a fertilisé la macrogamète. Après la fertilisation, les zygotes ont grandi rapidement et amassé d'importantes réserves sous forme d'inclusions lipidiques. Après le 16e jour, le noyau du jeune oocyste a subi des divisions multiples au cours du premier stade de la formation des sporoblastes. Les cristalloïdes apparaissent dans le cytoplasme de l'oocyste après le 20e jour. Le 24e jour, l'oocyste contient de nombreux sporoblastes, qui murissent tous après le 28e jour dans des sporocystes qui contiennent huit sporozoites et un grand corps résiduel. Les sporozoites contiennent un grand cristalloïde composé de petites particules en
arrangement paracristallin. L'usage des caractéristiques ultrastructurales pour la séparation des espèces d'Hepatozoon est présenté dans le contexte des hypothèses sur les phytogénies courantes des genres du sous-ordre Adeleorina.
MOTS CLÉS : Culex pipiens, gamétogonie, hémogrégarine, Hepatozoon, moustique, sporogonie, ultrastructure.
INTRODUCTION
S
p e c i e s o f the g e n u s Hepatozoon are h a e m o g r e - g a r i n e parasites c h a r a c t e r i z e d b y g a m e t o g e 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 , with the formation o f large multisporocystic o o c y s t s , in an a r t h r o p o d defi- nitive host, a n d m e r o g o n i c a n d g a m o n t o g o n i c d e v e - l o p m e n t in the internal organs a n d b l o o d cells o f a ver- t e b r a t e intermediate h o s t after an infected a r t h r o p o d is i n g e s t e d (Levine, 1 9 8 8 ) . O v e r 3 0 0 s p e c i e s o f Hepato-*Department of Zoology, University of Toronto, Toronto, Ontario, Canada M5S 3G5.
Correspondence: Sherwin S. Desser. Tel: (416) 978-6956 - Fax : (416) 978-8532 - E-mail <wired@zoo.toronto.edu>.
zoon h a v e b e e n r e p o r t e d from all g r o u p s o f t e t r a p o d v e r t e b r a t e s , a l t h o u g h o v e r 9 0 % o f t h e s e h a v e b e e n d e s c r i b e d s o l e l y o n the b a s i s o f b l o o d s t r e a m g a m o n t s (Smith, 1 9 9 6 ) . T h e m o r p h o l o g i c a l features o f intraery- throcytic a n d i n t r a l e u c o c y t i c stages are insufficient to s e p a r a t e m e m b e r s o f this g e n u s from t h o s e o f o t h e r g e n e r a o f h a e m o g r e g a r i n e s infecting terrestrial verte- brates, n a m e l y Haemogregarina, Hemolivia & Karyo- lysus ( M o h a m m e d & Mansour, 1 9 5 9 ; Ball et al., 1 9 6 7 ) , let a l o n e to c o n f e r specific status.
T h e m o s t useful criteria for the g e n e r i c p l a c e m e n t o f h a e m o g r e g a r i n e s and the differentiation o f Hepatozoon s p e c i e s are features o f g a m e t o g o n y a n d s p o r o g o n y in the invertebrate h o s t (Ball, 1 9 6 7 ) , w h i c h for m e m b e r s o f this g e n u s m a y b e e i t h e r a tick, mite, l o u s e , dip- teran, o r flea (Telford, 1 9 8 4 ) . However, with the e x c e p -
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141Article available athttp://www.parasite-journal.orgorhttp://dx.doi.org/10.1051/parasite/1997042141
SMITH T.G. & DESSER S.S.
tion o f a few ultrastructural studies (Vivier et al., 1 9 7 2 ; G ö b e l & Krampitz, 1 9 8 2 ; Bashtar et al, 1 9 8 4 b ; Lowi- chik et al., 1 9 9 3 ; D e s s e r et ai, 1 9 9 5 ) , data for sexual d e v e l o p m e n t are often i n c o m p l e t e o r restricted to stages o b s e r v e d by light m i c r o s c o p y . In this study, w e d e s c r i b e the ultrastructure o f the g a m e t o g e n i c and s p o r o g o n i c stages o f Hepatozoon sipedon, a s p e c i e s infecting Northern water s n a k e s in Culex pipiens, a potentially natural definitive host (Smith et al., 1 9 9 4 ) .
