MATERIAL A N D METHODS

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Ul t r a s t r u c t u r e o f s p e r m i o g e n e s is a n d s p e r m a t o z o a in Eu z e t r e m a k n o e p f f l e r i ( Pl a t y h e l m in t h e s, Mo n o g e n e a)

FOURNIER A.* ^and JUSTINE J.-L.**

Sum m ary :

Euzetrema knoepffleri is a monogenean parasite in the relict uro- dele amphibian Euproctus montanus in the island of Corsica.

Spermiogenesis w as studied by electron microscopy. The sperma

tid has a zone of differentiation containing two parallel centrioles, an elongating nucleus and mitochondrion, and no peripheral microtubule. The spermatozoon is formed by a lengthening of the zone of differentiation. The mature spermatozoon contains two axonemes of the platyhelminth 9 + " 1” pattern, a mitochondrion, a nucleus, and no peripheral microtubule. The spermatozoon corres

ponds to type 2 in the classification of Justine, Lambert & Mattei (1 9 8 5 ). This type 2 is characteristic of certain monopisthocotylean M onogenea. In the absence of supplementary apomorphies, it is not possible to specify the phylogenetic position of Euzetrema in the Monopisthocotylea from spermatozoal ultrastructure.

KEY WORDS : Euzetrema knoepffleri. ultrastructure, spermatozoon, phylo- geny. Monogenea.

MOTS CLES : Euzetrema knoepffleri. ultrastructure, spermatozoïde, phylogé

nie. Monogènes.

INTRODUCTION

R é s u m é : Ultrastructured elasperm io g en èseetd usperm ato z o ïd ed e Euzetremak n o epffler i( P l a t h e l m i n t h e s , M o n o g e n e a )

Euzetrema knoepffleri est un monogène parasite d e l'amphibien uro- dèle relicte Euproctus montanus en C orse. la spermiogenèse a été étudiée en microscopie électronique à transmission. La spermatide montre une zone d e différenciation contenant deux centrioles paral

lèles, le noyau et la mitochondrie en élongation, et aucun microtu- bule périphérique. Le spermatozoïde se forme par allongement de la zone de différenciation. Le spermatozoïde mûr contient deux axo- nèmes du type 9+ " 1 " d e Plathelminthes, une mitochondrie, le noyau, et aucun microtubule périphérique. Le spermatozoïde corres

pond au type 2 d e la classification des spermatozoïdes d e Justine, Lambert & Mattei ( 1985). C e type 2 est caractéristique d e certains M onogènes Monopisthocotylea. En l'absence d'apomorphies sup

plémentaires, la structure du spermatozoïde ne permet pas d e préci

ser la position phylogénétique d e Euzetrema à l'intérieur des M onogènes M onopisthocotylea.

MATERIAL A N D METHODS

S

permatozoal ultrastructure is one of the avai

lable tools for understanding the phylogeny of the Platyhelminthes (Justine, 1991a; Justine, 1991b; Justine, 1993; Justine, Lambert and Mattei, 1985a). We present here observations on spermioge

nesis and spermatozoa of a m onogenean, namely E u z e tr e m a k n o e p ffle r i Com bes, 1965. E u z e tr e m a belongs to the Iagotrematidae, a family comprising only three species with a very discontinuous geogra

phical distribution (Fournier, 1980). Moreover, the host of E u zetrem a kn oepffleri, the urodele amphibian Euproctus m on tan u s, is a relict species found in the island o f Corsica (Combes, 1965; Combes, Jourdane and Knoepffler, 1974).

* URA 6 9 8 CNRS, C en tre d e B io lo g ie et d 'É c o lo g ie T ro p ic a le et M é d ite rr a n é e n n e , L a b o ra to ire d e B io lo g ie A n im a le, U n iv ersité, A venue d e V illeneuve, F-66860 P erpignan C ed ex, France.

** URA 114 CNRS, Laboratoire d e B io lo g ie Parasitaire, Protistologie, H e lm in th o lo g ie , M u séu m N atio n al d 'H is to ire N a tu relle, 61 rue Bu ffon , F -75321 Paris C ed ex 05, France.

A dresse pou r co rresp o n d an ce et tirés-à-part : Jean -L ou Ju stin e, L aboratoire d e B io lo g ie Parasitaire, Protistologie, H elm inthologie M uséum national d'H istoire N aturelle, 61 rue Bu ffon F -75321 Paris C ed ex 05 , France.

