Belg. J. Zool. - Volume 128 ( 1998) - tssue 2 - pages 177-188 - Brussels 1998

Received: 22 April 1998

SPERMATOGENESIS OF HAPLOPHARYNX ROSTRATUS (PLATYHELMINTHES, )

KLAUS ROHDE (')AND ANNO FAU BEL(') (') Division of Zoology, School of Biological Sciences, University of New England, Armidale, NSW 2351, Australia (') Institut für Hydrobicilogie und Fischereiwissenschaft, Universitat Hamburg, D-2000, Hamburg 50, Germany e-mail: [email protected]

Abstract. During spermatogenesis, nuclei of spermatids are at first large with an irregular out­ li ne, containing chroma tin granules; then condense to become round containing dense chroma tin ; then elongate containing loose fibrillar chromatin. Golgi bodies produce dense granules. Immature sperm in the testis are amoeboid, aflagellate, with numerous peripheral microtub ~.tl es. Mature spenn in the sperm du ct have a nucleated part with a narrow layer of cytoplasm drawn out into two tl anges, and a thicker non-nucleated part. Peripheral mi crotubules and dense thickenings of th e plasma rnem­ brane are present in both parts. Two symmetrically arranged blunt bristle-like structures, apparently modifiee! large granules that do not reach the surface, are located in the nucleated part. Sperm struc­ ture supports tbe view that Haplopharyngida and Macrostomida are closely related. Key words : Platyhelminthes, Haplopharyngida, Haplophmynx rostratus, sperm, spennatogene­ sis, ultrastructure, phylogeny

INTRODUCTION

The Haplopharyngida are usually considered a taxon separate from but close to the Macrostomida (e.g. , CANNON, 1986). EHLERS ( 1985) includes them in the Macrostomida. DoE ( 1986) fou nd a ma tri x syncytium of the copula tory stylet in both taxa supporting the ir monophyly. Not a single electron-microscopie study of sperm and spermatogenesis of a haplophatyngid bas been made (WATSON & ROI-ID E, 1995), whereas severa! such studies of macrostomids have been published (BEDIN I & PAPI , 1970 ; N EWTON, 1980; ROH DE & WATSON, 1991 ; ROHD E & FAUBEL, 1997 a, b). ln this paper we describe the ultrastntcture of spenn and spermatogenesis of Haplophmy nx rostratus M EIXNER, 1938 with the aim of contributing to a better understanding of the phylogeny of the Platyhelminthes io general and of the Haplopharyngida in particular.

MATE RJALAND METHODS

One specimen of Haplopharynx rostratus was collected from the beach nea.r the old Research Station on the Island of Sylt, North Sea, and immediately fixed in 3% gluta:raldehyde 178 KLAUS ROI-IDE AND ANNO FAU BEL

in 0.1M sodium cacodylate buffer (pH 7.2) for about one week at room temperature. 1t was washed for 40 minutes at 4° C in 0.1 sodium-cacodylate buffer (pH 7.2) made up with seawa­ ter, postfixed for 1 hr in 1 % OsO. in the same buffer and dehydrated in a graded series of ethanol. It was embedded in Spun·'s resin. Seria! longitudinal sections were stained with uranyl acetate and lead citrate and examinee! under a JEOL 1200EX electron microscope at 60 kY. ""