MATERIALS AND METHODS
COLLECTION AND MAINTENANCE OF EXPERIMENTAL ANIMALS
N
o r t h e r n w a t e r s n a k e s (Nerodia sipedon sipedon) w e r e c o l l e c t e d from marshes near the Q u e e n ' s University Biological Station ( 4 4 ° 3 5 ' N ; 7 6 ° 1 5 ' W ) , north o f Kingston, Ontario, Canada in May, 1 9 9 2 . Smears m a d e o f b l o o d c o l l e c t e d from the caudal vein w e r e fixed and stained with Diff- Q u i k ® . and e x a m i n e d for h a e m o g r e g a r i n e s . S n a k e s w e r e h o u s e d in m e s h - c o v e r e d terraria and maintained on a diet o f c h o p p e d w o r m s and fish.Larvae o f the m o s q u i t o Culex pipiens, o b t a i n e d from Carolina Biological Supply C o m p a n y , w e r e fed a mix
ture o f TetraMin® fishfood and yeast, and maintained in dechlorinated tap water at 25 °C in shallow plastic- tubs. P u p a e in small jars o f water w e r e transferred to s c r e e n e d plastic b o x e s . Adults w e r e provided with distilled water and a 10 % sucrose solution, and main
tained at 25 °C at 7 5 - 8 0 % humidity o n a 1 4 : 1 0 hour day/night cycle.
EXAMINATION OF DEVELOPMENTAL STAGES
Mosquitoes deprived o f water and sucrose for 12 and 3 6 hours, respectively, w e r e allowed to feed o n an unrestrained infected water s n a k e in a glass feeding c a g e in the dark at 25 ° C Engorged m o s q u i t o e s w e r e maintained as described a b o v e .
Wet mounts o f the abdominal contents o f C. pipiens w e r e e x a m i n e d at 2 8 days PF. Single oocysts w e r e rup
tured by applying gentle pressure on an overlying c o v e r slip in order to o b s e r v e and count sporocysts.
A b d o m e n s o f blood-fed C. pipiens w e r e dissected at 1, 4 - 1 4 , 16, 18, 2 0 , 22, 24 and 2 8 days PF and fixed in 2.5 % ( v / v ) glutaraldehyde in 0 , 0 9 M S ö r e n s e n ' s p h o s p h a t e buffer ( B o z z o l a & Russell, 1 9 9 2 ) , pH 7 . 1 , for 7 2 h o u r s . S p e c i m e n s w e r e p o s t - f i x e d in 4 % osmium tetroxide in 0.13 M S ô r e n s e n ' s buffer contai
ning 0.8 % ( w / v ) potassium ferrocyanide and 0.15 M s u c r o s e , d e h y d r a t e d through an a s c e n d i n g g r a d e d ethanol series, and infiltrated and e m b e d d e d in Spurr's resin (Spurr, 1 9 6 9 ) . Semithin sections (0.5 μm), cut
using a R e i c h e r t Ultracut-E u l t r a m i c r o t o m e , w e r e stained with 1.0 % toluidine blue and post-stained with 1.0 % b a s i c fuchsin. Ultrathin s e c t i o n s w e r e stained using 2.0 % ( w / v ) uranyl acetate in 5 0 % m e t h a n o l , post-stained in Reynolds' lead citrate (Reynolds, 1 9 6 3 ) , and e x a m i n e d with a Hitachi H 7 0 0 0 e l e c t r o n micro
s c o p e .
All photomicrographs, including those o f w e t mounts and semithin sections, w e r e taken with a Zeiss Uni
versal 1 p h o t o m i c r o s c o p e using K o d a k T-MAX 100 film.
RESULTS
A
t 1 day PF, g a m o n t s o f Hepatozoon sipedon, w h i c h m a i n t a i n e d t h e e l o n g a t e d structure o b s e r v e d in b l o o d films (Fig. 1), had e m e r g e d from i n g e s t e d e r y t h r o c y t e s and w e r e o b s e r v e d in groups o f two, three or four (Fig. 2 ) a m o n g b l o o d cells in the gut o f the mosquito.At 4 days PF, gamonts that had paired in syzygy within a parasitophorous v a c u o l e w e r e o b s e r v e d undergoing g a m e t o g e n e s i s within the cytoplasm o f fat b o d y cells in the h a e m o c o e l o f the m o s q u i t o C. pipiens (Fig. 3 ) T h e m a c r o g a m e t e contained large mitochondria and a nucleus that a p p e a r e d similar to that o f a gamont. At the c o m p l e t i o n o f m i c r o g a m e t o g e n e s i s , m i c r o g a m e t e s w e r e o b s e r v e d adjacent to the large m a c r o g a m e t e and the residual b o d y o f the microgamont, an irregular structure still containing m i c r o n e m e s and the remains o f an apical c o m p l e x (Fig. 3 ) . Examination o f serial semithin sections revealed that two biflagellate micro- g a m e t e s w e r e formed from e a c h microgamont. Micro- g a m e t e s consisted almost c o m p l e t e l y o f an electron- d e n s e n u c l e u s ( F i g . 4 ) , s u r r o u n d e d b y a t y p i c a l trilaminar pellicle. Cross-sections through the flagella o f the m i c r o g a m e t e s typically revealed a 9 + 1 arran
g e m e n t o f singlet microtubules within a trilaminar fla
gellar sheath (Fig. 5 ) .
