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Ultrastructure of sperm and spermiogenesis in the monocotylid monogeneans Monocotyle helicophallus and Calicotyle australiensis (Platyhelminthes
hrernarional Journnlfor Parasimlogy. Vol. 24, No. 7, pp. 1019-1030, 1994 Copyright 0 1994 Australian Society for Parasitology Elwier Science Ltd
Pergamon
Printedin Great Britain.All rightsreserved ~2~7519/94 $7.00+ 0.0
0020-7519(94)30027-K
ULTRASTRUCTURE OF SPERM AND SPERMI~GENESIS IN THE MONOCOTYLID MONOGENEANS MONOCOTYLE HELICOPHALLUS AND CALICOTYLE AUSTRALIENSIS (PLATYHELMINTHES) N. A. WATSON*
and K. ROHDE
Department of Zoology, University of New England, Armidale, NSW 2351, Australia (Received 19 August 1993; accepted 15 February 1994)
N. A. and ROHDE K. 1994.Ultrastructure of sperm and spermiogenesis in the monogeneans Monocotyle helicophallus and Calicotyie australiensis (Platyhelminthes). ~~ier~ationai ~ournalfor Par~~tology 24: 1019-1030. Spermiogenesis in ~~~oco~y~e hei~caphall~ involves formation of free flagella which rotate to lie parallel before fusing. There is no median cytoplasmic process. A small number of microtubules (usually 2-4) is associated with the axonemes in the zone of differentiation. Nucleus and a fused mitochondrion migrate alongside the axonemes and one basal body moves distally. A coil of nucleus, originally in the cytophore., also moves out along the shaft. The mature spermatozoan has only a single axoneme for a short distance at each end, with two associated microtubules at the proximal end, as well as one axoneme, nucleus and mitochond~on throughout most of its length (the nucleus being enlarged and roughly coiled in one region), and a short region of two overlapping axonemes. We interpret our findings as two axonemes arranged almost end to end, one extending from the proximal end to some point in the mid-region, where the second axoneme begins and continues to the distal end of the sperm. Spermatozoa of Ca&otyfe austraiiensis develop from a zone of differentiation which has two basal bodies and a complete ring of corticat microtubules. Two initially free axonemes fuse with each other and there is no median cytoplasmic process. Spermatids form within parallel canals in the cytophore, by backward movement of the zone of differentiation. Prior to detachment, an electron-dense spiral end-piece forms around and proximal to the basal body region. Sixty-four spermatids are present in each isogenic group. With the study of spermiogenesis in more species of Monogenea Monopisthoc~tylea, it is apparent that the previously designated sperm patterns 2 and 3 are not distinct and should be combined and re-defined. Species previously designated as having sperm patterns 2 and 3 can all be accommodated by the description “two normal axonemes or one normal and one shortened, altered or displaced axoneme, and none, one or a few cortical microtubules remaining in a region of the sperm derived from the zone of differentiation in which a few or a complete ring of microtubules was present”. bstract-WATSON
monocotylid
INDEX KEY WORDS: Monogenea; Monopisthocotylea; phylogeny; spermatozoa; ultrastructure; Monocotyle helicophallus; Calicotyle australiensis.
INTRODUCTION ultrastructural investigations of sperm and spermiogenesis have been undertaken in species of Monogenea (see Justine 1991 for review, Justine, Mattei & Euzet, 1991; Tappenden & Kearn, 1991; Watson & Rohde, 1992; Justine, 1992; Justine, Afzelius, Malmberg & Mattei, 1993) and such studies are useful for phylogenetic considerations. Justine, Lambert & Mattei (1985) arranged the Monogenea into 4 phylogenetic groups based on spermatozoa1 patterns in the various families: pattern 1 characterised by the presence of 2 axonemes plus cortical microtubules, pattern 2 by 2 axonemes without NUMEROUS
*To whom all correspondence should be addressed.
spermiogenesis;
microtubules, pattern 3 by I axoneme plus I altered axoneme plus microtubules, and pattern 4 by 1 axoneme without microtubules. A fifth (aberrant) type was later defined for Di~lozo~~, which has no axoneme, and numerous cortical and cytoplasmic microtubules (Justine, Le Brun & Mattei, 1985). All Polyopisthocotylea examined so far have sperm pattern 1 except for the aberrant ~~~~~z~Q~, while types 2, 3 and 4 are found in various families of Monopisthocotylea. Sperm pattern 3 (1 normal axoneme, 1 altered axoneme and some cortical microtubules) was established from observations on one member of the family Monocotylidae, viz. HeterocotyIe (see Justine & Mattei, 1983b) and one of the family Loimoidae, viz. Loimosina (see Justine & Mattei, 1985). Subsequently Tappenden & Kearn
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N. A. WATSON and K.
(1991) found two complete axonemes in another monocotylid, Calicotyle kroyeri. We examined sperm and spermiogenesis in 2 more species of monocotylids in order to determine whether any further variation exists in the characteristics used to define sperm pattern and phylogenetic relationships in the Monopisthocotylea. MATERIALS AND METHODS Specimens of ~o~~oryle ~el~eo~~~i~~were removed from the gills of Himanrura sp. from Heron Island, off the coast of Queensland, Australia. They were fixed for 3-4 h in 3% ~utaraldehyde in 0. I M sodium cacodylate buffer, made with filtered seawater, washed several times in the same buffer, then stored in buffer for 2 days. Post fixation in 1% 0~0, in the same buffer for 1h at room temperature was followed by dehydration in alcohol and embedding in Spurr’s resin. Specimens of Calicotyle australiensis were obtained from a chimaerid host off the NE coast of Tasmania, fixed for 2 h in the same primary fixative as above, washed then stored in buffer for 21 days and further processed as for M. helicophallus. Sections were stained with uranyl acetate and lead citrate and examined under a JEOL 1200 EX electron microscope at 60 kV.
ROHDE
begins to migrate beside the axonemes (Figs. 5 and 13), which often appear bent back towards the spermatid body during this phase (Fig. 5). One axoneme then shifts distally (Figs. 6,8b and 14) while the nucleus and a long fused mitochondrion migrate along the shaft (Fig. 8c, d) A part of the nucleus still within the cytophore becomes coiled and this twisted region moves out along the spermatid shaft. Figures 7,9 and 10 show such coils in longitudinal and transverse sections. They may surround one or both axonemes, or form a twisted mass adjacent to them. A small variable number (14, most commonly 2) of microtubules lies for a short distance only along the periphery of the spermatid, initially in the zone of differentiation (ZD) (Figs. 2, 8a, b, c, 12, 14 and 15). A small dense region of ornamentation is usually present on the outer surface of the plasma membrane adjacent to each microtubule. It was not possible to determine the number of spermatids in each isogenic group because they do not develop in parallel canals as do the spermatids of many other monopisthocotylean monogeneans. Mature
RESULTS Spermiogenesis in Monocotyle The process of spermiogenesis is depicted diagrammatically in Fig. 42. Early spermatids, joined in cytophores, are wedge-shaped and develop two centrioles at right angles to each other near the apical cell membrane (Figs. 1 and 2). The nucleus is also wedge-shaped with the pointed end nearest the centrioles. Free flagella then grow from the basal bodies (Fig. 3) and subsequently rotate to lie parallel with each other, in a more or less straight line with the elongating body of the spermatid (Fig. 4). Chromatin begins to condense within the nucleus and the nucleus elongates. Cross-sections (Fig. 11) through the nuclear region of the spermatid at this stage show some microtubules around the nucleus. The two adjacent axonemes then fuse with each other and the nucleus
sperm of Monocotyle Mature sperm in the seminal vesicle have a single axoneme, nucleus and mitochondrion throughout much of their length (Figs. 16, 17,21 and 22). Two or three cortical microtubules associated with ornamentation of the plasma membrane can be seen in various cross sections, most of which show only a single complete or tapering axoneme (Figs. 17e and 22). Figure 16b, however, shows one sperm at the level of 2 axonemes, nucleus and mitochondrion, with 2 peripheral microtubules, although the presence of two profiles of mitochondrion may indicate that this is a folded or otherwise aberrant region. At both ends of the sperm a short region is devoid of nuclear and mitochondrial profiles; at one end the axoneme tapers and terminates over a short distance and has an electron dense cap (Figs. 19 and 21 arrowhead) (this may be the proximal end if the cap equates with a
FIGS. I-15. Sections of spermatids at various stages during spermiogenesis of ~o~oeor~~e heijcop~aft~s. 1. Early spermatid with centrioles (arrowheads) at right angles to each other. 2. Transverse section of centriole of early spermatid and 2 cortical microtubules (arrowhead). 3. Free flagella grow at right angles to each other from the apex of the early spermatid. 4. Flagella rotate to lie parallel with each other. 5. Flagella fuse with each other and the nucleus begins to migrate alongside them. 6. One axoneme shifts distally relative to the other (arrowheads indicate basal bodies). 7. Longitudinal section of the region of a spermatid with a coil of nuclear material. 8. Transverse sections through some developing spermatids. a -single axoneme and 2 cortical microtubules, b ~ one axoneme shifted relative to the other, c-nucleus migrates ahead of the mitochondrion, 2 cortical microtubules present, d-distal to the region with 2 cortical microtubules, e-one axoneme terminating within the spermatid. 9. Transverse section of the region of nuclear coil, with 2 axonemes separated by nuclear material. 10. Similar to Fig. 9 but axonemes remain adjacent. i 1. Transverse section through early outgrowing spermatid, proximal to the basal bodies of the axonemes. Note the “manschette” of microtubules around the nucleus. 12. Early free flagella, each with associated cortical microtubules. 13. Spermatid before one axoneme has shifted relative to the other. 14. Spermatid after shift of one axoneme. with 3 cortical microtubules (arrowh~d). 15.An early, free axoneme with 2 cortical mi~rotubules (arrowhead). n -nucleus, m - mitochondrion. Scale bars, 200 nm (2, 7, 8, l&15), 500 nm (1, 3, 4, 6, 9), 1 F (5).