RESULTS

Primary spermatocytes, clearly characterised by t~1eir synaptonemal complexes (Fig. 1 A, B) have large nuclei and light cytoplasm. Cells with much more electron-dense nucleoplasm and distinctly aggregated chromatin, and with dense cytoplasm containing much endoplasmic reticulum (Fig. 1 A) are interpretee! as spermatogonia, although the possibility cannot be excluded that they are secondary spermatocytes. The second alterna­ tive is not unlikely in view of the weil developed endoplasmic reticulum, which is unusual for spermatogonia. Spermatids contain Golgi complexes producing many vacuoles (Fig. 1 C, 0), their nuclei are at first large with light nuoleoplasm containing many chro­ matin granules, but later condense and become round, contai ning many small electron­ lucent a reas (Fig. 2 A-C). M itochondria aggregate around the nucleus (Fig. 2 B). Lack of membranes between some nuclei indicates that spermatids form syncytial clusters. At an even later stage of spermiogenesis, the chromatin becomes loosely fibrillar and the cells and nuclei elongate (Fig. 2 D). Sperm in the testis are amoeboid, with many deep invagi­ nations and lobe-like processes (Figs 3 A-C, 4 A). They conta in nu merous electron-dense granules of a range of sizes (Fig. 5), many of them observee! to be lined by distinct mem­ branes (Figs 3 A-E, 4 A). They a Iso conta in mitochondria (Figs 3 E, 4 A), a nucleus with fibrillar chromatin (Fig. 3 C, D), aggregations of dense ma teri al and more or less electron­ lucent vacuoles (Fig. 3 C), and their surface has a densely packed row ofperipheralmicro­ tubules below the plasma membrane (Figs 3 B, E, 4 A). The plasma membrane has many dense thickenings, some of which at !east are artefacts (e.g., Fig. 3 E). Mature or maturing sperm in the sperm duct have a nan·ow part containing the dense nucleus with very small lucent spaces, surrounded by a thin layer of cytoplasm with mito­ chondria and dense granules, and this narrow part is drawn out into two tlanges (Figs 4 C, 6 A-E). A row of microtubules is fou nd below the surface membrane (Figs 4 C, 6 A-0 ). Some microtubules are present below the peripheral row (Figs 4 A, 6 B). The non-nucleated pa1is of the spe1111 are mu ch lm-ger in cross-section, and they conta in a mu ch great er number of dense granules of various sizes; the smfate membrane al o has a row of microtubule (Fig. 4 B-D). Two symmetrically located curved and tapering structures ( « brist es») th at do not reach the surface and apparently are modifiee! granules, are located in the nucleated part of the sperm close to the nucleus (i.e., not at the tips of the tl anges), in the region close to the beginning of the tl anges (Figs 6 B, 7 A, 8). Dense structures een once at tbe tip of the tlange (Fig. 6 B) are of unknown nat11l'e but may representa di int grat' ng granule. The wall of the sperm duct is formee! by cells in contact by eptate junctions. C ilia are not densely packed but occLu- a li around the duct (Fig. 4 C, 0 ). Oiagrams of mature sperm are given in Fig. 8. SPERMATOGENESIS OF 1-/APLOPI-IARYNX 179

Fig. 1. A. - Secti on through testis with primaJy spermatocytes (?) on the right (p), and permatogo­ ni a or secondary spermatocytes on the left. Also note eDdoplasmic reticulum (e) and nuclei (n). B. Synaptonemal complexes (sy) in primary spermatocytes (p). C, D. Golgi complexes (g) in sper­ matids. Scale bars 2f.!m . 180 KLAUS ROH DE AND ANNO FAUBEL

Fig. 2. A. -Section through testis with spermatids containing unconden ed nuclei (n). Note rounded com­ pact nucl ei ofspermaticls on the right. B. Sperrnatids with rounded nuclei (n) and many mitochondria (m) near them. Note lack of membrane between nuclei. C. Two sperrnatids with rouncled nuclei (n). Note dense chromatin with some electron-lucent areas in B and C. D. Sli ghtly later stage of spennatid. Note nuclei (n) with loose-fi brillar confi guration, elongating on ri ght. Scale bar 1ÛiJ.!ll (A), 21-1-m (8-0). SPERMATOGENESIS OF HAPLOP!-!ARYNX 181

Fig. 3. - Sperm in testi s. Note amoeboid shape ( -C), nu merou elcctrort-den c granule (gr) of dif­ ferent sizes, some with distin ct membranes, nucleus (n), and mi tochondria (m). Also note theve ic­ ul ar regions (x) in C, th e peripheral mi crotubules (t) and den e thi kcning (d) (sorn e of them apparentl y artefacts) ... Scale bars 2pm (A-D), 1!lm (E). 00 N SPERMATOGENESIS OF HAPLOPHARYNX 183

90

80

. 70

60

50 z0 40

30

20

10

0 0.0 0. 1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 Mean diameter (!-lm) of sections through granules

Fig. 5 . - Frequency distribution of mean cliameters of sections through granules in sperm fi·om the testis.

Legend to the figure (see page 182)

Fig. 4. - A. Sperm in testis. Note peri pheralmicrotubules (t) and cl en ' e thiekenings (d}, mitochonclria (m) and gran ules (gr) of different sizes, ome with distinetmembrancs. B. perm in perm duel. Note marginal section through sperm on lelt, showing cl ensc ly packecl mierotu bule (l). C, O. perm in sperm cl uct. Note cl ense- fibrillar nucleus (n) with some lucent paces, granu les (gr). mitochonclria (m), peripheral microtubu les (t) and dense thickenings (cl). A iso note tha t nucleatcd part ofspenn has only a thin layer of cytoplasm cl rawn out in to two nange . the non-nuclcall:cl pa rt are th icker and packed with granules. The wall of the sperm duel con ists of ce lis in contact by eptate junction (j) ; ci lia (c) are not clense ly packecl bu t occLn· ail aro und the ciLLCt. ote nuclcu (n) oi' sperm duel in C, and two nue lei (n) in O. Scalc bar 1p m. 184 KLAUS ROHDE AND ANNO FAUBEL