S p o r o g o n i c d e v e l o p m e n t w a s o b s e r v e d b e t w e e n 5 and 28 days PF. Zygotes, e a c h contained within a para
sitophorous vacuole, rapidly increased in size after fer
tilization. At 6 days PF, immature oocysts c o n t a i n e d n u m e r o u s mitochondria around the periphery o f the cell and a few dispersed lipid inclusions (Fig. 6 ) . T h e nucleus c o n t a i n e d a granular nucleolus w h i c h w a s considerably m o r e c o n d e n s e d than that o b s e r v e d in m a c r o g a m e t e s . A substantial increase in the n u m b e r o f lipid inclusions and e l e c t r o n - d e n s e vesicles w a s evi
dent in immature oocysts at 13 days PF (Fig. 7 ) . T h e nucleus at this stage was proportionally smaller, with a highly-condensed nucleolus. T h e intracellular nature o f s p o r o g o n i c d e v e l o p m e n t w a s still apparent at this
GAMETOGENESIS AND SPOROGONY OF HEPATOZOON SIPEDON
Figs. 1-5. — Gametogenesis of Hepatozoon sipedon. 1. Light micrograph of gamont (arrow) in erythrocyte of Northern water snake. Bar
= 10 μm. 2. Two gamonts (arrows) that have emerged from erythrocytes in the gut of Culex pipiens at 1 day post-feeding (PF). Bar = 1 μm.
3. A macrogamete (ma) with diffuse chromatin, two microgametes (mi), and the residual body of the microgamont (r) in a fat body cell at 4 days PF. Cross-sections through the flagella of the microgametes are indicated by arrowheads. Bar = 1 μm. 4. Microgamete at higher magnification, revealing the large electron-dense nucleus (asterisk) that occupies most of the cell. Bar = 0.5 μm. 5. A higher magnification of a cross-section through the flagella of the microgamete, showing the 9 + 1 arrangement of singlet microtubules within a trilaminar fla
gellar sheath. Bar = 0.1 μm.
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1 4 3SMITH T.G. & DESSER S.S.
Figs. 6-9. — Early sporogonic development of H. sipedon. 6. Immature oocyst at 6 days PF, revealing the nucleus (n). peripherally-arranged mitochondria (m) and a few dispersed lipid inclusions (asterisks). Bar = 5 μm. 7. Immature oocyst at 13 days PF, showing an increase in the number of lipid inclusions (asterisks) and the appearance of electron-dense vesicles (arrow). Note the intracellular aspect of the deve
loping oocyst. Bar = 10 μm. 8. Immature oocyst at 14 days PF, with invaginations of the cytoplasmic membrane. Lipid inclusions (aste
risks) and electron-dense vesicles are numerous. Bar - 10 μm. 9. Immature oocyst at 16 days PF, revealing four nuclei (n), each with a diffuse nucleolus. The electron-dense vesicles are most numerous at this stage. Bar = 10 μm.
stage, although due to the increasing size o f the imma
ture oocyst, it w a s not o b v i o u s later in d e v e l o p m e n t . At 14 days PF, the cytoplasmic m e m b r a n e o f the imma
ture o o c y s t b e g a n to invaginate (Fig. 8 ) . Many small mitochondria occurred in the cytoplasm o f the oocyst b e t w e e n these invaginations. Serial sections revealed that the nucleus o f the o o c y s t had divided into four smaller nuclei at 16 days PF (Fig. 9 ) . O t h e r features included many lipid inclusions smaller in size than t h o s e s e e n at earlier s t a g e s , small e l e c t r o n - d e n s e vesicles, and numerous small b l e b s o f cytoplasm exten
ding from the oocyst. E a c h nucleus contained a dif
fuse granular nucleolus and regions o f heterochro- matin. At 2 0 days PF, the nuclei o f the oocyst had subdivided further and w e r e found near the periphery o f the o o c y s t (Fig. 1 0 ) . In addition to abundant mito
chondria and lipid inclusions, the cytoplasm o f the developing oocyst c o n t a i n e d small crystalloid b o d i e s consisting o f clumps o f round particles surrounded by rough e n d o p l a s m i c reticulum (Fig. 1 1 ) . A d e c r e a s e in the n u m b e r o f electron-dense inclusions was c o n c o mitant with the increase in the n u m b e r o f small crys
talloid bodies.
