Zoologica Scripta, Vol. 12, No. 4, pp. 257-266,1983 Printed in Great Britain

030&3256/83 $3.00 + .OO Pergamon Press Ltd.

The Norwegian Academy of Science and Letters

Spermatophore Transfer and Subsequent Sperm Development in a Homalorhagid Kinorhynch

ROSEMARY BROWN

School of Biological Sciences, Macquarie University, North Ryde, N.S.W., Australia

Accepted 24 August 1983

Brown, Rosemary. 1983. Spermatophore transfer and subsequent sperm development in a homalorhagid kinorhynch.-Zool. Scr. 12: 257-266.

The cuticular morphology and precise location of male and female gonopores and penile spines of the homalorhagid kinorhynch Kinorhynchus phyllotropis Brown & Higgins, 1983 are described and illustrated. In this species spermatozoa are transferred from male to female by a sper- matophore. This is the first record of the mechanism of sperm transfer in a kinorhynch. The spermatophore is presumably extruded through the male gonopore and directed towards the female by the ductless penile spines. Spermatozoa in the spermatophore are rod-shaped and catenulate. The spermatophore is pressed directly against the cuticular plates of the female, and usually covers the female gonopores. The spermatophore contains a mass of intertwined spermatids and spermatozoa surrounded by clear material covered with a layer of debris. Spermatozoa are found in the female lodged in the seminal receptacle tissue applied to the dorsal aspect of posterior oocytes. There the spermatozoa complete their development. Nuclei change from filiform to geniculate, and oval corpuscles surrounding the nuclei disappear, so that the spermatozoa are seen as densely-packed, polyhedral cells. These observations conform with literature reports of aberrant spermatozoa of unknown origin seen in female Pycnophyes. The fertilization process remains unknown.

Rosemary Brown, School of Biological Sciences, Macquarie University, North Ryde, N . S . W., 2113, Australia

Introduction

Literature on the anatomy of kinorhynch reproductive organs spans nearly one hundred years (Reinhard 1887; Zelinka 1928; Remane 1936; Boykin 1965; Higgins 1974) and has been based entirely on light microscope observa- tions. No observations have been published of kinorhynch fertilisation, insemination or copulation. Observation of the reproductive practices of kinorhynchs is rendered difficult by their size of less than one millimetre, their marine mud milieu, and by the exoskeletal plates which may obscure the genitalia.

Male genitalia consist of penile spines of unknown function mounted adjacent to a pair of gonopores, which are associated with bristles in some cyclorhagid kino- rhynchs. According to Higgins (1974), the cyclorhagid genus Echinoderes is the only cyclorhagid genus in which external primary sex characters have been observed. In Echinoderes the gonopores are visible at the lateral mar- gins of the junction of the last two terminal segments. In homalorhagid kinorhynchs the gonopores are located ventrally near the anterolateral margins of each terminal sternal plate, except in the neotenic genus Neocen- trophyes in which the gonopores and penile spines are located laterally as in the cyclorhagids. In the genus Paracentrophyes, of family Neocentrophyidae, males and some juveniles possess flexible penile spines near the anterolateral margins of the terminal segment (Higgins 1983). Boykin (1965) recorded that in Kinorhynchus ilyocryptus one penile spine is located dorsal and one ventral to a plate, shaped like a quarter circle, which projects from the intersegmental membrane between the sternites of segment 12 and segment 13. A semi-circular

shelf is formed by these plates on each side of the body, with a gap in the centre where the plates are separated.

There is no record in the published literature of whether homalorhagid gonopores and penile spines are located dorsal, lateral, or ventral to the anterolateral margins of the sternal plates of segment 13. Nor is there any descrip- tion of the cuticular morphology of the genitalia. It is not known whether penile spines act as sperm conduits, or whether they function like nematode copulatory spicules (Higgins 1974) as intromittent organs serving to expand the female orifice.