Fig. 6. - Transverse sections through nucleated pa rt of one permatozoon in perm luet. A. ection through wide part with many granules and end of nu clcu (n). B. Section through part at beginning of ft anges (fl), with el en e structur s of unknown nature(?). Note pe ripheral r w or microtubule (t), and so me microtu bul es below the peripheral row, granules (gr) and mitoch nclrion (m). . Section through part with flanges ( fi ). O. Secti on through part nea r Lh e end of nucleu s (n). E. ection through Lip without nucleus. Sca le bar ! pm. •

SPERMATOGENESIS OF HAPLOPHARYNX 185

Fig. 7. - Longitudinal sections th.rough sperm atlcvel ofbeginning of Oanges (11), witJ1 nucleus (n), granu les (gr), and two symmetrically located «bri tles» (br). A lso note longitud inal sections thro ugh peripheral microtubules. Scale bar 1~ Lill. 186 KLAUS ROH DE AND ANNO FAUBEL

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• •..,. -

gr

m

Fig. 8. - Diagrams of cros -sec tions th rough sperm (left), and of longitudinal sec tion in the region of the fl anges and bri stl es . Note «bristl es» (br), den c thi ckenings (d), flangcs (Il), granu les (gr), mito­ chonclria (111) , nu cleus (n), and microtubul es (l). SPERMATOGENESIS OF HA PLOPHA R YNX 187

DISCUSSION

Spermatozoa of Haplophg;_'JlnX rostratus resemble ali macrostomids examined in the lack of flagella, and most macrostomids in the presence of bristle-like structures, which are absent in Paromalostomum fitsculum. They are apparently modified granules, which do not reach the surface of sperm in Haplophwy nx rostratum. Di fferences between the two taxa are the number and kind of symmetry of the peripheral microtubules. In tuba Graff, 1882 and M pusillum Ax, 1954 (Macrostomatidae), micro­ tubules are arranged in two contralateral rows (ROHDE & WATSON, 1991; ROHDE & FAUBEL, 1997b ). ln Paromalastomum fuscu/um A x, 1952 (Doli chomacrostamidae), the number of microtubules in each row is small (usuall y four) , and a conclusion concerning· the ki nd of symmetry is not possibl e (ROH DE & FAU BEL, 1997a). Furthermore, centrioles were found during spermi ogenes is, apparentl y resorbed by the cytopl as m during sperm formation (ROI-IDE & FAUB EL, 1997a). Sperm of Haplopharynx rostratum differ from th ose of the macrostom ids in the mu ch lm·ger number of peripheral microtubules. Immature sperm from the testi s differ fro m those of macrostomids in the di stinctly amoeboid shape. Other aspects of spermi ogenesis have no pec uli ar features. Altogether, similarities of sperm structure in Haploph01ynx rostratum and macrosto­ mid s support the view that haplopharyngids and macrostom ids are closely related, alth ough one of the synapomorphi es, lack of fla gell a in mature sperm , is a purely nega ti ve characteri stic du e to secondary reduction (as indicated by the presence of centri oles dur­ ing certain stages of spermiogenes is in P .fitsculum), also found in ome other platy­ helminths (WATSON & ROHD E, 1995). Large bri stl es are present in IV! tuba (ROHDE & WATSON, 199 1), and rudimentary ones reachin g the surfa ce of sperm in IV! pusillum (ROI-IDE & FAUBE L, 1997b). P .fitsculum lacks bristl es (ROI-ID E & FAUBEL, 1997a). The bri stles of IV! pusillum and H. rostratus clea rl y are not fla gell ar derivatives, whereas the much larger bri stles of IV! tuba ha ve been interpreted as modified fl age ll a. lt is poss ibl e that dense granules in ali three spec ies co ntribute to bri tle form ation , but that the large size and more complex structure of bristles in IV! tuba are due to a fi age ll ar component. However, evid ence for thi s assumpti on is lack ing. The pos ibility mu ta iso be considered that the very small bri stles of IV! pusillum and H rostratus are not homo logous with the much larger and more co mpl ex ones of M. tul;;a. Al o, there is a certain simi larity betvvee n th e bri stles of IV! pusillum and the crested bod ies of cestode (BÂ & MAR HAN D, 1995), but homology is unlik.ely in view of th e different hape (spirall in g aro uBd the anteri or end of sperm at !east in some cestodes). The fun ction of the bri stles is unkn ow n. That the curved dense structures in H rostratus ma y act as « bri stl es » is indi cated by their taper­ in g tips close to th e surface. Conceivab ly, th e soft surface membrane, when pu shed aga inst a hard or elastic surface, permits the tips to fi nd a hold and act as « bri stles ». Th e observation th at many granules (from very mali to mediu m large) in spenn of H. rostral us are lin ed by a di stin ct membrane, whereas many other of the ame size are not, may indi cate that loss of a membrane is a fixation artefact. The frequcncy distribution of diameters of section s through th e granules uggest that thcre arc a ize class of ma li granul es (average diameter approx im ately 0.2 ~Lm , sli ght ly grcater than t h ~ p~..:nk of m a- 188 KLAUS ROHDE AND ANNO FAUBEL surements of sections because most sections will not be exact! y through the middle of the granules) and many larger granules which probably belong to a size class oftheir own. The observation that some curved structures occur among the granules in the in teri or of sperm from the testis, resembling the subsurface "bristles" in mature spenn, suggests that the « bristles » are formed in the in teri or and migra te to the surface du ring maturation of the sperm.