B y 24 days PF, n u m e r o u s sporoblasts (Fig. 1 2 ) w e r e found within developing oocysts. D e v e l o p i n g sporo
blasts, e a c h invested by a trilaminar pellicle, included large crystalloid b o d i e s , amylopectin and lipid inclu
sions, and o n e or more nuclei that were often observed near the periphery o f the sporoblast. Crystalloid bodies in s p o r o b l a s t s w e r e o r g a n i z e d into paracrystalline arrays (Fig. 1 3 ) and lacked the enclosing rough e n d o plasmic reticulum s e e n in immature oocysts. Sporoblast nuclei contained a d e n s e nucleolus, unlike the granular nature o b s e r v e d in earlier stages. O t h e r distinctive fea
tures o f sporoblasts included large projections o f cyto
plasm (Fig. 1 4 ) , and the p r e s e n c e o f micropores o n the sporoblast surface (Fig. 14, inset).
By 2 8 days PF, most oocysts w e r e mature, consisting o f hundreds ( m e a n = 5 9 1 , n = 5 ) o f differentiated spo- rocysts e n c l o s i n g fully-formed s p o r o z o i t e s . Mature oocysts (Fig. 1 5 ) w e r e spherical and averaged 2 6 2 . 6
± 2 3 0 μm (n = 2 0 ) in diameter; mature sporocysts (Fig. 1 6 ) w e r e spherical and averaged 18.2 ± 1 . 0 μm (n = 4 0 ) in diameter. Individual sporocysts (Fig. 1 7 ) c o n t a i n e d eight sporozoites and a large residual b o d y e n c l o s e d within a thick sporocyst wall c o m p o s e d o f two layers (Fig. 17, inset A ) . T h e residual body, which e n c l o s e d large amylopectin and lipid inclusions, was b o u n d e d by a single m e m b r a n e , in contrast to the tri
laminar pellicle surrounding the sporozoites (Fig. 17, inset B ) . Sporozoites (Figs. 17, 1 8 ) contained o n e large crystalloid body, n u m e r o u s a m y l o p e c t i n inclusions, and a typical apical c o m p l e x consisting o f a c o n o i d , polar ring c o m p l e x , rhoptries, m i c r o n e m e s and sub- pellicular microtubules.
DISCUSSION
T
h e observation that g a m o n t s o f Hepatozoon sipedon associate in c l o s e proximity to e a c h other after emerging from erythrocytes within the b l o o d meal o f the m o s q u i t o is reported for a species o f the genus for the first time. Lowichik et al.
( 1 9 9 3 ) reported widely-dispersed gamonts o f H. mocas- sini that travelled independently through the peritro- phic m e m b r a n e and penetrated the gut wall o f the m o s q u i t o . W h e t h e r or not g a m o n t s o f H. sipedon maintain this c l o s e association as they penetrate the gut wall is not known. Syzygy, gametogenesis, the for
mation o f a Plasmodium-like o o k i n e t e , and fertiliza
tion, w h i c h has b e e n reported, perhaps erroneously (Siddall, 1 9 9 5 ) for s o m e H e p a t o z o o n s p e c i e s to o c c u r in the gut lumen o f arthropods (Miller, 1 9 0 8 ; Robin,
Figs. 10, 11. — Late sporo
gonic development of H.
sipedon at 20 days PF.
10. Immature oocyst with small nuclei (n) near the periphery.
Bar = 10 μm. 11. Higher magnification of a developing crystalloid body consisting of lipoprotein particles sur
rounded by rough endo
plasmic reticulum. Bar
= 0.2 μm.
