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ZOOLOGY
Zoology 108 (2005) 211216 www.elsevier.de/zool

How can nematodes mate without spicules? Function of the male gonoduct glands in the roundworm Myolaimus
Alexander Furst von Lieven, Verena Barmann, Walter Sudhaus
AG Evolutionsbiologie, Institut fu Biologie/Zoologie, Freie Universita Berlin, Ko r t nigin-Luise-Str. 1-3, D-14195 Berlin, Germany Received 1 March 2005; received in revised form 6 April 2005; accepted 11 April 2005

Abstract
Males of roundworms (Nematoda) usually possess cuticular copulatory organs (spicules) that are inserted in the females vulva to attach the male to the female and to widen the vulva against the inner body pressure for sperm transfer. Among free-living nematodes, the only exception of this rule is Myolaimus where the males lack spicules. Until now there exist no reports on how mating is achieved in Myolaimus. Here we show that sperm transfer in Myolaimus apparently involves at least six different secretions of the male gonoduct that are pumped into a sack-like cuticular protrusion of the females vulva to form a spermatophore-like capsule. The role of gonoduct glands in male nematodes (even in the model organism Caenorhabditis elegans) is poorly understood. Here we present the rst study explaining the role of different vas deferens gland products in nematodes and argue that Myolaimus males lost their spicules as a result of sperm competition. r 2005 Elsevier GmbH. All rights reserved.
Keywords: Mating plug; Spermatophore; Spicula; Vas deferens

Introduction
Male nematodes usually possess a pair of cuticular copulatory organs that are named spicules. During mating spicules are inserted in the females vulva to attach the male to the female and to widen the vulva against the inner body pressure for sperm transfer. Out of the approximately 20 000 described nematode species (Ax, 2001), the only known examples where male nematodes lack spicules are the closely related parasites Anatrichosoma and Trichosomoides (Bird and Bird, 1991), and among the free-living nematodes the species of the genus Myolaimus (De Ley and Blaxter, 2002). The thin male of Anatrichosoma buccalis inserts up to half of
Corresponding author.

its entire length into the vagina and uterus of the female, whereas the tiny Trichosomoides male lives within the uterus. Until now there exist no reports on how mating is achieved in Myolaimus. Apart from the absence of spicules, other features that are important for understanding the mating process in Myolaimus were used to characterize this taxon (De Ley and Blaxter, 2002):

 

The rear end that bears the genital opening (cloacal opening; Fig. 1a) of Myolaimus males forms a bursalike structure. The outer layer of the body cuticle in Myolaimus specimens is not rmly attached to the inner layer, but accid, and has a wrinkled appearance.

E-mail address: lieven@zedat.fu-berlin.de (A. Furst von Lieven). 0944-2006/$ - see front matter r 2005 Elsevier GmbH. All rights reserved. doi:10.1016/j.zool.2005.04.002

In the midbody region of the female, the loose outer cuticle layer of the vulva lips forms a sack-like

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Fig. 1. Idealized drawing of the mating process in Myolaimus. (a) Rear end of male with gonad attached to the cloaca (posterior left-hand side, anterior right-hand side), colored circles indicate secretion vesicles of different kinds. (b) Same as in (a), but rear end attached to vulval sack of female. Secretion vesicles in valve region of male gonad are expelled. (c) Vulval sack is inated by glue. (d) Two portions of capsule substance are injected into the vulval sack. (e) Capsule substance is inated by seminal uid and sperm.

protrusion (vulval sack; Fig. 1b). This study shows that males attach their rear end to the opening of the vulval sack (Fig. 1b). Thus there is no direct contact between the cloaca and the vulva proper. How then can the male widen the vulva opening for sperm transfer, a function that is normally performed by the spicules? Because the inside of nematodes is under higher pressure than the outside medium (Harris and Crofton, 1957), all body openings such as the vulva act as valves that must be opened actively. We show that in Myolaimus the vulva is opened by the pressure of male gonoduct uids. Pressure is generated by transforming the vas deferens into a pump. The distance between cloaca and vulva is bridged by a spermatophore-like capsule. Formation of pump and capsule result from secretion of a cocktail of at least six different vas deferens gland products (Figs. 1ce). Secretions play an important role in nematode mating. This is indicated by the morphology of the vas deferens, the cells of which are usually lled conspicuously with secretion granules (Chitwood and Chitwood,