Sperm and spermiogenesis in monocotylids
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N. A. WATSONand K. ROHDE
FIGS. 16-2.5. Sections through mature sperm from the seminal vesicle of Monocoryle hel~c~pha~lus. 16. a - most common profile containing 1 axoneme, nucleus and mitochondrion, b - 2 axonemes, nucleus, 2 mitochondrial profiles and 2 cortical microtubules with associated ornamentations (arrowhead), c- near oneend with only nucleus and a single axoneme, d - the region of transition, 1 axoneme terminating and the other beginning, nucleus and mitochundrion present, e-tapering microtubular doublets, probably the distal extremity of the sperm. 17. a -most common profile, b - 2 complete axonemes, nucleus and 1 or 2 mitochondrial profiles, c - region of transition, 1 axoneme normal, the other tapering, d - both axonemes tapering, e - proximal end with a single axoneme and 2 cortical microtubules with associated ornamentations (arrowhead). 18. distal extremity of sperm, nucleus terminates (arrowhead) and axoneme tapers. 19. Proximal extremity of sperm where axoneme ends in a ‘bulb (arrowhead) and the nucleus commences (arrow). 20. Region of transition with continuous nucleus and tapering axoneme (arrowh~d). 21. Transverse section depicting both proximal (arrowhead) and distal (arrow) regions. 22. Transverse section through the proximal region containing a single axoneme and 4 cortical microtubules with associated ornamentations (arrowhead). 23-25. Transition regions showing varying degrees of overlap of the tapering axonemes, nucleus (n) and mitochondrion (mf present. Scale bars, 200 nm (21-25), 500 nm (14-20).
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Sperm and spermiogenesis in monocotylids centriolar adjunct), while at the other end the microtubules diminish gradually and the axoneme tapers to a fine end (Figs. 16e, 18 and 21). The nucleus and mitochondrion are of almost equal length since few profiles show only one or the other present beside the single axoneme. Detailed examination of a large number of mature sperm revealed a small percentage of profiles showing two complete axonemes or one complete axoneme and one with a reduced number of doublets or with only single microtubules present (Figs. 16b and 17b, c). This could be interpreted as one axoneme present throughout the whole length of the sperm and a second much shorter and tapering one along a short region. However, some longitudinal sections (e.g. Fig. 20) suggested that one axoneme may terminate and another begin at some point along the body of the sperm, i.e. that two normal axonemes were present with a short region of overlap between them. This suggestion was confirmed by finding sections where the region of overlap was so short that one axoneme was terminating and the other beginning in the one profile (Figs. 16d, 17d and 23-25). We therefore propose that the mature sperm has the structure depicted in Fig. 43. Spermiogenesis in Caliocotyle
The process of spermiogenesis is depicted diagrammatically in Fig. 44. Clusters of spermatogonia are scattered within the testis, not restricted to the wall region. Primary spermatocytes, identified by the presence of synaptonemal complexes, have a large nucleus with a prominent nucleolus (Fig. 26), and many small mitochondria. Early spermatids are joined in a rosette formation and a zone of differentiation develops at the apex of each spermatid, containing two centrioles at right angles to each other. Two free cilia develop and microtubules radiating from their basal bodies. extend alongside the elongating nucleus (Fig. 27). Microtubules may be present in dense cytoplasm beneath the apical cell membrane (Fig. 27 arrows). The basal bodies then rotate (probably by means of the attached microtubules) to lie parallel with one another, and the nucleus and fused mitochondrion move into the apical region, which is now surrounded by a complete ring of microtubules (Fig. 28). Basal bodies then migrate proximally, deeper into the cytoplasmic mass, and the accompanying backward movement of an arching membrane results in the spermatid shaft lying in a canal, deeper and deeper within the spermatid mass. One basal body shifts a short distance in relation to the other during this stage of elongation of the shaft. When most of the cytoplasmic mass has been incorporated into the elongate spermatids and they lie in parallel canals, a complex spiral end-piece is formed around the basal
body and adjacent axonemal region. Figures 29-31 depict stages in this development, which involves condensation of cytoplasm and contraction and rounding of the contours of the spiral end-piece. Figure 32 indicates that growth of spermatids has involved backward movement of the ZD deep into the cytoplasmic mass. There are 64 spermatids in each isogenic group. Spermatozoa then detach from the residual cytoplasm. Mature sperm of Calicotyle
Transverse sections of mature sperm from the sperm duct indicate the following arrangement of its elements: at the most proximal end, an open spiral of fine granular to amorphous, electron-dense material is joined to the rest of the sperm by a more coarsely granular, electron-dense region in which the two basal bodies and the end of the mitochondrion are embedded (Figs. 33-35,37 and 38). Distal to this endpiece region, the mitochondrial profile is greatly enlarged (Figs. 36 and 39) a thin tail of the nucleus is seen and two peripheral longitudinal microtubules lie adjacent to the nucleus (Fig. 39). In this region the mitochondrion has numerous, prominent cristae (Fig. 36). More distal, nucleus and mitochondrion are approximately equal in diameter (Fig. 40), both taper (Fig. 41) and disappear, leaving two axonemes and finally a single tapering axoneme in the most distal extremity. There are no peripheral microtubules in the more distal regions. Figure 45 is a diagram of the proposed structure of the mature sperm. DISCUSSION Calicotyle australiensis
Features of spermiogenesis
and mature sperm in
Calicotyle australiensis resemble those of C. kroyeri (see Tappenden & Kearn, 1991) although some
differences were noted. Thus, in early spermiogenesis axonemes of C. australiensis initially extend at right angles to each other before they rotate to lie parallel, and the proximal spiral end-piece which develops late in spermiogenesis exhibits 34 turns in C. australiensis compared with 7 or 8 turns in C. kroyeri. The mature sperm of C. australiensis retains the spiral end-piece (compacted to three turns), the basal bodies of the 2 axonemes are close together, so that no proximal region has only a single axonemal profile, there is no coiling of the nucleus in the distal extremity and both axonemes continue beyond termination of the nucleus and mitochondrion. By comparison, the spiral endpiece of spermatids of C. kroyeri was not observed in mature sperm and the distal extremity showed many nuclear profiles suggesting that the nucleus is coiled in this region, beyond the termination of both axonemes. The spiral end piece has not been described in any
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N. A. WATSONand K. ROHDE
Sperm and spermiogenesis in monocotylids other monogeneans and is clearly an autapomorphy for the genus Calicotyle. The only possible equivalent among monogeneans may be the lateral dense body of Calceostoma (see Justine & Mattei, 1986) although in that species the dense body forms a short helix coiled around the shaft in the region about one third the total sperm length from the proximal end, and the sperm body is regularly bent at this point. The location of a similar electron-dense lateral crest in some cestodes more closely resembles that of the structure found in Calicotyle. A spiralling body has been observed, irregularly distributed in a large number of cestodes in the Diphyllidea, Cyclophyllidea and Pseudophyllidea (Justine, 1986) and, at least in tetraphyllideans, it is located at the proximal end (Euzet, Swiderski & Mokhtar-Maamouri, 1981). However, in the cestodes it extends for much longer and wraps around the shaft containing the axoneme, whereas in Calicotyle it is restricted to the end proximal to the axonemes. The chemical composition and function of these bodies are unknown. C. australiensis shares the following features with C. kroyeri and Heterocotyle (also Monocotylidae, see Justine & Mattei, 1983b) and with Loimosina wilsoni (Loimoidae, see Justine & Mattei, 1985). Two initially free axonemes develop at the beginning of spermiogenesis and soon fuse with each other (i.e. a median cytoplasmic process is not formed). The early ZD is surrounded by longitudinal cortical microtubules in dense cytoplasm, with some degree of ornamentation external to the plasma membrane. These microtubules subsequently become reduced in number and restricted in length to a short region of the sperm. Development of the spermatids results in proximal movement of basal bodies and the arching membrane, so that maturing spermatids lie in parallel canals within the residual cytoplasm. C. kroyeri, Heterocotyle and L. wiLsonialso have in common a terminal coiled nucleus, whereas in C. australiensis the nucleus is neither coiled nor terminal. The four species differ in the relative lengths of the two axonemes, from two normal axonemes of almost equal length in both the Calicotyle species, to one long and one shortened but otherwise normal axoneme in L. wilsoni, to one long and one much reduced and altered axoneme in Heterocotyle.