T é l: (1 ) 4 0 7 9 35 0 3 - Fax: (1 ) 4 0 7 9 34 9 9 - E-m ail: ju stine@ m nhn.fr

T

he host Euproctus m on tan u s was collected in Corsica. Adult monogeneans were fixed in 3%

glutaraldehyde in a pH 7.3, 0.1 M cacodylate buffer at 4°C for 3 hrs, then rinsed and post-fixed in 1% osmium tetroxide in the same buffer. Dehydration was performed in an acetone- propylene oxide series, blocks w ere em bedded in Epon, and grids were con trasted with uranyl acetate and lead citrate.

Glycogen demonstration was performed after Thiéry (1967).

OBSERVATIONS

I

n contrast to many parasitic Platyhelminthes, the testis of E u zetrem a contains only few stages of spermiogenesis and spermatozoa; this could be related to the annual cycle of this species.

Early spermatids are attached to a cytophore. Nurse cells send extensions between the germinal cells (Fig.

a). The early sperm atid is round, with a round nucleus and mitochondria grouped at one pole of the cell (Fig. a). Then the nucleus becomes pyriform with the mitochondria grouped at its base, and two axo-

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Article available athttp://www.parasite-journal.orgorhttp://dx.doi.org/10.1051/parasite/1994012123

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FOURNIER A. a n d JUSTINE J.-L.

nemes growing near its pointed extremity (Figs. b, c).

The axonemes, at this stage, show no central core and thus have a 9+0 structure (Fig. c). Two centrioles are present (Fig. d) and thus two free axonemes are probably present in spermatids, but sections showing two elements were not found.

Later, the spermatid lengthens and free flagella are no longer seen. A zone of differentiation with two free axonemes and a middle cytoplasmic process, such as seen in the Polyopisthocotylea and Digenea, was never observed. Transverse sections of the lengthening spermatid exhibit two axonemes of the trepaxo- nematan 9+“1” type, the elongating nuclr us and the elongating mitochondrion (Fig. e). In the proximal part of the spermatid, transverse sections show two centrioles made up of nine triplets (Fig. f). No peripheral microtubule was seen in any stage of sper

miogenesis. During the r longation process, dynein arms of the immature axonemes are indistinct (Fig.

g). In som e spermatids, the elongating extrem ity shows more than two sections of axonemes (Fig. h), thus indicating that the extremity makes a loop on itself, and axonemal tips have their central core on the outside o f the circle of nine doublets (Fig. h).

The mature sperm atozoon, observed in the testis (Figs. i, j) and vas deferens (Fig. k) show s two regions: a) a nuclear region (Fig. i), with nucle us, mitochondrion, two axonem es and abundant beta glycogen demonstrated with Thiéry’s reaction (Fig. j);

and b) a region without nucleus (Fig. k), containing only two axonemes with a section of mitochondrion in some sections. Exceptionally, a section showing one single peripheral microtubule (Fig. k) was found among many observed sections.

DISCUSSION

T

he phylogenetic value of monogenean sper

m atozoon ultrastructure is currently well accepted and the number of species descri- bed is growing rapidly. A recent review (Justine, 1993) listed 68 species, and since, nine studies have b e e n p u b lish ed , on O c t o m a c r u m la n c e a t u m (H ath aw ay, H athaw ay and K ritsky, 1 9 9 3 a ), L agarocotyle sa la m a n d r a e (Hathaway, Hathaway and Kritsky, 1993b), Isa n cistru m s u b u la t a e (Malmberg and Lilliemark, 1993), P seu d od ip lorch is a m erica n u s (C a b le and T in sle y , 1 9 9 3 ), E n t o b d e l l a s o le c te (T a p p e n d e n , K earn and E v an s-G ow in g , 1 9 9 3 ), M onocotyle h elicop ballu s and C alicotyle australiensis (Watson and Rohde, 1994a), G onoplasius sp. (Rohde and Watson, 1994a), P olylabroides au stralis (Rohde and Watson, 1994b) and N eopolystom a sp. (Watson and Rohde, 1994b). The present observations were

included in an unpublished thesis (Fournier, 1980) and because of the current interest in monogenean spermiogenesis we thought it was useful to allow oth er research ers a ccess to the ob serv atio n s on E u zetrem a kn oep ffleri by publishing them.