1974). In many cases the rst attachment to the female is achieved by a sticky secretion that glues the genital openings of male and female together (Rehfeld and Sudhaus, 1985). The source and role of secretions involved in mating is mostly unkown, even in Caenorhabditis elegans, where three or more cell types are assumed to exist in the vas deferens (White, 1988; Barker, 1994).

Material and methods

Myolaimus sp. (strain PDL0023), an unidentied, possibly new species was cultured in Petri dishes (10 cm diam., 0.5 cm high, lled with a 0.5 cm layer of 1% water agar) provided with a small piece of potato to stimulate growth of bacteria. Mating was observed in the culture dishes with a LEITZ MZ16 dissecting microscope equipped with a PLANAPO 2.0 objective and, after

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transferring couples on slides provided with a 1 mm layer of 5% water agar, with a ZEISS AXIOPLAN2 microscope equipped with NOMARSKI optics at highest magnication (100 objective). Observations were documented with a HITACHI KP-D20BP video camera attached to the microscope and a SONY DC-PC110E Camcorder attached to the dissecting microscope.

Results
In contrast to other nematodes that use spicules and secretions in mating, copulation in Myolaimus depends solely on secretions. Microscopic examination of mating suggests that at least six different substances are exuded from the vas deferens cells. The posterior portion of the vas deferens (36 granulated cells) forms a lumen lled with glue (substance 1 in Figs. 1a and 2a, b) produced by cells adjacent to the lumen. Most of the glue is stored in an anterior and a posterior extension of the lumen. Anteriorly the remaining vas deferens cells constrict the gonoduct lumen and form a valve that seals off the spermatids from the lumen. Adjacent to the anterior extension of the lumen the valve possesses three so-called piston glands (two subdorsal and one ventral; Fig. 3) lled with small vesicles (substance 2; Figs. 1a and 2a, b). Between the three piston glands longitudinal crystals can be seen in the dorsal and subventral planes (Fig. 3d, asterisks in Figs. 2a and b). In the remaining valve cells four further types of secretion vesicles can be distinguished. Entrance of sperm into the vas deferens lumen occurs after secretion vesicles of all four types have been expelled. In the posterior valve region little secretion granules surrounding the core of the valve are expelled (substance 3; Figs. 1a and 2a, b). Noticeably refractive secretion vesicles that are arranged in three rings within the valve cells are extruded into the lumen to form the so-called capsule substance. The vesicles of the anterior ring (substance 5; Figs. 1a and 2a) are more refractive than those of the posterior ones (substance 4; Figs. 1a and 2a), and consequently the capsule substance consists of two components with different refractive indexes. Distally, where the valve is in contact with the spermatids, a clear substance is secreted that takes up the sperm and therefore is named seminal uid (substance 6; Figs. 1a, 2a, b). Precopulatory behavior begins with the male searching for the cuticular vulval sack of the female by apposing his rear end against her body and moving backwards in the same way as is described for C. elegans (Emmons and Sternberg, 1997). When a male reaches the opening of the vulval sack he attaches himself to the female by expelling a little of the glue from the cloaca. Approximately 5 min after successful attachment, substances 36 are emitted from the valve cells

Fig. 2. Video images of sperm transfer in Myolaimus (compare with Fig. 1). (a) Male, valve region of vas deferens before mating, showing secretion vesicles of six different kinds. Asterisks indicate crystals surrounding the anterior extension of the vas deferens lumen (posterior left-hand side, anterior right-hand side). (b) Same as in (a), after attachment to female. Secretion vesicles are expelled to ll the vas deferens lumen with substances 1, 3, 4, 5, and 6. (c, d) Formation of capsule within vulval sack. (c) Injection of capsule substance (4, 5). (d, e) Capsule substance (4, 5) is inated by seminal uid (6). (f) Sperm transfer. The capsule bursts and sperm and seminal uid (6) are pumped into the female. Scale bars: (a, b) 25 mm, (ce) 20 mm, (f) 35 mm.