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Figs. 12-14. — Late sporogonic development of H. sipedon at 24 days PF. 12. Sporoblast, surrounded by a trilaminar membrane, contai
ning large crystalloid bodies (c), amylopectin (arrow) and lipid (asterisks) inclusions, and a nucleus («). Bar = 1 μm. 13. Crystalloid bodies are organized into paracrystalline arrays of small lipoprotien particles. Bar - 0.5 μm. 14. Developing sporoblasts with a trilaminar pellicle (arrow), large projections of cytoplasm, and micropores (arrowheads). Bar = 1 μm. Inset shows a micropore on the outer surface of the sporoblast membrane. Bar = 0.2 μm.
GAMETOGENESIS AND SPOROGONY OF HEPATOZOON SIPEDON
Figs. 15-18. — Mature oocysts and sporocysts of H. sipedon at 30 clays PF. 15. Light micrograph of mature oocyst, containing over 500 spo
rocysts. Bar = 50 μm. 16. Phase contrast micrograph of a mature sporocyst with 8 sporozoites. Bar = 10 μm. 17. Cross-section through mature sporocyst. revealing sporozoites (arrows) that contain a single large crystalloid body (c), and a residual body (r) with amylopectin and lipid inclusions. Bar = 0.5 μm. Inset A shows the thick outer wall of the sporocyst composed of two layers. Bar = 0.2 μm. Inset B reveals that the residual body (r) is surrounded by a single membrane unlike the trilaminar pellicle (arrowhead) enclosing the mature spo- rozoite. Bar = 0.2 μm. 18. Mature sporozoite with an apical complex consisting of a conoid (C), polar ring complex (P), rhoptries (R) and micronemes (M). Bar = 0.2 μm.
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1936; Lewis & Wagner, 1 9 6 4 ) , have not b e e n observed in this o r other ultrastructural studies o f the g a m e t o - g e n e s i s o f Hepatozoon s p e c i e s (Vivier et al, 1 9 7 2 ; G ö b e l & Krampitz, 1 9 8 2 ; Bashtar et al., 1984b; Lowi- chik étal, 1 9 9 3 ; D e s s e r et al., 1 9 9 5 ) .
T h e ultrastructural features o f the g a m e t o g e n i c stages o f H. sipedon in its definitive host, Culex pipiens, w e r e generally similar to those o f other species o f Hepato
zoon, including H. domerguei in Anopheles stephensi (see Vivier et al., 1 9 7 2 ) , H. aegypti in Culex pipiens
molestus ( s e e Bashtar et al., 1984b), and H. mocassini in Aedes aegypti ( s e e Lowichik et al., 1 9 9 3 ) , which uti
lize snakes as vertebrate hosts, H. cateshianae in Culex territans, which infects the erythrocytes o f frogs (Desser et al., 1 9 9 5 ) , and H. erhardovae in the flea Xenopsylla cheopis, w h i c h infects the leucocytes o f voles ( G ö b e l
& Krampitz, 1 9 8 2 ) . In e a c h o f these species, syzygy involved the pairing o f a microgamont and a m a c r o - gamont in the s a m e parasitophorous v a c u o l e and sub
sequent transformation o f e a c h gamont to a roughly spherical shape. Microgametogenesis resulted in the for
mation o f microgametes that are characterized b y a large electron-dense nucleus and, d e p e n d i n g o n the species, the p r e s e n c e o f o n e or two flagella. T h e fate o f those microgametes not involved in fertilization and the residual b o d y o f the microgamont is unknown, des
pite the fact that such residual structures are never s e e n after fertilization is c o m p l e t e , unlike that observed for Haemogregarina balli, in which such a residuum is pre
sent in the mature oocyst (Siddall & Desser, 1990).