The morphology and the ultrastructure of homalorhagid spermatozoa have been described (Zelinka 1908, 1928; Remane 1936; Boykin 1965; Nyholm 1976; Nyholm & Nyholm 1982). Only animals which retain the method of sperm discharge into the ambient water have the primitive type of spermatozoon, which usually means animals with external fertilisation (Baccetti & Afzelius 1976). FranzCn (1956) commented that the kinorhynch sperm is not a primitive sperm, but a strongly modified form which does not have a typical head as the nucleus is surrounded by a large amount of cytoplasm. Zelinka (1928, figs. 21-23, plate 28) showed kinorhynch sperm with an elongate tip or “perforatorium”, a sausage-shaped head structure containing a filiform nucleus surrounded by densely-staining rounded bodies, no demarcated mid- dle piece and a flagellum 10-15 pm long. Zelinka described groups of filaments lying along the nucleus in cyclorhagid sperm, and homalorhagid sperm which reach a length of 70-90 pm, about one tenth of the kinorhynch body length. Nyholm (1976) described spermatozoa of homalorhagid Pycnophyes species with elongate nuclei surrounded by spherical organelles (termed platelets

257 Zoologica Scripta 12

258 Rosemary Brown

because of their suggested similarity to the yolk platelets of oocytes). Tracts of filaments run alongside the nuclei to flagellar axonemes and participate in flagellar attachment. Nyholm & Nyholm (1982) described the locomotory activity of spermatozoa from living testis tissue, the ultras- tructural changes during spermiogenesis of Pycnophyes comrnunis. They observed that forward locomotion of sperm is achieved by rapid bending and looping of the anterior third of the head while the flagellum remains inactive. The elongate nucleus and bundle of filaments are surrounded by three types of oval corpuscles or granules (termed platelets). Type 1 has a matrix of fish-bone pattern and often has small membrane vesicles around the rounded edge. Type 2 has a fibrous matrix and an irregular edge, while type 3, of electron density intermediate to the other two types, is found on the opposite side of the nucleus to the tract of filaments.

Spermatozoa with normal morphology were described in the seminal receptacles of female homalorhagids (Nyholm 1976). In Kinorhynchus ilyocryptus the seminal receptacle is a solid body connected by a compact stalk to the medial aspect of the posterior oviduct (Boykin 1965). The presence in the seminal receptacle of spermatozoa of reduced size with bent or geniculate nuclei has also been recorded (Zelinka 1928; Nyholm 1977). The atypical sperm described in these last two observations have been considered as secretion granules (Remane 1936), chromo- somes (Boykin 1965) and the “abnormal sperm of sper- matogonia in morphological hermaphrodites” (Nyholm 1977). Nyholm defined morphological hermaphrodites thus: “The chaotic sperm mass, developed by amitosis from spermatogonia at the apical end of the female gonad, constitutes a morphological hermaphroditism in homalorhagids.” Part of the evidence for this conclusion was the absence of visible sperm nuclear membranes or cell membranes in the sperm mass.

There is no record in the published literature of the mode of sperm transfer in any kinorhynch.

An examination of the reproductive structures of the homalorhagid kinorhynch Kinorhynchus phyllotropis Brown & Higgins, 1983 has been undertaken with the following objectives: firstly, sectioning males to establish

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the exact location and cuticular morphology of the genitalia and to look for ducts that could function as sperm conduits; secondly, sectioning females to establish the exact location and cuticular morphology of the genitalia, and to look for male gametes with particular reference to the seminal receptacle tissue; thirdly, searching females for any evidence of the mode of sperm transfer.

Material and methods From spring 1980 to winter 1981, three-monthly samples of K . phyllot- ropis were collected from Hunter Bay, Sydney Harbour, Australia (33”49”30S, 151”15”24”E) in sandy mud at 6 m depth using a Higgins epibenthic dredge (Higgins 1961).