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(Figs. 1b and 2b). Contraction of longitudinal body muscles increases the inner body pressure and pushes the valve products posteriorly until the capsule substance passes the region of the crystals. The secretion of the piston glands is exuded when the capsule substance passes. As a consequence, the piston glands decrease dramatically in volume, leading to the formation of three pouches that surround the orice of the valve (Figs. 3a,b and d). The orice of the valve can thereby be opened easily when sperm is pushed posteriorly by the increased inner body pressure produced by additional contractions of longitudinal body muscles. When the body muscles relax, the valves orice closes to hinder return ow of sperm. Additionally, formation of these three pouches transforms the valve into the piston of a piston pump: when the male contracts its body the valves proximal tip moves against the uid pressure within the pouches until they collapse and uid is pumped out of the vas deferens (Fig. 3c). When a portion of sperm has passed the valve, the displaced glue is pumped into the vulval sack which thereby becomes inated (Fig. 1c). Approximately 10 min after the rst attachment of the male, the capsule substance is also injected into the vulval sack by subsequent body contractions (Figs. 1d and 2c). Judging from the constant shape that the capsule substance retains throughout its journey from the valve to the cloaca, it appears to have a much higher viscosity than the other uids. The capsule substance is not injected entirely into the vulval sack; its distal portion remains attached to the opening of the cloaca. Thereafter, the seminal uid is pumped into the capsule substance, which becomes inated and transforms into a capsule extending from the cloaca to the vulva opening (Figs. 1e and 2d, e). By further body contractions of the male, sperm is pumped into the capsule, facilitated by the exible walls of the capsule. The capsule eventually bursts at its distal tip next to the vulva opening (Fig. 2f) and decreases a little in volume. It is the sudden increase of pressure directed against the vulva opening that opens the vulva; in other nematodes this is accomplished by the penetration of spicules. The whole process of mating takes 2030 min. After the male has detached mechanically, the capsule can be seen within the females vulval sack for hours until it nally disappears by an unknown mechanism.

Fig. 3. Detail of male gonad in Myolaimus showing function and orientation of piston glands. (a) Secretion vesicles of piston glands. (b) Formation of pouches by retracted piston glands after expelling vesicles. (c) Collapsed pouches during contraction of body muscles. (d) Idealized transverse section (dorsal top, ventral bottom) through pouches showing arrangement of two subdorsal and one ventral pouch as well as dorsal and subventral crystals.

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Discussion
The ndings reported here provide unique insight into the function of male gonoduct glands in nematodes. In Myolaimus we observed ve different functions that can be assigned to six different substances: (1) The glue is used for initial attachment to the female and for inating the vulval sack. (2) Expelling the piston gland substance enhances the valve function and transforms the gonoduct into a pump that is able to eject its exudations when the body muscles contract. The timing for emptying the piston glands ensures that the pump is only formed during mating and is not functional in any other situation where body muscle contractions occur. (3) The substance exuded at the core of the proximal valve cells opens the valve for passage of capsule substance and sperm. (4) The capsule substance that comprises two distinguishable secretions keeps the sperm separated from the sticky glue and forms a spermatophore-like capsule when injected into the vulval sack, where it displaces the glue outwardly. (5) The seminal uid takes up spermatids that must be suspended in some kind of matrix for transfer and inates the capsule substance. As mating in Myolaimus is basically a matter of male gonoduct secretions, the function of vas deferens glands is more obvious in this species than in other nematodes. However, judging from our own observations of C. elegans and various species of Teratorhabditis, a similar variety of vas deferens secretions can also be observed in nematodes that possess spicules. The mating type in Myolaimus is obviously a derived condition within Nematoda. It is challenging to explain under what conditions it was advantageous to reduce the mating tools that work perfectly in thousands of other nematode species. There are a variety of possible adaptive explanations for the Myolaimus mating type. One possibility is that females could have evolved the protruding vulval sack to reduce spicule-related trauma to internal organs and males might have evolved the capsule in response. However, we favor a scenario that involves malemale competition. The vas deferens substance which Myolaimus uses for attachment and ination of the vulval sack is most likely homologous with the glue or cement used by males of other nematodes for attaching to females and forming the so-called mating plugs. At the end of mating roundworm males often plug the females genital opening with sticky secretions to hinder mating attempts of subsequent males which assures their sole paternity of the offspring (Rehfeld and Sudhaus, 1985; Barker, 1994). Competition for paternity might have driven the evolution of the mating type in Myolaimus. The vulval sack lled with glue could represent an enormous mating plug that increased the distance between the vulva proper and the cloaca so