G a m e t o g e n i c and s u b s e q u e n t s p o r o g o n i c d e v e l o p ment o f H. sipedon o c c u r r e d in the h a e m o c o e l o f the definitive invertebrate host ( s e e also Smith et al., 1994), the most c o m m o n site for sexual development o f other m e m b e r s o f this genus, including all o f those infecting s n a k e s (Landau et al., 1 9 7 0 ; Ball & O d a , 1 9 7 1 ; Bashtar et al., 1 9 8 4 a ; Lowichik et ai, 1 9 9 3 ) , and many o f those parasitizing lizards (Mackerras, 1 9 6 2 ; Bashtar et al.,
1 9 8 7 ) and m a m m a l s (Furman, 1966; Krampitz, 1 9 8 1 ) . Consistent with this localization o f d e v e l o p m e n t is the formation o f a non-motile zygote following syzygy and g a m e t o g e n e s i s . O t h e r m e m b e r s o f the g e n u s u n d e r g o sexual d e v e l o p m e n t and s p o r o g o n y in the gut wall o f a mite, as is the c a s e with the lizard parasite, H. lygo- somarum ( s e e Allison & Desser, 1 9 8 1 ) or a tick, as o b s e r v e d with the tortoise h a e m o g r e g a r i n e , H. mau-
ritanicum (see Michel, 1 9 7 3 ) . T h e gut as a d e v e l o p mental location for g a m e t o g o n y and s p o r o g o n y is similar to that o f m o r e derived adeleorins (Barta, 1 9 8 9 ; Siddall & Desser, 1991 ; Siddall, 1 9 9 5 ) , including s p e cies o f Karyolysus ( s e e Svahn, 1975), Desseria ( s e e Sid
dall & Desser, 1 9 9 2 ) , Cyrilia ( s e e Lainson, 1 9 8 1 ) , Hae
mogregarina ( s e e S i d d a l l & D e s s e r , 1 9 9 0 ) , a n d Babesiosoma ( s e e Barta & Desser, 1 9 8 9 ) . In a phylo- g e n e t i c analysis o f the g e n u s Hepatozoon and related
h a e m o g r e g a r i n e taxa (Smith & Desser, in press), in w h i c h the d e v e l o p m e n t a l location o f s p o r o g o n y w a s used as a character, it was found that H. lygosomarum, H. mauritanicum, the remaining h a e m o g r e g a r i n e taxa and the dactylosomatids form a m o n o p h y l e t i c group that is the sister clade to a m o n o p h y l e t i c g r o u p o f Hepatozoon s p e c i e s that d e v e l o p in the h a e m o c o e l o f the invertebrate host.
T h e n u m b e r o f m i c r o g a m e t e s formed from a micro
g a m o n t also varies a m o n g s p e c i e s o f Hepatozoon and has b e e n used as a character in the p h y l o g e n e t i c ana
lysis o f the g e n u s (Smith & Desser, in press). Four m i c r o g a m e t e s per m i c r o g a m o n t have b e e n c o m m o n l y o b s e r v e d (Mackerras, 1 9 6 2 ; Ball & O d a , 1 9 7 1 ; Michel, 1 9 7 3 ; Bashtar et al., 1 9 8 4 b ; Bashtar et al., 1 9 8 7 ; Lowi
chik et al., 1 9 9 3 ) , w h e r e a s o t h e r studies, including this one, h a v e revealed only t w o (Landau et al., 1 9 7 2 ; G ô b e l & Krampitz, 1 9 8 2 ; Desser et al, 1995). However, the inconsistency o f this character a m o n g Hepatozoon s p e c i e s , c o u p l e d with the difficulty in determining the n u m b e r o f m i c r o g a m e t e s formed from e a c h microga
mont, precludes this character from b e i n g used to dif
ferentiate groups within the genus.
T h e n u m b e r o f flagella b o r n e by e a c h m i c r o g a m e t e also provides p h y l o g e n e t i c data for separating Hepa
tozoon species, as again there is variation within m e m bers o f the genus. Biflagellated m i c r o g a m e t e s are the most c o m m o n state (Mackerras, 1 9 6 2 ; Landau et al., 1 9 7 0 ; Ball & O d a , 1 9 7 1 ; Lowichik et al, 1 9 9 3 ; D e s s e r et al., 1 9 9 5 ) , but uniflagellated m i c r o g a m e t e s have b e e n reported ( G ô b e l & Krampitz, 1982 ; Bashtar et al., 1 9 8 4 b ; Bashtar et al., 1 9 8 7 ) , as have aflagellated forms (Michel, 1 9 7 3 ) . T h e biflagellated state is shared with the m i c r o g a m e t e s o f eimeriorins, resulting in a less derived position for Hepatozoon in the p h y l o g e n e t i c analyses performed by Barta ( 1 9 8 9 ) , Siddall & D e s s e r ( 1 9 9 1 ) & Siddall ( 1 9 9 5 ) . T h e uniflagellated condition is similar to that observed in haemospororins (Garnham et al., 1 9 6 7 ; Desser, 1 9 7 0 ) and l o w e r adeleorins o f the g e n u s Klossia ( s e e Moltmann, 1 9 8 1 ) , while the afla
gellated forms are characteristic o f m o r e highly derived adeleorins (Lainson, 1 9 8 1 ; Siddall & D e s s e r , 1 9 9 0 , 1 9 9 2 ; Barta, 1 9 9 D .