Mature adults from these samples were fixed in 2% glutaraldehyde with 0.1M sodium cacodylate and 1.5% sucrose in sea water, post-fixed in cold 1% osmium tetroxide in sodium cacodylate buffer, and stored in a buffer of 0.1M sodium cacodylate, 1.5% sucrose and sea water. Specimens for transmission electron microscopy were dehydrated and transferred in graduated steps to Spurr’s embedding medium. They were polymerised, sectioned and stained with uranyl acetate for 30 min, and lead citrate for 10 min, then photographed through a JEOL JEM lOOCX transmission electron microscope. Specimens for scanning electron microscopy were stored in the sodium cacodylate buffer, transferred to distilled water, frozen in liquid nitrogen and dried under vacuum. Some specimens were mounted dorsally or ventrally on aluminium stubs with double-sided tape. A second strip of tape was placed over these speci- mens and then lifted off so that the specimens were torn in two. This latter tape was placed on a second stub. Both fractured and whole- mounted specimens were coated with gold and photographed using a JEOL T20 scanning electron microscope.

Spermatophore incidence was determined using only the March sam- ple. Kinorhynchs were extracted by Higgin’s (1964, 1969, 1971) non- quantitative method of passing air bubbles through the sediment.

P D 12

A

Fig. 1 . Kinorhynchus phyllotropis, male. Schematic representation ot reproductive organs of terminal segments 12 and 13.

VlP An. v i 3 v. P’n.Sp.

Fig. 2. Kinorhynchusphyllotropis, male. Posterodorsal view of terminal segments 12 and 13 showing reproductive

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structures

Kinorhynch reproduction 259

Fig. 3. Kinorhynchus phyllotropis. Reproductive tissues of terminal segments 12 and 13.-A. Male, left sagittal section. Transmission electron micrograph.-B. Male, extreme left sagittal section. Transmission electron micrograph.-C. Female, sagittal section. Transmission electron micrograph.

Results

Cuticular morphology of genitalia of K. phyllotropis

Male genitalia (Figs. 1, 2 , 3a-b). Male genitalia of K . phyllotropis are located between the small sternites of segment 13, which are 10-30 pm long and 1 pm thick, and the large sternites of segment 12 which are 90-100 pm long and 4 p m thick. These genitalia consist of a penile spine mounted on the intersegmental ligament, or arthrocorium, dorsomedially to the gonopore on each side of the body, and a penile spine mounted ventrome- dially to each gonopore. The four penile spines project below the sternites of segment 13, ventrolateral to the

anus. These spines are 30 pm long, and are shaped with slight sigmoid curvature. The spine cuticle is striated externally and consists of a darkly-staining outer layer, 0.7 pm thick, and a fibrous inner layer 1.5 pm thick, with the fibres running around the spine circumference. The penile spine contains cytoplasm and cell membranes which extend into the spine from the hypodermal cells. There is no duct in the penile spine. The spine diameter thins from 3 pm at the base to less than 1 pm near the tip. A flexible joint is formed at the base of the penile spine with a rounded protuberance of the arthrocorial ligament. Projecting into the body cavity from the arthrocorial ligament supporting the penile spine is a process with a bifid tip which has no muscle fibre associated with it.

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260 Rosemary Brown

There is no shelving plate projecting from the arthroco- rial ligament, but there is a gonopore on each side of the anus. The gonopore is 14 p m wide, with a lipped collar 6 pm long. This collar is bordered by a fringe of cuticular hairs up to 30 pm long. The gonopore connects with the testis by a cuticle-lined duct containing intervals dilated up to 1.5 pm diameter, the diameter of a sperm.

Female genitalia and associated structures (Figs. 3c, 4, 5) . The female gonopore is located in the arthrocorium between the segment 12 and segment 13 sternites, ven- trolateral to the unarticulated lateroterminal spinous pro- cess of this species. The gonopore, 15 p m wide, is closed by two lips of fibrous cuticle, each 4 pm long. The gono- pore connects to the squamous epithelium of the oviduct by a curving duct of dense cuticle 0.75 p m thick. The oviduct is connected to the seminal receptacle, a body of spongy tissue bound by a basement membrane, with a stalk of flecked compact tissue 5 pm in diameter. The seminal receptacle contains pockets of spermatozoa.