much that spicules could not reach the genital opening of the female any more and became superuous after mechanisms to generate enough pressure to open the vulva had evolved. This idea would, however, require that secretion-based sperm transfer was present as a precursor of the capsule substance. The postulated homologue of the capsule substance remains to be detected in other nematodes that possess spicules in combination with a variety of secretions. As the capsule in Myolaimus is reminiscent of a primitive spermatophore, the Myolaimus mating type could be a model for the evolution of true spermatophores in nematodes such as those of parasitic Rhigonematidae (Hunt, 2001) and the marine monhysterid Prorhynchonema (Gourbault and Renaud-Mornant, 1983).

Acknowledgements
We are indebted to Prof. P. De Ley, Riverside, CA, for providing our laboratory with cultures of Myolaimus. Thanks to Prof. D. Fitch, New York, for critical comments on the manuscript. This study was a project of a summer class on nematology held in 2004 at the Freie Universitat Berlin. As a participant of this class, V. Barmann videotaped the mating behavior of Myolaimus. The sequences were analyzed and interpreted under the guidance of A. Furst von Lieven and W. Sudhaus.

References
Ax, P., 2001. Das System der Metazoa III. Spektrum Akademischer Verlag, Heidelberg. Barker, D.M., 1994. Copulatory plugs and paternity assurance in the nematode Caenorhabditis elegans. Anim. Behav. 48, 147156. Bird, A.F., Bird, J., 1991. The Structure of Nematodes. Academic Press Inc., San Diego. Chitwood, B.G., Chitwood, M.B., 1974. Introduction to Nematology. University Park Press, Baltimore. De Ley, P., Blaxter, M., 2002. Systematic position and phylogeny. In: Lee, D.L. (Ed.), The Biology of Nematodes. Taylor & Francis, London, pp. 130. Emmons, S.W., Sternberg, P.W., 1997. Male development and mating behavior. In: Riddle, D.L., Blumenthal, T., Meyer, B.J., Priess, J.R. (Eds.), C. elegans II. Cold Spring Harbour Laboratory Press, Cold Spring Harbour, pp. 295334. ` Gourbault, N., Renaud-Mornant, J., 1983. Systeme reproduc ` teur dun Nematode marin a fecondation par spermato phore. Rev. Nematol. 6, 5156. Harris, J.E., Crofton, H.D., 1957. Structure and function in the nematodes: internal pressure and cuticular structure in Ascaris. J. Exp. Biol. 34, 116130. Hunt, D.J., 2001. The African Carnoyidae (Nematoda: Rhigonematida). 1. Brumptaemilius brevispiculus sp. n.

ARTICLE IN PRESS
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from Ghana with observations on copulatory plugs and spermatophore development. Nematology 3, 313323. Rehfeld, K., Sudhaus, W., 1985. Vergleichende Untersuchung des Sexualverhaltens eines Zwillingsarten-Paares von Rhabditis (Nematoda). Zool. Jahrb. Syst. 112, 435454.

White, J., 1988. The anatomy. In: Wood, W.B. (Ed.), The Nematode Caenorhabditis elegans. Cold Spring Harbour Laboratory Press, Cold Spring Harbour, pp. 81122.