T h e flagella o f Hepatozoon s p e c i e s have b e e n s h o w n in the present study and those b y Bashtar et al. (1984b), Lowichik et al. ( 1 9 9 3 ) and D e s s e r et al. ( 1 9 9 5 ) to consist o f unusual arrangements o f single microtu
bules, including 9 + 1, 8 + 2, 8 + 1, 7 + 1, and 4 + 0 forms, all o f w h i c h deviate from the classical 9 + 2 arrangement o f doublet microtubules s e e n in the m i c r o g a m e t e s o f other a p i c o m p l e x a n s ( S c h o l t y s e c k et al., 1 9 7 2 ) . As suggested b y Siddall & D e s s e r ( 1 9 9 0 ) , such unusual arrangements o f microtubules indicate that these structures are vestigial in nature, as such organelles w o u l d b e u n n e c e s s a r y for adeleorin para-
sites that associate in c l o s e proximity during syzygy.
In any c a s e , the highly variable microtubule arrange
ments recorded for a single s p e c i e s o f Hepatozoon likely precludes this feature for s p e c i e s description o r differentiation.
U l t r a s t r u c t u r a l f e a t u r e s o f s p o r o g o n i c s t a g e s o f H. sipedon are consistent with t h o s e o b s e r v e d for H. domerguei ( s e e Vivier et al., 1 9 7 2 ) , H. erhardovae ( s e e G ô b e l & Krampitz, 1 9 8 2 ) H. aegypti ( s e e Bashtar et al., 1 9 8 4 © , H. mocassini ( s e e Lowichik et al., 1 9 9 3 ) and H. catesbianae ( s e e D e s s e r et al., 1 9 9 5 ) , despite the fact that for each species o f parasite a different spe
cies o f intermediate host is utilized. T h e early stages o f nuclear division, o b s e r v e d as four nuclei c o n t a i n e d within the immature o o c y s t (Fig. 9 ) , have b e e n illus
trated previously b y Vivier et al. ( 1 9 7 2 ) . T h e y inter
preted this stage as the final result o f meiotic division o f the zygote, a prelude to the mitotic division o f nuclei that marks the first stage o f sporoblast formation. Sub
sequently, several rounds o f mitosis p r o d u c e many sporocyst nuclei that are distributed n e a r the per
iphery o f the oocyst. D e e p invaginations o f the plasma m e m b r a n e result in the formation o f cytoplasmic pro
trusions, e a c h containing o n e nucleus, that eventually b u d off to form immature sporocysts (Bashtar et al.,
1 9 8 4 b ) .
In m a n y studies o f the s p o r o g o n i c d e v e l o p m e n t o f Hepatozoon species, small crystalloid bodies b o u n d b y rough e n d o p l a s m i c reticulum a p p e a r e d prior to the division o f t h e i m m a t u r e o o c y s t into s p o r o b l a s t s ( 2 0 days P F ) . It s e e m s that these crystalloid b o d i e s are the precursors o f the larger forms s e e n in sporoblasts and sporocysts, as Bashtar et al. ( 1 9 8 4 b ) o b s e r v e d d e e p invaginations o f the o o c y s t m e m b r a n e prior to sporoblast differentiation, with e a c h resulting projec
tion containing o n e nucleus and a n u m b e r o f small crystalloid b o d i e s . Mature crystalloid b o d i e s s e e n in mature sporozoites are c o m p o s e d o f paracrystalline arrays o f small lipoprotein-containing particles des
cribed originally from sporozoites o f L e u c o c y t o z o o n and Haemoproteus s p e c i e s ( D e s s e r & Trefiak, 1 9 7 1 ; Trefiak & Desser, 1 9 7 3 ) . Small electron-dense inclu
sions that w e r e abundant prior to the formation o f crys
talloid b o d i e s d e c r e a s e d in n u m b e r and eventually disappeared w h e n crystalloid bodies w e r e fully formed.
T h e s e inclusions may serve as the lipoprotein precursor that is p a c k a g e d into the particles that constitute e a c h crystalloid body.
T h e p r e s e n c e o f a trilaminar pellicle surrounding the d e v e l o p i n g s p o r o c y s t w a s r e p o r t e d in H. sipedon (Fig. 1 3 ) and H. domerguei ( s e e Vivier et al, 1 9 7 2 ) . T h e present study revealed that the mature sporozoites are invested b y a trilaminar pellicle, w h e r e a s the resi
dual b o d y remaining after c o m p l e t i o n o f sporocyst d e v e l o p m e n t w a s e n c l o s e d b y a single plasma m e m
brane. T h e formation o f sporozoites from sporoblasts has b e e n o b s e r v e d for other species o f Hepatozoon ( B a s h t a r et al, 1984b; Lowichik et al, 1 9 9 3 ) , and involves concurrent nuclear division o f the sporoblast nucleus into presumptive sporozoite nuclei and the for
mation o f sporozoite anlagen from cytoplasmic pro
trusions o f the sporocyst.