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Monocotyle helicophallus
This species, although closely related to Calicotyle and Heterocotyle in conventional taxonomy (both family Monocotylidae), differs in several important aspects of spermiogenesis and sperm structure from those two genera. In Monocotyle there are only 2-4 peripheral microtubules in the zone of differentiation (although a manschette of microtubules does surround. the nucleus proximal to the zone of differentiation), spermiogenesis does not proceed by a backward movement of basal bodies within the cytoplasm so that parallel canals are not formed, a coil of nucleus develops but is not located in a terminal position in the mature sperm, and most importantly, the mature sperm has two axonemes arranged almost end to end, with only a short or almost no region of overlap between them. Justine, Lambert & Mattei (1985) grouped Monocotylidae and Loimoidae together on the basis of a common sperm pattern, designated pattern 3 and characterised by the presence of one axoneme plus one altered axoneme, plus some microtubules. It was distinguished from sperm pattern 2, characterised by 2 axonemes and without microtubules (found in Capsalidae, Dionchidae, Euzetrema, Udonellidae and Gyrodactylidae). Acanthocotyle and Myxinidocotyle were subsequently included in the group with sperm pattern 2 (despite the presence of a single longitudinal microtubule in some regions of mature sperm) (Malmberg & Afzelius, 1990; Justine et al., 1993) and subsequently other capsalids and a gyrodactylid were also noted to have a single microtubule. Thus the definition of pattern 2 was amended to “spermatozoa with two axonemes and no cortical microtubules, except a single microtubule much shorter than the spermatozoon” (Justine et al., 1993). Table 1 summarises information regarding the presence and persistence of microtubules in the proximal region of spermatids and sperm, and the nature of axonemes in the mature sperm of taxa classified as belonging to sperm patterns 2 and 3. Within pattern 3, there may be a small number or a more or less complete ring of microtubules in the ZD, with from 1 to 6 remaining in a short region of the mature sperm. Axonemes vary from 1 normal and one altered, to 2 normal, to two shifted end-to-end. Within pattern 2, there may also be
FIGS.2632. Sections depicting various stages during spermatogenesis of Calicoryleaustraliensis.26. Part of the nucleus of a primary spermatocyte, showing nucleolus (nc) and synaptonemal complex (arrowhead). 27. Early spermatid protuberance, showing 2 basal bodies, microtubules associated with 1 basal body (arrowhead) and extending alongside the nucleus (n), electron-dense region at the apical membrane (arrows) and numerous mitochondria (m). 28. Zone of differentiation in transverse section, showing 2 basal bodies (b), migrating nucleus (n) and mitochondrion (m), a complete ring of cortical microtubules (arrowhead) and the spermatid lying in a canal within the cytoplasm ofthe cytophore. 29-31. Successive stages in the formation of the complex spiral end-piece of the spermatid. 32. Cytophore containing late spermatids with their proximal ends lying deep within parallel canals. Scale bars, 500 nm (27-31), 1 m (26), 2 q (32).
N. A. WATSON and K. ROHDE
36
2
38
FIGS. 3341. Sections of mature sperm from the sperm duct of Culicoryle austrafiensis. 33. Part of the spiral end-piece in transverse section. 34-35. Spiral end-piece in longitudinal section, depicting three turns of fine granular (almost amorphous) material, with coarse granular material (arrowhead) surrounding the axonemal attachment region and the mitochondrion (m). 36. Longitudinal section in the proximal region where the mitochondrion (m) has a wide profile. Note the numerous, closely spaced transverse cristae. 37-38. Sections through the same spermatozoan near its proximal end, showing the continuation of the mitochondrion (m) into the end-piece region and the beginnings of the 2 axonemes (b). Arrowhead points to the coarse granular material and arrows to the fine granular (almost amorphous) material. 39. Section through a spermatozoan near the proximal end, showing 2 axonemes, a large mitochondrial profile (m), small nuclear profile (n) and 2 cortical microtubules (arrowheads). 4C41. More distal regions of spermatozoa, with nuclear (n) and mitochondrial (m) profiles almost equal and tapering. Scale bars, 200 nm (3741) 500 nm (33-36).
:..,.: .,.::,: u
1027
Sperm and spermiogenesis in monocotylids
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FIGS. 42,43. Diagrams depicting spermiogenesis and mature sperm respectively in Monocotyle helicophallus. a, axoneme; c, cap on (presumed) proximal end of spermatozoan; m, mitochondrion; mt, microtubules; n, nucleus; ta, tapering axoneme.
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N. A. WATSONand K. ROHDE
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FIGS. 4% 45. Diagrams depicting spermiogenesis and mature sperm respectively in cu/icoty/e austru/iensis. a, axoneme; am, arching membrane; cc cytoplasmic canal; ep, spiral end-piece on proximal end of spermatid and spermatozoan; m, mitochondrion; mt, microtubules; n, nucleus.
Sperm and s~rmiogenesis
1029
in mon~otylids
TABLE ~--CHARACTERISTKSOFSPERMIOGENESISANDMATIJRESPERM INSOMESPECIESOFMONOGENEAMONOPISTHOCOTYLEA.SPERMPATTERNSFOLLOWING JUSTINE'STERMINOLOGY(JUSTINE,LAMBERT& MA~EI, 1985) Family
Sperm
Taxon
pattern Monocotylidae
3
~5noco~yle helicophalluv Calicotyle kroyeri Calicotyle australiensis Heterocot$e
Monocotylidae
3
Monocotylidae
3
Mon~tylidae
3
Loimoidae
3
Dionchidae
2
Capsalidae
2
CapsaIidae Capsalidae Acanthocotylidae
2 2 2
Loimosina wilsoni Dionchtu remorae Caballerocotyle manteri ~n~otyl~~e ~e~a~ocotyie Acanthocotyle
Acanthocotylidae
2
Myxinidocotyle
Microtubules Microtubules in ZD in mature sperm 14
1 or 2
ring
Axonemes in mature sperm
Origin of data
This paper
2
2-shifted end to end 2 normal
ring
2
2 normal
This paper
ring
approx. 6
ring
5
ring
none seen
ring
none seen
2 normal
Justine & Mattei, 1987
ring ring
1 1
1
1
2 normal 2 normal 2 normal
1 or 2
1
2 normal
Justine, Mattei di Euzet, 1991 Justine & Mattei, 1983a Malmberg & Afzelius, 1990, Justine et al., 1993 Malmberg % Afielius, 1990; Justine et al., 1993
Tappenden & Keam, 1991
1 normal Justine & Matteri, 1983b I altered and short 1 normal Justine & Mattei, 1985 1 short 2 normal Justine St Mattei, 1987
Note: we consider that there is no consistent difference between types 2 and 3 and they should be combined and redefined. a small number or a more or less complete ring of microtubules in the ZD, with 1 or none persisting, and all species appear to have 2 normal axonemes. It must be emphasised that detection of a small number of persistent microtubules in a very short region of mature sperm, and certainty of the normality of two axonemes, depends on very thorough examination of a large number of sections of mature sperm. Therefore the possibility cannot be ruled out that persistent microtubules are present in all taxa with sperm pattern 2, or that one axoneme may be altered or shortened in some of these taxa. Thus the distinction between patterns 2 and 3 is becoming less defined, with regard to peripheral microtubules and relative lengths, positions and normality of the two axonemes, and their separation can no longer be justified. The two patterns do have in common some or a complete ring of microtubules in the zone of di~erentiation, one or a few of these persisting in a short region of the mature sperm, and two normal axonemes present at the start of spermiogenesis, and at least the remnants or a shortened or displaced second axoneme remaining in the mature spermatozoon. Thus, collectively, patterns 2 and 3 are clearly distinguished from pattern 1, in which two normal axonemes and a complete row of peripheral microtubules are present throughout the principal region of the mature sperm, and from pattern 4 in which only one axoneme is present from the start of spermiogenesis and there are no cortical microtubules at any stage.