The oocyte in the oviduct contains vitellus stratified into osmiophilic droplets of lipid yolk and nonstaining droplets of proteid yolk. The disc-shaped germinal vesi- cle, 5 4 pm in diameter and 1 pm thick, contains a 2 pm diameter nucleolus and patchy heterochromatin. The ooc- yte, 130 p m long, is constricted by the pachycyclus (Fig. 4).

P nn

Fig. 4 . Kinorhynchus phyllotropis, female. Schematic representation of reproductive organs of terminal segments 12 and 13.

Spermatophores (Figs. 6,7,8) Genital segments of some females carry spermatophores, rounded brown bodies containing spermatids and sper- matozoa. Other females carry spermatophores on the dorsal and ventral plates of segments 7-11. (Sper- matophore incidence in the autumn sample of March 1981 was 1 per 298 kinorhynchs-93 males, 88 females and 117 juveniles).

These spermatophores, up to 180 pm wide and 190 pm long, are covered by a thick detrital coating containing clay particles, algal fragments and diatoms. This coating is not seen next to the kinorhynch cuticle. A 5-10 pm thick region underneath the detritus is fairly free of gametes, but can be seen to contain bodies identified as protists, which are presumably picked up during spermatophore extrusion. The spermatophore centre contains about 140 intertwined spermatozoa and a similar number of sper- matids (Figs. 7, 8a,d). The rod-shaped spermatozoa are 1.3-1.5 p m in diameter. The condensed filiform nucleus, 0.3 pm in diameter, is surrounded by a single layer of densely-staining oval corpuscles (see Discussion), 0.7- 0.8 pm in diameter. Some oval corpuscles show a laminate matrix with a laminar periodicity of 4&60 nm (also seen in testicular oval corpuscles (Fig. 8c). Spermatozoa in transverse section show 3-5 oval corpuscles around the nucleus. The filament tract laying parallel to the nucleus contains up to 22 hollow filaments with electron-dense cores, 55 nm in diameter.

In these characteristics, spermatozoa in spermatophores resemble spermatozoa in the testis. Scanning electron micrography of testis tissue (Fig. 8b) shows the catenulate, or elongate tubercular, surface appearance imparted to the rod-shaped spermatozoa by the oval corpuscles.

Spermatophores contain rounded spermatids, pear- shaped spermatids and elongate spermatids. Rounded spermatids are mulberry-like cells with cytoplasm packed with 13-23 rounded bodies when viewed in transverse section. Rounded spermatids have a diameter of 3 4 pm and an irregular nucleus containing partially condensed chromatin. As the opaque perinuclear bodies condense into oval corpuscles they show wedges of fine laminae with a periodicity of 10 nm, and some are associated with small vesicular outgrowths of the cell membrane.

Spermatophores attached around the posterior seg- ment of the female contain intertwined gametes, but in spermatophores attached to the middle of the trunk gametes are arranged in parallel, perpendicular to the cuticle.

10 urn

Fig. 5. Kinorhynchusphyl[otropis, female. Posterodorsal view of terminal segments 12 and 13 showing reproductive structures

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Kinorhynch reproduction 261

1 0-m

6 A

6 B

Fig. 6 . Kinorhynchus phyllotropis, female.-A. Dorsal view of trunk segments, with head retracted, showing spermatophore attached to terminal segments. Light microscope brightfield micrograph.-B. Outline drawing of female with attached spermatophore showing tergite margins.

Fig. 7. Kinurhynchus phyllotropis. Longitudinal section of spermatophore attached to terminal plates of female, showing central mass of spermatozoa and spermatids surrounded by clear material containing a protist of some kind, with the external layer of debris indicated in outline.

