how can nematodes mate without spicules function of the male gonoduct

7/31/2019 How Can Nematodes Mate Without Spicules Function of the Male Gonoduct

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ZOOLOGYZoology 108 (2005) 211216

How can nematodes mate without spicules? Function of the male gonoductglands in the roundworm Myolaimus

Alexander Fu rst von Lieven, Verena Ba rmann, Walter Sudhaus

AG Evolutionsbiologie, Institut fur Biologie/Zoologie, Freie Universitat Berlin, Konigin-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 first 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 ofAnatrichosoma buccalis inserts up to half of

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 under-

standing 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 bursa-

like structure.

The outer layer of the body cuticle in Myolaimus

specimens is not firmly attached to the inner layer,

but flaccid, and has a wrinkled appearance.

In the midbody region of the female, the loose outer

cuticle layer of the vulva lips forms a sack-like

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doi:10.1016/j.zool.2005.04.002

Corresponding author.

E-mail address: [email protected] (A. Fu rst von Lieven).
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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 bodyopenings 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 fluids. 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 filled conspicu-

ously with secretion granules (Chitwood and Chitwood,

1974). In many cases the first 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 Caenor-

habditis 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 unidentified,

possibly new species was cultured in Petri dishes (10 cm

diam., 0.5 cm high, filled 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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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 isinflated by glue. (d) Two portions of capsule substance are injected into the vulval sack. (e) Capsule substance is inflated by seminal

fluid and sperm.

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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 high-

est magnification (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 filled

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) filled 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 deferenslumen 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 thespermatids, a clear substance is secreted that takes

up the sperm and therefore is named seminal fluid

(substance 6; Figs. 1a, 2a, b).

Precopulatory behavior begins with the male search-

ing 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 attach-

ment, substances 36 are emitted from the valve cells

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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, ante-

rior right-hand side). (b) Same as in (a), after attachment to

female. Secretion vesicles are expelled to fill 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 inflated by seminal fluid

(6). (f) Sperm transfer. The capsule bursts and sperm and

seminal fluid (6) are pumped into the female. Scale bars:

(a, b) 25mm, (ce) 20mm, (f) 35mm.

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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 ofthree pouches that surround the orifice of the valve

(Figs. 3a,b and d). The orifice of the valve can thereby

be opened easily when sperm is pushed posteriorly by

the increased inner body pressure produced by addi-

tional contractions of longitudinal body muscles. When

the body muscles relax, the valves orifice closes to

hinder return flow 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 fluid pressure

within the pouches until they collapse and fluid 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 inflated (Fig. 1c). Approximately 10 min after

the first 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

fluids. 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 fluid is

pumped into the capsule substance, which becomesinflated 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 flexible 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 hoursuntil it finally disappears by an unknown mechanism.

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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 findings reported here provide unique insight into

the function of male gonoduct glands in nematodes. In

Myolaimus we observed five different functions that can

be assigned to six different substances: (1) The glue is

used for initial attachment to the female and forinflating 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 fluid takes up spermatids that must be

suspended in some kind of matrix for transfer and

inflates 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 withinNematoda. 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 Myolai-

mus uses for attachment and inflation of the vulval sackis 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 filled 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 superfluous 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 bedetected in other nematodes that possess spicules in

combination with a variety of secretions. As the capsule

in Myolaimus is reminiscent of a primitive spermato-

phore, 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 Universita t Berlin. As a participant of this

class, V. Ba rmann videotaped the mating behavior of

Myolaimus. The sequences were analyzed and inter-

preted under the guidance of A. Fu rst von Lieven and

W. Sudhaus.

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how can nematodes mate without spicules function of the male gonoduct

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