Utilising an enlarged data set, Justine (1993) no longer distinguished the Loimoidae/Monocotylidae on the basis of sperm structure but still considered it the sister group to all the other Monogenea, being “weakly defined by a homoplasic s~apomo~hy -the existence of a region without nucleus in the distal extremity of the sperm”. The present study has revealed that this characteristic may not be constant even within a single genus (Calicotyle kroyeri versus Calicotyle australiensis), hence it is of dubious usefulness. Justine (ibid) also states “in fact this group Loimoidae/Monocotylidae is defined by the absence of synapomorphies which characterise the other Monopisthocotylea”. The only two of these synapomorphies listed as being without homoplasy or reversal are characters 7 and 18. Character 7, cytoplasmic middle process and flagella fused from the start, does in fact show reversal (see ~c~~~~~coty~e spermiogenesis diagram in Tappenden & Kearn, 1990) and character 18 (microtubules in the zone of differentiation persisting or disappearing) can also be seen from Table 1 to be unstable. Thus, there are no clear differences in sperm or spermiogenesis between Loimoideae/~onocotylidae and the other Monopisthocotyleans. We therefore propose that the categories of sperm pattern found within the Monogenea be modified to combine the previously distinguished patterns 2 and 3. Justine et al. (1985) suggested that patterns 2 and 3 were derived from pattern 1 found in polyopisthocoty-
1030
N. A. WATSONand K. ROHDE
leans. He also suggested that pattern 3 with one altered axoneme, may be an intermediate stage between patterns 1 and 4. However, with the distinction between patterns 2 and 3 no longer recognisabte, it is equally possible that pattern 4 has evolved from a stage exhibiting two normal axonemes. This is a valid supposition considering that most of the species which have been examined and characterised as having either pattern 2 or 3, have a more or less complete ring of peripheral microtubules in the ZD. Within this pattern 213 state, Capsalidae and Dionchidae are characterised by the presence of a giant, bead-like mitochondrion which is probably synapomorphic for these two families. However, it appears that the other taxa cannot presently be divided along valid taxonomic lines by any of the known characteristics of spermiogenesis or mature sperm. Acknowledgements-This work was supported by a grant from the Australian Research Council. Dr I. Whittington, University of Queensland, collected and identified the specimens of M. helicophallus and perfonned the primary fixation. The specimens of C. uustrufiensis were collected and primary fixed by Craig Hayward during an expedition of F.R.V. “Southern Surveyor” (Australian Antarctic Division), in June-July, 1991. We thank P. Garlick for use of the facilities of the Electron Microscope Unit, University of New England, R. Porter for developing and Z. Enoch for printing the micrographs. REFERENCES Eu~ET’L., SWIDERSKIZ. & MOKHTAR-~AAMOURIF. 1981. Ultrastructure cornpa& du spermatozojide des cestodes. Relations avec la phylogenese. Annales de Parasitoiogie Humaine et Comparde 56: 241-259.
JUSTINEJ.-L. & MATTEIX. 1983a. Etude ultrastructurale comparee de la spermiogenese des monogenes. 1. Megulocotyte (Monop~stho~otyl~ Capsalidae). Journal of U&mstructure Research 82: 296308.
JUSTINEJ.-L. & MATTEIX. 1983b. Comparative ultrastructural study of spermiogenesis in monogeneans (flatworms). 2. Heteroco6yle (Monopisthocotylea Monocotylidae). Journal of Ultrasiructure Research 84: 213-223. JUSTINEJ.-L., LAMBERTA. & MATTEIX. 1985. Spennatozoon ultrastructure and phyiogenetic relationships in the mo~ogeneans (Platyhelminthes). ~nternutjo~~~Journ~ifor Parasitology 15: 601-608. JUSTINEJ.-L., LE BRUN N. & MATTEIX. 1985. Theflagellate spermatozoon of Diplozoon (Platyhelminthes: Monogenea: Polyopisthocotylea): A demonstrative case of relationship between sperm ultrastructure and biology of reproduction. Journal of U~trastru~ture Research 92: 47-54. JUSTINE J.-L. & MATTEI X. 1985. Ultrastructure de la spermiogentse et du spermatozoYde de Loimosina wilsoniet
affinit&s phyletiques des Loimoidae (Platyhelminthes, Monogenea, Monopisthocotylea). Zoologica Scripta 14: 169-175. JUSTINEJ.-L. 1986. Ultrastructure of the spermatozoon of the cestode Duthiersiu fimbriara Diesing, 1854 (Pseudophyllid~, DiphylIoboth~idae). Cunudiun Journal of Zoology 64: 154551548.
’
JUSTINEJ.-L. & MAI-~EIX. 1986. Comparative ultrastructural study of spermiogenesis in monogeneans (flatworms) 5. Calceostoma (Monopisthocotylea Calceostomatidae) Journal of UItrastructure and Molecular Structure Research. 96: 54-63.
JUSTINEJ.-L. & MATI’EIX. 1987. Phylogenetic relationships between the families Capsalidae and Dionchidae (Platyhelminthes, Monogenea, Monopisthocotylea) indicated by the comparative ultrastructural study of spenniogenesis. Zoologica Scripta 16: 11l-l 16. JUSTINE J.-L. 1991. Cladistic study in the Monogenea (Piatyhelminthes), based upon a parsimony analysis of spermiogenetic and spermatozoa1 characters. ~nfernufion~~ Journal for Parasitology 21: 821-838.
JUSTINEJ.-L., MAI”I’EIX. & EUZETL. 1991. Ultrastructure of spermatozoa in two monopisthocotylean monogeneans: EncotyNabe sp. (Capsalidae) and Z’etraonchoides sp. (Tetraonchoidae). Annaies de Parasito~ogie H~aine et Comparee 66 173-178. JUSTINEJ.-L. 1992. Ultrastructure of spermiogenesis, spermatozoa and the tegument in Atriuster sp. (Platyhelminthes, Monogenea, Polyopisthocotylea, Microcotylidae). Zoologica Scripta 21: 231-238. JUSTINEJ.-L. 1993. Phylogenie des Mono&es basee sur une analyse de parcimonie des caracttres de l’ultrastru~ture de la spermiogenese et des spermatozdides incluant les risultats r¢s. Bulletin Fran@s de la P&he et de la Pisriculture 328. 137-l 55.
JUSTINEJ.-L., AFZELIUSB. A., MALMEERGG. & MATTEIX. 1993. Ultrastructure of spermiogenesis in Acanthocotyle and Nyxinidocotyte (Platyhelminthes, Monogenea, Acantocotylidae). Acta Zoologica {Stockholm) 74: 119-l 26. MALMBERG G. & AFZELIUSB. A. 1990. Sperm ultrastructure in h4yxinidocotyle and Acanthocotyle (Platyhelminthes, Monogenea, Acanthocotylidae). Zoologica Scripfa 19: 129-132. TAPP~NDENT. & KEARN G. C. 1990. Spermiogenesis and sperm ultrastructure in the monogenean parasite Acanthocotyie lobi~chi. Znternat~on~iJournu~for P~r~jtology 20: 747-753.
TAPPENDENT. & KEARN G. C. 199t. Spermiogenesis and sperm ultrastructure in the monocotylid monogenean Calicotyle kroyeri. international Journal for Parasitology 21: 5763. WATSONN. A. & ROHDEK. 1992. Ultrastructure of sperm and s~rmatogenesis of Anop~od~~s ~~rr~sp~ra~~(Monogenea, Monopis~ocotylea). Annales de Parasitoiogie Humaine et Comparee 67: 131-140.