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262 Rosemary Brown

Fig. 8. Kinorhynchus phyllotropis. Spermatozoa in testis and spermatophore.-A. Longitudinal section of head of sperm in spermatophore. Transmission electron micrograph.-B. Fragment of testis showing oval corpuscles of sperm heads. Scanning electron micrograph.-C. Transverse section of heads of sperm in testis. Transmission electron micrograph.-D. Gametes in spermatophore showing sperm head in transverse section and flagellum of spermatid in longitudinal section. Transmission electron micrograph.

Spermatozoa in seminal receptacle tissue (Figs. 8,9) Females with no spermatophores attached to the gono- pore (Fig. 9a) and females with terminally attached spermatophores (Fig. 9b) both contain pockets of sperm chromatin in the seminal receptacle tissue.

Spermatozoa in female tissue differ from spermatozoa in spermatophores. The atypical head had an irregular, polyhedral cellular outline, not a rounded or rod-shaped section. Sperm heads in the female have a diameter of 3 pm, compared with 1.5 pm diameter in the testis. Oval corpuscles, with a diameter of 500 nm compared with 700-800nm in spermatophore sperm, are seen in few sperm cells found in the female. The oval corpuscles are

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separated from the nucleus by areas of clear cytoplasm not seen in spermatophore or testis sperm. The nucleus is folded, not filiform, always contains electron-lucent spots, and is not closely apposed to the filament tract as in spermatophore or testis sperm. Fjlament tracts, contain- ing about 20 filaments of 45 nm diameter, are adpressed to the sperm cell membrane.

Sperm nuclei are found next to the oocyte 30 pm from the germinal vesicle. Eight to eleven sperm heads could be counted on one section of a sperm pocket, but sections taken further posteriorly cut through the flagella of 24 sperm.

Kinorhynch reproduction 263

Fig. 9. Kinorhynchus phyllorropis. Spermatozoa in the female.-A. Seminal receptacle of female without spermatophore attached to gonopore. Transmission electron micrograph.-E. Seminal receptacle of female with spermatophore attached to gonopore. Transmission electron micrograph.

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264 Rosemary Brown

Discussion

Location and cuticular morphology of the genitalia In the homalorhagid Kinorhynchus phyllotropis the gono- pores and penile spines are located in the intersegmental ligament, ventral to the sternal plates of terminal segment 13. The penile spines contain cytoplasmic extensions from the hypodermis, but no duct. There is no shelving plate between the spines, but there is a gonopore with aproject- ing collar which is fringed. Boykin (1965, fig. 87) shows a thin cuticular plate between the penile spines of K. ilyoc- ryptus. A possible explanation for the presence of this plate may be that it is a projection from a lipped gonopore, or that it may be formed from the hairs of a fused gonopore fringe. The female gonopore of K. phyllotropis is guarded by two cuticular lips.

Function of the penile spines The kinorhynch penile spine is ductless like the intromit- tent copulatory spicule of the nematode, but the nematode spicule and its associated gubernaculum are furnished with muscle fibres, unlike the equivalent struc- tures in the kinorhynch. Without the leverage imparted by muscle the kinorhynch penile spines are unlikely to function as intromittent probes.

It is possible that penile spine function to prevent loss of the spermatophore after its extrusion, because, during fixation processes, male kinorhynch specimens often relax, with eversion of the head accompanied by protru- sion of segment 13, causing the four curved penile spines to fan out. In this position the penile spines would direct the extruding spermatophore towards the ventral aspect of the male kinorhynch. There the spermatophore would contact the female if, during copulation, the female is secured by the ventral adhesive tubes of the male, as postulated by Zelinka (1928).

It is unlikely that penile spines act as coupling devices as normal spermatophores are observed attached to the smooth mid-dorsal and midventral plates of females. Spermatophore transfer is direct, for no debris is observed between the spermatophore and the female cuticle. Sperm placed on the midtrunk region of the female are arranged in parallel. Sperm placed over the female gono- pore are twisted into an intertwined mass, presumably because sperm migration is initiated by substances pro- duced through the female gonopore.