The spermatozoa of monogeneans have been classi- fied into four patterns (Justine et al., 1985a) and a fifth pattern was later added (Justine, Le Brun and Mattei, 1985b). In this classification, E. kn oepffleri cor

responds to type 2 (two axonemes, peripheral micro

tubules absent). Type 2 was separated into several subtypes, and E. kn oep ffleri corresponds to subtype 2c (Justine, 1991a). Recently, a single peripheral microtubule was found in some cases (Justine, Mattei and Euzet, 1991) and the de finition o f type 2 has b een am ended (Justine, Afzelius, M alm berg and Mattei, 1993) as “spermatozoa with two axonem es and no cortical microtubules, except a single microtu

bule much shorter than the spermatozoon”. The pat

tern o f E. k n o e p f f l e r i c o rre sp o n d s w ell to the definition of type 2; the single microtubule exceptio

nally found is probably much shorter than the sper

matozoon. Cladistic analyses of the Monogenea based on sperm ultrastructure (Justine, 1991a; Justine, 1993) use many characters, and not only the classification o f sperm patterns. In these analyses, the pattern found in E. kn oepffleri corresponds to the most ple- siomorphic pattern found in the Monopisthocotylea, and lack of other apomorphies does not allow its use for u nderstanding the p h y lo g en etic p o sitio n o f E u zetrem a within the Monopisthocotylea.

Watson and Rohde (1994a) recently claimed that the limits between patterns 2 and 3 were not clear and thus proposed a type 2/3. It is true that C alicotyle au stralien sis and C. kroyeri have spermatozoa sho

wing two axonemes and several cortical microtubules (Tappenden and Kearn, 1991; Watson and Rohde, 1994a) and, thus, this should leacl to a re-examination of the définition of type 3. However, microtubules of mature spermatozoa in type 3 clearly originate from the cortical microtubules, with associated ornamenta

tion, located in the spermatid’s early zone of diffe ren

tiation, while the origin o f the single (som etim es absent) microtubule in type 2 is less clear. In other words, the single microtubule of type 2 might be not homologous with the cortical microtubules with orna

mentation of type 3- The combination of types 2 and 3 into a single type would require a demonstration of homology.

A study of spermiogenesis in Iagotrem a, a parasitic monogenean found in a south American turtle and supposedly related to E u zetrem a, would be useful to clarify the p h y lo g en etic p o sitio n o f the fam ily Iagotrematidae.

124 Parasite,1994, ^1, 123-126

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SPERMATOZOON OF A MONOGENEAN

Figs. a-k. — Sperm iogen esis in E u zetrem a kn oep ffleri

a. Early sperm atid w ith round n ucle us. M itochondria (M ) are grouped at o n e p o le o f th e cell. N ote p ro cesses o f nurse cells.

x 20 000. b Early sperm atid, with pyriform nucle us and axo n em e (A), x 15 000. c . T w o early sperm atids, sam e stage as b. T he a x o n em e (A ) has n o central core. x 15 000. d . Early sperm atid, sectio n sh ow in g tw o cen trioles (C ). x 15 000. e . Elongating late sperm atid, transverse sectio n sh ow in g tw o ax o n em es and elon gatin g n ucle us and m itochon drion. x 36 000. f . Late sperm atid, transverse sectio n at th e b a se o f th e zo n e o f diffe rentiation, sh ow in g tw o cen trio les (C ). x 3 6 000. Late sperm atid, elongating extrem ity. D ynein arms are indistinct, x 36 000. h . T ransverse sectio n o f distal extrem ities o f sperm atids. x 25 000. L Transverse sectio n o f m ature sperm atozoa in testis, sh ow in g n u clear re gion with nucle us, and a re gion w ithout nucle us, x 4 0 000. j. Beta-gly- co g e n in m ature sp erm atozoo n , ev id en ced by T hiéry’s reaction, x 30 000. k. T ransverse sectio n o f m ature sperm atozoa in vas deferen s. Note single m icrotubu le in o n e sectio n (arrow ). x 30 000.

Parasite, 1994, 1. 123-126 M é m o ire ---125

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FOURNIER A. a n d JUSTINE J.-L.

ACKNOWLEDGEMENTS

Dr. Nikki Watson and Prof. Klaus Rohde provided papers in press. Andre a Bullock kindly edited the English.

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Accepté le 28 avril 1994

126 Mémoire Parasite. 1994, 1, 123-126