Pergamon
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ULTRASTRUCTURE OF SPERM AND SPERMI~GENESIS IN THE MONOCOTYLID MONOGENEANS MONOCOTYLE HELICOPHALLUS AND CALICOTYLE AUSTRALIENSIS (PLATYHELMINTHES) N. A. WATSON*
and K. ROHDE
Department of Zoology, University of New England, Armidale, NSW 2351, Australia (Received 19 August 1993; accepted 15 February 1994)
N. A. and ROHDE K. 1994.Ultrastructure of sperm and spermiogenesis in the monogeneans Monocotyle helicophallus and Calicotyie australiensis (Platyhelminthes). ~~ier~ationai ~ournalfor Par~~tology 24: 1019-1030. Spermiogenesis in ~~~oco~y~e hei~caphall~ involves formation of free flagella which rotate to lie parallel before fusing. There is no median cytoplasmic process. A small number of microtubules (usually 2-4) is associated with the axonemes in the zone of differentiation. Nucleus and a fused mitochondrion migrate alongside the axonemes and one basal body moves distally. A coil of nucleus, originally in the cytophore., also moves out along the shaft. The mature spermatozoan has only a single axoneme for a short distance at each end, with two associated microtubules at the proximal end, as well as one axoneme, nucleus and mitochond~on throughout most of its length (the nucleus being enlarged and roughly coiled in one region), and a short region of two overlapping axonemes. We interpret our findings as two axonemes arranged almost end to end, one extending from the proximal end to some point in the mid-region, where the second axoneme begins and continues to the distal end of the sperm. Spermatozoa of Ca&otyfe austraiiensis develop from a zone of differentiation which has two basal bodies and a complete ring of corticat microtubules. Two initially free axonemes fuse with each other and there is no median cytoplasmic process. Spermatids form within parallel canals in the cytophore, by backward movement of the zone of differentiation. Prior to detachment, an electron-dense spiral end-piece forms around and proximal to the basal body region. Sixty-four spermatids are present in each isogenic group. With the study of spermiogenesis in more species of Monogenea Monopisthoc~tylea, it is apparent that the previously designated sperm patterns 2 and 3 are not distinct and should be combined and re-defined. Species previously designated as having sperm patterns 2 and 3 can all be accommodated by the description “two normal axonemes or one normal and one shortened, altered or displaced axoneme, and none, one or a few cortical microtubules remaining in a region of the sperm derived from the zone of differentiation in which a few or a complete ring of microtubules was present”. bstract-WATSON
monocotylid
INDEX KEY WORDS: Monogenea; Monopisthocotylea; phylogeny; spermatozoa; ultrastructure; Monocotyle helicophallus; Calicotyle australiensis.
INTRODUCTION ultrastructural investigations of sperm and spermiogenesis have been undertaken in species of Monogenea (see Justine 1991 for review, Justine, Mattei & Euzet, 1991; Tappenden & Kearn, 1991; Watson & Rohde, 1992; Justine, 1992; Justine, Afzelius, Malmberg & Mattei, 1993) and such studies are useful for phylogenetic considerations. Justine, Lambert & Mattei (1985) arranged the Monogenea into 4 phylogenetic groups based on spermatozoa1 patterns in the various families: pattern 1 characterised by the presence of 2 axonemes plus cortical microtubules, pattern 2 by 2 axonemes without NUMEROUS
*To whom all correspondence should be addressed.
spermiogenesis;
microtubules, pattern 3 by I axoneme plus I altered axoneme plus microtubules, and pattern 4 by 1 axoneme without microtubules. A fifth (aberrant) type was later defined for Di~lozo~~, which has no axoneme, and numerous cortical and cytoplasmic microtubules (Justine, Le Brun & Mattei, 1985). All Polyopisthocotylea examined so far have sperm pattern 1 except for the aberrant ~~~~~z~Q~, while types 2, 3 and 4 are found in various families of Monopisthocotylea. Sperm pattern 3 (1 normal axoneme, 1 altered axoneme and some cortical microtubules) was established from observations on one member of the family Monocotylidae, viz. HeterocotyIe (see Justine & Mattei, 1983b) and one of the family Loimoidae, viz. Loimosina (see Justine & Mattei, 1985). Subsequently Tappenden & Kearn
1020
N. A. WATSON and K.
(1991) found two complete axonemes in another monocotylid, Calicotyle kroyeri. We examined sperm and spermiogenesis in 2 more species of monocotylids in order to determine whether any further variation exists in the characteristics used to define sperm pattern and phylogenetic relationships in the Monopisthocotylea. MATERIALS AND METHODS Specimens of ~o~~oryle ~el~eo~~~i~~were removed from the gills of Himanrura sp. from Heron Island, off the coast of Queensland, Australia. They were fixed for 3-4 h in 3% ~utaraldehyde in 0. I M sodium cacodylate buffer, made with filtered seawater, washed several times in the same buffer, then stored in buffer for 2 days. Post fixation in 1% 0~0, in the same buffer for 1h at room temperature was followed by dehydration in alcohol and embedding in Spurr’s resin. Specimens of Calicotyle australiensis were obtained from a chimaerid host off the NE coast of Tasmania, fixed for 2 h in the same primary fixative as above, washed then stored in buffer for 21 days and further processed as for M. helicophallus. Sections were stained with uranyl acetate and lead citrate and examined under a JEOL 1200 EX electron microscope at 60 kV.
ROHDE
begins to migrate beside the axonemes (Figs. 5 and 13), which often appear bent back towards the spermatid body during this phase (Fig. 5). One axoneme then shifts distally (Figs. 6,8b and 14) while the nucleus and a long fused mitochondrion migrate along the shaft (Fig. 8c, d) A part of the nucleus still within the cytophore becomes coiled and this twisted region moves out along the spermatid shaft. Figures 7,9 and 10 show such coils in longitudinal and transverse sections. They may surround one or both axonemes, or form a twisted mass adjacent to them. A small variable number (14, most commonly 2) of microtubules lies for a short distance only along the periphery of the spermatid, initially in the zone of differentiation (ZD) (Figs. 2, 8a, b, c, 12, 14 and 15). A small dense region of ornamentation is usually present on the outer surface of the plasma membrane adjacent to each microtubule. It was not possible to determine the number of spermatids in each isogenic group because they do not develop in parallel canals as do the spermatids of many other monopisthocotylean monogeneans. Mature
RESULTS Spermiogenesis in Monocotyle The process of spermiogenesis is depicted diagrammatically in Fig. 42. Early spermatids, joined in cytophores, are wedge-shaped and develop two centrioles at right angles to each other near the apical cell membrane (Figs. 1 and 2). The nucleus is also wedge-shaped with the pointed end nearest the centrioles. Free flagella then grow from the basal bodies (Fig. 3) and subsequently rotate to lie parallel with each other, in a more or less straight line with the elongating body of the spermatid (Fig. 4). Chromatin begins to condense within the nucleus and the nucleus elongates. Cross-sections (Fig. 11) through the nuclear region of the spermatid at this stage show some microtubules around the nucleus. The two adjacent axonemes then fuse with each other and the nucleus
sperm of Monocotyle Mature sperm in the seminal vesicle have a single axoneme, nucleus and mitochondrion throughout much of their length (Figs. 16, 17,21 and 22). Two or three cortical microtubules associated with ornamentation of the plasma membrane can be seen in various cross sections, most of which show only a single complete or tapering axoneme (Figs. 17e and 22). Figure 16b, however, shows one sperm at the level of 2 axonemes, nucleus and mitochondrion, with 2 peripheral microtubules, although the presence of two profiles of mitochondrion may indicate that this is a folded or otherwise aberrant region. At both ends of the sperm a short region is devoid of nuclear and mitochondrial profiles; at one end the axoneme tapers and terminates over a short distance and has an electron dense cap (Figs. 19 and 21 arrowhead) (this may be the proximal end if the cap equates with a
FIGS. I-15. Sections of spermatids at various stages during spermiogenesis of ~o~oeor~~e heijcop~aft~s. 1. Early spermatid with centrioles (arrowheads) at right angles to each other. 2. Transverse section of centriole of early spermatid and 2 cortical microtubules (arrowhead). 3. Free flagella grow at right angles to each other from the apex of the early spermatid. 4. Flagella rotate to lie parallel with each other. 5. Flagella fuse with each other and the nucleus begins to migrate alongside them. 6. One axoneme shifts distally relative to the other (arrowheads indicate basal bodies). 7. Longitudinal section of the region of a spermatid with a coil of nuclear material. 8. Transverse sections through some developing spermatids. a -single axoneme and 2 cortical microtubules, b ~ one axoneme shifted relative to the other, c-nucleus migrates ahead of the mitochondrion, 2 cortical microtubules present, d-distal to the region with 2 cortical microtubules, e-one axoneme terminating within the spermatid. 9. Transverse section of the region of nuclear coil, with 2 axonemes separated by nuclear material. 10. Similar to Fig. 9 but axonemes remain adjacent. i 1. Transverse section through early outgrowing spermatid, proximal to the basal bodies of the axonemes. Note the “manschette” of microtubules around the nucleus. 12. Early free flagella, each with associated cortical microtubules. 13. Spermatid before one axoneme has shifted relative to the other. 14. Spermatid after shift of one axoneme. with 3 cortical microtubules (arrowh~d). 15.An early, free axoneme with 2 cortical mi~rotubules (arrowhead). n -nucleus, m - mitochondrion. Scale bars, 200 nm (2, 7, 8, l&15), 500 nm (1, 3, 4, 6, 9), 1 F (5).