Sperm development in the female This study has confirmed Zelinka’s (1928) observation that the seminal receptacle tissue of hornalorhagid kino- rhynchs contains spermatozoa of reduced size derived from males. He observed 20-30 sperm in the seminal receptacle of Pycnophyes communis. This compares with 24 sperm sectioned in K. phyllotropis. Sperm maturation in the female may also occur in P. jlaveolatus. Nyholm (1977) observed a female specimen, with no spermato- phore attached to it, that carried pockets of sperm chromatin in the seminal receptacle. This sperm showed the same features as seen in K . phyllotropis-bent or geniculate nuclei with electron-lucent spots surrounded by swathes of flagella (Nyholm 1977, fig. 4). Nyholm commented on the absence of cell membranes, which have been observed in the present study (Fig. 9b).

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The elongate, catenulate spermatozoa of the spermato- phore complete their development in the female with marked change of cellular organisation. The linear nuc- leus becomes irregularly bent, with concomitant changes in cell outline. Dense nuclear chromatin develops elec- tron-lucent spots, and perinuclear oval corpuscles become reduced in size and number. The filament tract loses its close association with the nucleus and is found adjacent to the cell membrane. There is an increase in the volume of clear cytoplasm around the nucleus, so that the sperm cell increases to twice its original diameter in the testis, i.e. to 3 pm from 1.5 pm. FranzCn’s (1956, p. 456) observation that, in its “large amount of plasm”, the kinorhynch sperm “resembles here an ordinary cell” is a particularly apt description of the final stage of maturation.

Oval corpuscles In the sperm of the acoel turbellarian Diopisthoporus longitubus black dense bodies are arranged around an elongate nucleus in 1-2 layers (Hendelberg 1977). This arrangement is similar to that found in the homalorhagid kinorhynchs Pycnophyes communis (Nyholm 1976) and Kinorhynchus phyllotropis. Although Nyholm & Nyholm (1982) employed the terms “oval corpuscles” and “granules” for these bodies, they preferred the term “platelets” in reference to their possible analogy to the yolk platelets of oocytes (Nyholm 1976).

Nyholm & Nyholm (1982) found three types of these perinuclear bodies in P. communis. Type I shows a fish- bone matrix comparable to the linear matrix seen in some of these corpuscles in K. phyllotropis. The Nyholms suggest that these bodies may function to store nutrient material for the zygote or the female, or may be a form of mitochondrion. The absence of a middle piece in the kinorhynch sperm suggests that an alternative site for energy transfer reactions has evolved. This suggestion is compatible with the Nyholm’s observation that in the mature sperm locomotory activity occurs in the anterior third of the head and not in the tail.

The sperm of K. phyllotropis have to undergo extrusion from the rope-like arrangement in the testis, through the narrow cuticular opening of the gonopore, and then pre- sumably make a similarly abrasive journey from the spermatophore up through the female gonopore. The perinuclear bodies may perform a secondary function of giving protective covering to the thread-like nucleus dur- ing sperm migration.

Spermatophore reproduction Other members of the meiofauna besides kinorhynchs produce spermatophores. These include the acochlid mol- luscs Microhedyle milaschewitchii, M . lactea, Ganitus evelinae and Hedylopsis brambelli (Swedmark 1968), the interstitial polychaetes Hesionides arenaria and H. gohari (Westheide & Ax 1965; Westheide 1969, 1970), the archiannelids Nerilla antennata (Magnani 1965; Jouin 1968), Protodrilus rubropharyngeus (Jagersten 1952) and P. albicans (Jouin 1970), the gastrotrich Dactylopodola baltica (Teuchert 1968) and the marine nematode Prorhynchonema warwicki (Gourbault & Renaud-Mor- nant 1982). Spermatophores are a means of ensuring fertilization in animals of low cell number and sparing gamete production (Swedmark 1964).