Micropores o n the surface o f the sporoblast (Fig. 1 7 ) have b e e n o b s e r v e d previously b y Vivier et al. ( 1 9 7 2 ) . Their p r e s e n c e indicates that sporoblasts are still acti
vely assimilating nutrients from the external environ
ment, in this c a s e the residuum c o n t a i n e d within the walls o f the oocyst, or perhaps material s e q u e s t e r e d from the remains o f the fat b o d y o f the mosquito host.
In fact, mosquitoes containing 100 or m o r e oocysts are usually devoid o f eggs, indicating that resources nor
mally allotted to egg production are probably utilized b y the developing parasites.
Mature sporozoites o f H. sipedon contain only o n e crys
talloid body, similar to t h o s e o f H. catesbianae ( s e e D e s s e r et al., 1 9 9 5 ) . However, sporozoites o f other Hepatozoon species (Bashtar et al., 1 9 8 4 b ; Lowichik et al., 1 9 9 3 ) and o t h e r a d e l e o r i n s (Siddall & D e s s e r , 1991 ; Siddall & Desser, 1993), contain two distinct crys
talloid bodies. Hepatozoon sipedon, w h i c h requires both an anuran and ophidian host (Smith et al., 1 9 9 4 ) and H. catesbianae, w h i c h exclusively utilizes frogs as vertebrate hosts ( D e s s e r et al., 1 9 9 5 ) , w e r e h y p o thesized to b e sister taxa in a p h y l o g e n e t i c r e c o n s truction o f Hepatozoon s p e c i e s (Smith & Desser, in press), and h e n c e the reduction to o n e crystalloid b o d y might represent an a p o m o r p h i c feature o f this clade. T h e ultrastructure o f these b o d i e s is similar to those reported from other Hepatozoon species and ade
leorins.
Given the variability in the site o f gametogenic and spo
rogonic d e v e l o p m e n t and the n u m b e r and structure o f the microgametes, as well as the conclusions made from phylogenetic hypotheses o f the adeleorin genera, there is substantial information with which to justify splitting the genus Hepatozoon, an observation previously made by other workers (Ball & Oda, 1 9 7 1 ; Barta, 1 9 8 9 ) and reiterated b y Smith & D e s s e r (in press). T h e r e is little doubt that groups o f Hepatozoon species displaying such variety in life history could b e elevated to generic- status, although the features that would constitute viable characters for separation would have to b e determined.
T h e lack o f c o m p l e t e , reliable life cycle and ultra- structural data for the majority o f Hepatozoon species, especially for those m e m b e r s which infect mammals, lizards, birds and crocodilians, currently precludes any division o f this genus. Siddall & D e s s e r ( 1 9 9 2 ) stress the necessity for ultrastructural studies to fully document the life cycle o f adeleorin parasites. Although ultra- structure may b e useful in distinguishing a m o n g exis-
Parasite, 1997, 2, 141-151
149 GAMETOGENESIS AND SPOROGONY OF HEPATOZOON SIPEDON
S M I T H T . G . & D E S S E R S . S .
ting genera, the similarity o f many o f these stages a m o n g the Hepatozoon species investigated s o far indi- cates that only certain features, including the n u m b e r o f m i c r o g a m e t e s p r o d u c e d p e r m i c r o g a m o n t , t h e number o f flagella borne by each microgamete, a n d the n u m b e r and fine structure o f crystalloid bodies, will b e useful in further t a x o n o m i c studies.
ACKNOWLEDGEMENTS
W
e t h a n k H e n r y H o n g , B e t t y K i m a n d C h o n g x i e X i a o for their c o m m e n t s o n ear- lier drafts o f the paper. Further recognition is d u e Henry Hong for his technical assistance a n d Andrea Lawson for h e r help in collecting m o s q u i t o e s at the Wildlife Research Station. This research w a s sup- p o r t e d b y T h e Natural S c i e n c e s a n d E n g i n e e r i n g Research Council o f Canada.REFERENCES
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Reçu le 1
e rfévrier 1997 Accepté le 14 mars 1997
Mémoire 151
Parasite, 1997, 2, 141-151