Sperm and spermiogenesis in monocotylids
1021
1022
N. A. WATSONand K. ROHDE
FIGS. 16-2.5. Sections through mature sperm from the seminal vesicle of Monocoryle hel~c~pha~lus. 16. a - most common profile containing 1 axoneme, nucleus and mitochondrion, b - 2 axonemes, nucleus, 2 mitochondrial profiles and 2 cortical microtubules with associated ornamentations (arrowhead), c- near oneend with only nucleus and a single axoneme, d - the region of transition, 1 axoneme terminating and the other beginning, nucleus and mitochundrion present, e-tapering microtubular doublets, probably the distal extremity of the sperm. 17. a -most common profile, b - 2 complete axonemes, nucleus and 1 or 2 mitochondrial profiles, c - region of transition, 1 axoneme normal, the other tapering, d - both axonemes tapering, e - proximal end with a single axoneme and 2 cortical microtubules with associated ornamentations (arrowhead). 18. distal extremity of sperm, nucleus terminates (arrowhead) and axoneme tapers. 19. Proximal extremity of sperm where axoneme ends in a ‘bulb (arrowhead) and the nucleus commences (arrow). 20. Region of transition with continuous nucleus and tapering axoneme (arrowh~d). 21. Transverse section depicting both proximal (arrowhead) and distal (arrow) regions. 22. Transverse section through the proximal region containing a single axoneme and 4 cortical microtubules with associated ornamentations (arrowhead). 23-25. Transition regions showing varying degrees of overlap of the tapering axonemes, nucleus (n) and mitochondrion (mf present. Scale bars, 200 nm (21-25), 500 nm (14-20).
1023
Sperm and spermiogenesis in monocotylids centriolar adjunct), while at the other end the microtubules diminish gradually and the axoneme tapers to a fine end (Figs. 16e, 18 and 21). The nucleus and mitochondrion are of almost equal length since few profiles show only one or the other present beside the single axoneme. Detailed examination of a large number of mature sperm revealed a small percentage of profiles showing two complete axonemes or one complete axoneme and one with a reduced number of doublets or with only single microtubules present (Figs. 16b and 17b, c). This could be interpreted as one axoneme present throughout the whole length of the sperm and a second much shorter and tapering one along a short region. However, some longitudinal sections (e.g. Fig. 20) suggested that one axoneme may terminate and another begin at some point along the body of the sperm, i.e. that two normal axonemes were present with a short region of overlap between them. This suggestion was confirmed by finding sections where the region of overlap was so short that one axoneme was terminating and the other beginning in the one profile (Figs. 16d, 17d and 23-25). We therefore propose that the mature sperm has the structure depicted in Fig. 43. Spermiogenesis in Caliocotyle
The process of spermiogenesis is depicted diagrammatically in Fig. 44. Clusters of spermatogonia are scattered within the testis, not restricted to the wall region. Primary spermatocytes, identified by the presence of synaptonemal complexes, have a large nucleus with a prominent nucleolus (Fig. 26), and many small mitochondria. Early spermatids are joined in a rosette formation and a zone of differentiation develops at the apex of each spermatid, containing two centrioles at right angles to each other. Two free cilia develop and microtubules radiating from their basal bodies. extend alongside the elongating nucleus (Fig. 27). Microtubules may be present in dense cytoplasm beneath the apical cell membrane (Fig. 27 arrows). The basal bodies then rotate (probably by means of the attached microtubules) to lie parallel with one another, and the nucleus and fused mitochondrion move into the apical region, which is now surrounded by a complete ring of microtubules (Fig. 28). Basal bodies then migrate proximally, deeper into the cytoplasmic mass, and the accompanying backward movement of an arching membrane results in the spermatid shaft lying in a canal, deeper and deeper within the spermatid mass. One basal body shifts a short distance in relation to the other during this stage of elongation of the shaft. When most of the cytoplasmic mass has been incorporated into the elongate spermatids and they lie in parallel canals, a complex spiral end-piece is formed around the basal
body and adjacent axonemal region. Figures 29-31 depict stages in this development, which involves condensation of cytoplasm and contraction and rounding of the contours of the spiral end-piece. Figure 32 indicates that growth of spermatids has involved backward movement of the ZD deep into the cytoplasmic mass. There are 64 spermatids in each isogenic group. Spermatozoa then detach from the residual cytoplasm. Mature sperm of Calicotyle
Transverse sections of mature sperm from the sperm duct indicate the following arrangement of its elements: at the most proximal end, an open spiral of fine granular to amorphous, electron-dense material is joined to the rest of the sperm by a more coarsely granular, electron-dense region in which the two basal bodies and the end of the mitochondrion are embedded (Figs. 33-35,37 and 38). Distal to this endpiece region, the mitochondrial profile is greatly enlarged (Figs. 36 and 39) a thin tail of the nucleus is seen and two peripheral longitudinal microtubules lie adjacent to the nucleus (Fig. 39). In this region the mitochondrion has numerous, prominent cristae (Fig. 36). More distal, nucleus and mitochondrion are approximately equal in diameter (Fig. 40), both taper (Fig. 41) and disappear, leaving two axonemes and finally a single tapering axoneme in the most distal extremity. There are no peripheral microtubules in the more distal regions. Figure 45 is a diagram of the proposed structure of the mature sperm. DISCUSSION Calicotyle australiensis
Features of spermiogenesis
and mature sperm in
Calicotyle australiensis resemble those of C. kroyeri (see Tappenden & Kearn, 1991) although some
differences were noted. Thus, in early spermiogenesis axonemes of C. australiensis initially extend at right angles to each other before they rotate to lie parallel, and the proximal spiral end-piece which develops late in spermiogenesis exhibits 34 turns in C. australiensis compared with 7 or 8 turns in C. kroyeri. The mature sperm of C. australiensis retains the spiral end-piece (compacted to three turns), the basal bodies of the 2 axonemes are close together, so that no proximal region has only a single axonemal profile, there is no coiling of the nucleus in the distal extremity and both axonemes continue beyond termination of the nucleus and mitochondrion. By comparison, the spiral endpiece of spermatids of C. kroyeri was not observed in mature sperm and the distal extremity showed many nuclear profiles suggesting that the nucleus is coiled in this region, beyond the termination of both axonemes. The spiral end piece has not been described in any
1024
N. A. WATSONand K. ROHDE
Sperm and spermiogenesis in monocotylids other monogeneans and is clearly an autapomorphy for the genus Calicotyle. The only possible equivalent among monogeneans may be the lateral dense body of Calceostoma (see Justine & Mattei, 1986) although in that species the dense body forms a short helix coiled around the shaft in the region about one third the total sperm length from the proximal end, and the sperm body is regularly bent at this point. The location of a similar electron-dense lateral crest in some cestodes more closely resembles that of the structure found in Calicotyle. A spiralling body has been observed, irregularly distributed in a large number of cestodes in the Diphyllidea, Cyclophyllidea and Pseudophyllidea (Justine, 1986) and, at least in tetraphyllideans, it is located at the proximal end (Euzet, Swiderski & Mokhtar-Maamouri, 1981). However, in the cestodes it extends for much longer and wraps around the shaft containing the axoneme, whereas in Calicotyle it is restricted to the end proximal to the axonemes. The chemical composition and function of these bodies are unknown. C. australiensis shares the following features with C. kroyeri and Heterocotyle (also Monocotylidae, see Justine & Mattei, 1983b) and with Loimosina wilsoni (Loimoidae, see Justine & Mattei, 1985). Two initially free axonemes develop at the beginning of spermiogenesis and soon fuse with each other (i.e. a median cytoplasmic process is not formed). The early ZD is surrounded by longitudinal cortical microtubules in dense cytoplasm, with some degree of ornamentation external to the plasma membrane. These microtubules subsequently become reduced in number and restricted in length to a short region of the sperm. Development of the spermatids results in proximal movement of basal bodies and the arching membrane, so that maturing spermatids lie in parallel canals within the residual cytoplasm. C. kroyeri, Heterocotyle and L. wiLsonialso have in common a terminal coiled nucleus, whereas in C. australiensis the nucleus is neither coiled nor terminal. The four species differ in the relative lengths of the two axonemes, from two normal axonemes of almost equal length in both the Calicotyle species, to one long and one shortened but otherwise normal axoneme in L. wilsoni, to one long and one much reduced and altered axoneme in Heterocotyle.