ACKNOWLED6 MENTS

The work was supported by grants from the Australian Research Council and the University of New England. Nikki Watson did the postfixipg, sectioning and staining and critically commented on the manusc ript. We thank Peter Garlick for mak in g fac ilities at th e E.M. Unit, UNE, available tous, Rick Porter for developing and Zoltan Enoch for printing the micrographs. Li sa Donaldson typed the manuscript, and Becky Francis helped with th e drawings in Fig. 8.

REFERENCES

BÂ, C. T. & B. MARCHAND ( 1995) - Spermiogenes is, sperm atozoa and ph yletic aff-inities in the Cestoda. ln: B.G.M. JAM IESON, J. Aus10 and J.-L. JuS TI NE (eds.), Ad va nces in Spermatozoal Phyloge ny and , Mémoires du Muséum Na tional d'Histoire !Yaturelle, Paris, 87-95. BEDI NI, C. , & F. PAPI ( 1970) - Pec uli ar pattern s of mi crotubul ar organiza tion in sperm atozoa of lower Turbell aria. ln: B. BACCEHI (ed.), Comparative Spermato logy, Accad Naz. dei Lincei, Rom e, 363-366. CANNO N, L.R.G. ( 1986) - of th e World. A Guide to Fam ili es and Genera, Queensland Museum , Brisbane, 136 pp. DoE, D. A. ( 1982) - Ultrastructure of th e copul atory orga ns of Haplop hCII ynx quadristimulus and its phylogenetic significance. (Pl atyhelminthes, l-lapl op haryngida), Zoomorphology, 106: 163 -17 3. EHL ERS, U. ( 1985) - Oas Ph ylogeneti sche System der Pl athelminthes, Gusta v Fischer, Stuttga rt, New York, 3 17 pp. N EWTON , W. O. ( 1980) - Ultrastructural analys is of th e motil e apparatu s of th e aflage ll ate sperm ato­ zoon of Macrostomum tu bum, J. Ultras/rue/. Res., 73: 3 18-330. ROHD E, K. & A. FAU BEI_ ( 1997a) - Sperm atogenes is of Paromalostomumfusculum (P iatyhelminthes, Mac rostomid a, Doli chomac rostomid ae) . lnvertebr Repmd and De v. , 32: 75 -84 . ROHDE , K. & A. FAU I3E I_ ( 1997b) - Spennat:o ge nes is of !vlacmstomum pus ilium (Pi atyhelminthes, Macrostomatid ae), lnvertebr Reprod and Dev. , 32: 209-215. ROHDE , K. & N. WATSON ( 199 1) - Ultra structure of sperm atogenes is and sperm of ivlacmstomum tuba, J. Submicrosc. Cytol. Pathol. , 23: 23 -32 . WATSON , N.A. & K. ROI-ID E ( 1995) - Sperm and spermiogenes is of the «Turbell ari a» and imp li ca­ tions for th e ph ylogeny of the ph ylum Pl atyhelq1, in thes . ln : B.G.M . JAM IESON, J. Aus10 and J. ­ L. .J uST INE (eds.), Advances in Spermatozoa l Ph ylogeny and Taxonomy, Mémoires du Muséum Na tional d'Histoire Naturelle, Pa ri s, 37-54 .