Kinorhynch reproduction 265

In kinorhynchs sperm transfer might be considered to be handicapped by lack of locomotory organs. When Kinorhynchus phyllotropis adults do succeed in finding a member of the same species of appropriate sex and matur- ity, the- hazards of copulatory alignment are such that sperm may be delivered to an inappropriate destination on the middle of the body. The effects of these disadvan- tages may have been overcome by a device which ensures that insemination is followed by a prolonged period of fertility. This device is the storage of a highly modified male gamete in the organ termed a “seminal receptacle”. Gamete storage may enhance the possibility of laying a fertile egg when favourable conditions encourage the accretion of yolk in the oocyte.

This study has shown that, in the male of this homalorhagid, the sperm duct passes from the testis to a gonopore opening between two cytoplasm-filled cuticular spines, the penile spines. Sperm are transferred to the female in a spermatophore, which is usually placed over the terminal segments of the female. The plates of seg- ment 13 are telescoped inside the plates of segment 12 and the female gonopores are situated at the sides of the body between the ventral plates of segment 12 and segment 13. In the female, the seminal receptacle, a body of spongy tissue applied to the posterior oviduct, contains sperm which have different cellular organisation from that seen in the spermatophore and the testis.

It is not known how the spermatophore is passed to the female, nor what route the sperm take to reach the seminal receptacle, nor how sperm reach the ovum from the seminal receptacle. The stage at which fertilisation occurs is unknown. Whether the eggs are laid freely into the mud or into the spermatophore remains yet another question to be solved. Although it is not known whether other kinorhynchs produce spermatophores, it might be predicted that they do. Those female gonopores which have been observed are so deeply recessed beneath cuticu- lar plates that direct sperm transfer involving stiff cuticular penile spines is difficult to imagine.

Acknowledgements This project has been carried out with assistance from many sources. I wish to thank the staff of Macquarie University School of Biological Sciences, and, in particular, my supervisor, Dr D. F. Hales, as well as members of the Microscopy Department, Mr R . J . Oldfield, Mrs J . F. Gregory, Miss S. Doyle and Miss D. Clucas for advice and technical assistance. Early drafts of this manuscript benefitted from the criticisms of Dr E. E. Ruppert of Clemson University, South Carolina, Dr S. Gardiner of the Smithsonian Institution, Washington, and Dr D. S. Horning of the Macleay Museum, University of Sydney. I thank Dr Reinhardt Kristensen of the Universityof Copenhagen for hisgcnerosity in encouraging me to publish these observations which he, himself, had made independently. Sample collections were carried out in the rescue boat of the Balmoral Sailing Club, and I thank the Commodore, Mr Eric Hart, and drivers Mr John McFarlane, Mr Ron Thompson and Mr Ken Warby.

Finally, grateful acknowledgement is made of the generosity of Dr R. P. Higgins of the Smithsonian Institution, who sustained this project for nearly two years with a stream of letters, photographs and diagrams.

Abbreviations used in figures A arthrocorium, ligament connecting cuticular plates

between segments An anuscontainingfaecal material C H G cuticular hairs fringinggonopore CM cell membrane surrounding sperm head structures

C Proc

D Pn Sp D 12 D 13 D-V M E F FT

G P I LOC N OC OD ov P

PS Rg S SP SR ST T V Pn Sp v 12 V 13

c SP

GP

lateroterminal cuticular process cuticular sensory spot dorsal penile spine dorsal plate, tergite, ofsegment 12 dorsal plate, tergite, of segment 13 dorsoventral muscle nuclei of squamous epithelium investing testis flagellum filament tract passing alongside sperm nucleus gonopore gonopore containing duct passing to testis intestine oval corpuscle showing laminar matrix nucleus of sperm oval corpuscles of sperm head squamous epithelium of oviduct ovum pachycyclus, ring of thickened cuticle, component of exoskeleton penile spine mid-dorsal cuticular ridge hollow seta spermatophore seminal receptacle tissue spermatid testis ventral penile spine ventral plate, sternite, ofsegment 12 ventral plate, sternite, of segment 13

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