1025
Monocotyle helicophallus
This species, although closely related to Calicotyle and Heterocotyle in conventional taxonomy (both family Monocotylidae), differs in several important aspects of spermiogenesis and sperm structure from those two genera. In Monocotyle there are only 2-4 peripheral microtubules in the zone of differentiation (although a manschette of microtubules does surround. the nucleus proximal to the zone of differentiation), spermiogenesis does not proceed by a backward movement of basal bodies within the cytoplasm so that parallel canals are not formed, a coil of nucleus develops but is not located in a terminal position in the mature sperm, and most importantly, the mature sperm has two axonemes arranged almost end to end, with only a short or almost no region of overlap between them. Justine, Lambert & Mattei (1985) grouped Monocotylidae and Loimoidae together on the basis of a common sperm pattern, designated pattern 3 and characterised by the presence of one axoneme plus one altered axoneme, plus some microtubules. It was distinguished from sperm pattern 2, characterised by 2 axonemes and without microtubules (found in Capsalidae, Dionchidae, Euzetrema, Udonellidae and Gyrodactylidae). Acanthocotyle and Myxinidocotyle were subsequently included in the group with sperm pattern 2 (despite the presence of a single longitudinal microtubule in some regions of mature sperm) (Malmberg & Afzelius, 1990; Justine et al., 1993) and subsequently other capsalids and a gyrodactylid were also noted to have a single microtubule. Thus the definition of pattern 2 was amended to “spermatozoa with two axonemes and no cortical microtubules, except a single microtubule much shorter than the spermatozoon” (Justine et al., 1993). Table 1 summarises information regarding the presence and persistence of microtubules in the proximal region of spermatids and sperm, and the nature of axonemes in the mature sperm of taxa classified as belonging to sperm patterns 2 and 3. Within pattern 3, there may be a small number or a more or less complete ring of microtubules in the ZD, with from 1 to 6 remaining in a short region of the mature sperm. Axonemes vary from 1 normal and one altered, to 2 normal, to two shifted end-to-end. Within pattern 2, there may also be
FIGS.2632. Sections depicting various stages during spermatogenesis of Calicoryleaustraliensis.26. Part of the nucleus of a primary spermatocyte, showing nucleolus (nc) and synaptonemal complex (arrowhead). 27. Early spermatid protuberance, showing 2 basal bodies, microtubules associated with 1 basal body (arrowhead) and extending alongside the nucleus (n), electron-dense region at the apical membrane (arrows) and numerous mitochondria (m). 28. Zone of differentiation in transverse section, showing 2 basal bodies (b), migrating nucleus (n) and mitochondrion (m), a complete ring of cortical microtubules (arrowhead) and the spermatid lying in a canal within the cytoplasm ofthe cytophore. 29-31. Successive stages in the formation of the complex spiral end-piece of the spermatid. 32. Cytophore containing late spermatids with their proximal ends lying deep within parallel canals. Scale bars, 500 nm (27-31), 1 m (26), 2 q (32).
N. A. WATSON and K. ROHDE
36
2
38
FIGS. 3341. Sections of mature sperm from the sperm duct of Culicoryle austrafiensis. 33. Part of the spiral end-piece in transverse section. 34-35. Spiral end-piece in longitudinal section, depicting three turns of fine granular (almost amorphous) material, with coarse granular material (arrowhead) surrounding the axonemal attachment region and the mitochondrion (m). 36. Longitudinal section in the proximal region where the mitochondrion (m) has a wide profile. Note the numerous, closely spaced transverse cristae. 37-38. Sections through the same spermatozoan near its proximal end, showing the continuation of the mitochondrion (m) into the end-piece region and the beginnings of the 2 axonemes (b). Arrowhead points to the coarse granular material and arrows to the fine granular (almost amorphous) material. 39. Section through a spermatozoan near the proximal end, showing 2 axonemes, a large mitochondrial profile (m), small nuclear profile (n) and 2 cortical microtubules (arrowheads). 4C41. More distal regions of spermatozoa, with nuclear (n) and mitochondrial (m) profiles almost equal and tapering. Scale bars, 200 nm (3741) 500 nm (33-36).
:..,.: .,.::,: u
1027
Sperm and spermiogenesis in monocotylids
..’ ..:
p
4 :I(b .’ :.
‘.
.‘;;;.
; .;
,!! . :
‘. ..‘. .. .:
‘.‘.
.:. .. :
a
a
I
a
FIGS. 42,43. Diagrams depicting spermiogenesis and mature sperm respectively in Monocotyle helicophallus. a, axoneme; c, cap on (presumed) proximal end of spermatozoan; m, mitochondrion; mt, microtubules; n, nucleus; ta, tapering axoneme.
1028
N. A. WATSONand K. ROHDE
v
v
_... . (pi: :.. .!,.,.
1.g
:. ‘; _ :
m
P
-am-
I-- 1”_ L
AYarn mt-
a
-cc
a
a
a
-
m-
-n
1111&11111 a a mt
FIGS. 4% 45. Diagrams depicting spermiogenesis and mature sperm respectively in cu/icoty/e austru/iensis. a, axoneme; am, arching membrane; cc cytoplasmic canal; ep, spiral end-piece on proximal end of spermatid and spermatozoan; m, mitochondrion; mt, microtubules; n, nucleus.
Sperm and s~rmiogenesis
1029
in mon~otylids
TABLE ~--CHARACTERISTKSOFSPERMIOGENESISANDMATIJRESPERM INSOMESPECIESOFMONOGENEAMONOPISTHOCOTYLEA.SPERMPATTERNSFOLLOWING JUSTINE'STERMINOLOGY(JUSTINE,LAMBERT& MA~EI, 1985) Family
Sperm
Taxon
pattern Monocotylidae
3
~5noco~yle helicophalluv Calicotyle kroyeri Calicotyle australiensis Heterocot$e
Monocotylidae
3
Monocotylidae
3
Mon~tylidae
3
Loimoidae
3
Dionchidae
2
Capsalidae
2
CapsaIidae Capsalidae Acanthocotylidae
2 2 2
Loimosina wilsoni Dionchtu remorae Caballerocotyle manteri ~n~otyl~~e ~e~a~ocotyie Acanthocotyle
Acanthocotylidae
2
Myxinidocotyle
Microtubules Microtubules in ZD in mature sperm 14
1 or 2
ring
Axonemes in mature sperm
Origin of data
This paper
2
2-shifted end to end 2 normal
ring
2
2 normal
This paper
ring
approx. 6
ring
5
ring
none seen
ring
none seen
2 normal
Justine & Mattei, 1987
ring ring
1 1
1
1
2 normal 2 normal 2 normal
1 or 2
1
2 normal
Justine, Mattei di Euzet, 1991 Justine & Mattei, 1983a Malmberg & Afzelius, 1990, Justine et al., 1993 Malmberg % Afielius, 1990; Justine et al., 1993
Tappenden & Keam, 1991
1 normal Justine & Matteri, 1983b I altered and short 1 normal Justine & Mattei, 1985 1 short 2 normal Justine St Mattei, 1987
Note: we consider that there is no consistent difference between types 2 and 3 and they should be combined and redefined. a small number or a more or less complete ring of microtubules in the ZD, with 1 or none persisting, and all species appear to have 2 normal axonemes. It must be emphasised that detection of a small number of persistent microtubules in a very short region of mature sperm, and certainty of the normality of two axonemes, depends on very thorough examination of a large number of sections of mature sperm. Therefore the possibility cannot be ruled out that persistent microtubules are present in all taxa with sperm pattern 2, or that one axoneme may be altered or shortened in some of these taxa. Thus the distinction between patterns 2 and 3 is becoming less defined, with regard to peripheral microtubules and relative lengths, positions and normality of the two axonemes, and their separation can no longer be justified. The two patterns do have in common some or a complete ring of microtubules in the zone of di~erentiation, one or a few of these persisting in a short region of the mature sperm, and two normal axonemes present at the start of spermiogenesis, and at least the remnants or a shortened or displaced second axoneme remaining in the mature spermatozoon. Thus, collectively, patterns 2 and 3 are clearly distinguished from pattern 1, in which two normal axonemes and a complete row of peripheral microtubules are present throughout the principal region of the mature sperm, and from pattern 4 in which only one axoneme is present from the start of spermiogenesis and there are no cortical microtubules at any stage.
Utilising an enlarged data set, Justine (1993) no longer distinguished the Loimoidae/Monocotylidae on the basis of sperm structure but still considered it the sister group to all the other Monogenea, being “weakly defined by a homoplasic s~apomo~hy -the existence of a region without nucleus in the distal extremity of the sperm”. The present study has revealed that this characteristic may not be constant even within a single genus (Calicotyle kroyeri versus Calicotyle australiensis), hence it is of dubious usefulness. Justine (ibid) also states “in fact this group Loimoidae/Monocotylidae is defined by the absence of synapomorphies which characterise the other Monopisthocotylea”. The only two of these synapomorphies listed as being without homoplasy or reversal are characters 7 and 18. Character 7, cytoplasmic middle process and flagella fused from the start, does in fact show reversal (see ~c~~~~~coty~e spermiogenesis diagram in Tappenden & Kearn, 1990) and character 18 (microtubules in the zone of differentiation persisting or disappearing) can also be seen from Table 1 to be unstable. Thus, there are no clear differences in sperm or spermiogenesis between Loimoideae/~onocotylidae and the other Monopisthocotyleans. We therefore propose that the categories of sperm pattern found within the Monogenea be modified to combine the previously distinguished patterns 2 and 3. Justine et al. (1985) suggested that patterns 2 and 3 were derived from pattern 1 found in polyopisthocoty-
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N. A. WATSONand K. ROHDE
leans. He also suggested that pattern 3 with one altered axoneme, may be an intermediate stage between patterns 1 and 4. However, with the distinction between patterns 2 and 3 no longer recognisabte, it is equally possible that pattern 4 has evolved from a stage exhibiting two normal axonemes. This is a valid supposition considering that most of the species which have been examined and characterised as having either pattern 2 or 3, have a more or less complete ring of peripheral microtubules in the ZD. Within this pattern 213 state, Capsalidae and Dionchidae are characterised by the presence of a giant, bead-like mitochondrion which is probably synapomorphic for these two families. However, it appears that the other taxa cannot presently be divided along valid taxonomic lines by any of the known characteristics of spermiogenesis or mature sperm. Acknowledgements-This work was supported by a grant from the Australian Research Council. Dr I. Whittington, University of Queensland, collected and identified the specimens of M. helicophallus and perfonned the primary fixation. The specimens of C. uustrufiensis were collected and primary fixed by Craig Hayward during an expedition of F.R.V. “Southern Surveyor” (Australian Antarctic Division), in June-July, 1991. We thank P. Garlick for use of the facilities of the Electron Microscope Unit, University of New England, R. Porter for developing and Z. Enoch for printing the micrographs. REFERENCES Eu~ET’L., SWIDERSKIZ. & MOKHTAR-~AAMOURIF. 1981. Ultrastructure cornpa& du spermatozojide des cestodes. Relations avec la phylogenese. Annales de Parasitoiogie Humaine et Comparde 56: 241-259.
JUSTINEJ.-L. & MATTEIX. 1983a. Etude ultrastructurale comparee de la spermiogenese des monogenes. 1. Megulocotyte (Monop~stho~otyl~ Capsalidae). Journal of U&mstructure Research 82: 296308.
JUSTINEJ.-L. & MATTEIX. 1983b. Comparative ultrastructural study of spermiogenesis in monogeneans (flatworms). 2. Heteroco6yle (Monopisthocotylea Monocotylidae). Journal of Ultrasiructure Research 84: 213-223. JUSTINEJ.-L., LAMBERTA. & MATTEIX. 1985. Spennatozoon ultrastructure and phyiogenetic relationships in the mo~ogeneans (Platyhelminthes). ~nternutjo~~~Journ~ifor Parasitology 15: 601-608. JUSTINEJ.-L., LE BRUN N. & MATTEIX. 1985. Theflagellate spermatozoon of Diplozoon (Platyhelminthes: Monogenea: Polyopisthocotylea): A demonstrative case of relationship between sperm ultrastructure and biology of reproduction. Journal of U~trastru~ture Research 92: 47-54. JUSTINE J.-L. & MATTEI X. 1985. Ultrastructure de la spermiogentse et du spermatozoYde de Loimosina wilsoniet
affinit&s phyletiques des Loimoidae (Platyhelminthes, Monogenea, Monopisthocotylea). Zoologica Scripta 14: 169-175. JUSTINEJ.-L. 1986. Ultrastructure of the spermatozoon of the cestode Duthiersiu fimbriara Diesing, 1854 (Pseudophyllid~, DiphylIoboth~idae). Cunudiun Journal of Zoology 64: 154551548.
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JUSTINEJ.-L. & MAI-~EIX. 1986. Comparative ultrastructural study of spermiogenesis in monogeneans (flatworms) 5. Calceostoma (Monopisthocotylea Calceostomatidae) Journal of UItrastructure and Molecular Structure Research. 96: 54-63.
JUSTINEJ.-L. & MATI’EIX. 1987. Phylogenetic relationships between the families Capsalidae and Dionchidae (Platyhelminthes, Monogenea, Monopisthocotylea) indicated by the comparative ultrastructural study of spenniogenesis. Zoologica Scripta 16: 11l-l 16. JUSTINE J.-L. 1991. Cladistic study in the Monogenea (Piatyhelminthes), based upon a parsimony analysis of spermiogenetic and spermatozoa1 characters. ~nfernufion~~ Journal for Parasitology 21: 821-838.
JUSTINEJ.-L., MAI”I’EIX. & EUZETL. 1991. Ultrastructure of spermatozoa in two monopisthocotylean monogeneans: EncotyNabe sp. (Capsalidae) and Z’etraonchoides sp. (Tetraonchoidae). Annaies de Parasito~ogie H~aine et Comparee 66 173-178. JUSTINEJ.-L. 1992. Ultrastructure of spermiogenesis, spermatozoa and the tegument in Atriuster sp. (Platyhelminthes, Monogenea, Polyopisthocotylea, Microcotylidae). Zoologica Scripta 21: 231-238. JUSTINEJ.-L. 1993. Phylogenie des Mono&es basee sur une analyse de parcimonie des caracttres de l’ultrastru~ture de la spermiogenese et des spermatozdides incluant les risultats r¢s. Bulletin Fran@s de la P&he et de la Pisriculture 328. 137-l 55.
JUSTINEJ.-L., AFZELIUSB. A., MALMEERGG. & MATTEIX. 1993. Ultrastructure of spermiogenesis in Acanthocotyle and Nyxinidocotyte (Platyhelminthes, Monogenea, Acantocotylidae). Acta Zoologica {Stockholm) 74: 119-l 26. MALMBERG G. & AFZELIUSB. A. 1990. Sperm ultrastructure in h4yxinidocotyle and Acanthocotyle (Platyhelminthes, Monogenea, Acanthocotylidae). Zoologica Scripfa 19: 129-132. TAPP~NDENT. & KEARN G. C. 1990. Spermiogenesis and sperm ultrastructure in the monogenean parasite Acanthocotyie lobi~chi. Znternat~on~iJournu~for P~r~jtology 20: 747-753.
TAPPENDENT. & KEARN G. C. 199t. Spermiogenesis and sperm ultrastructure in the monocotylid monogenean Calicotyle kroyeri. international Journal for Parasitology 21: 5763. WATSONN. A. & ROHDEK. 1992. Ultrastructure of sperm and s~rmatogenesis of Anop~od~~s ~~rr~sp~ra~~(Monogenea, Monopis~ocotylea). Annales de Parasitoiogie Humaine et Comparee 67: 131-140.