C. Cuoc á D. Defaye á M. Brunet á R. Notonier á J. Mazza

Female genital structures of Metridinidae (Copepoda: Calanoida)

Received: 30 December 1996 /Accepted: 11 February 1997

Abstract The female genital structures of six calanoidcopepod species, belonging to the genera Gaussia, Met-ridia and Pleuromamma, were studied using light andscanning electron microscopy (SEM). The copulatorypores and seminal receptacles are paired in Gaussia andMetridia, but unpaired in Pleuromamma. A thin ep-icuticle and a spermatophoral plug are the mechanismsby which the pores are closed before and after copula-tion. The pores open directly into the receptacles, whichare reduced to shallow integumentary depressions inGaussia. The mode of insemination suggests two matingsin females of Gaussia and Metridia, but only one inPleuromamma. Paired gonopores and egg-laying ductsare present in the three genera, with a characteristic,closed semicircular con®guration; these are openedduring egg-laying by the action of retractor muscles ofthe gonoporal plates. The seminal ducts, which openinto the gonopores, are long, thin and paired in Gaussiaand Metridia, whereas in Pleuromamma they are short,broad and unpaired. The opening of the seminal andegg-laying ducts is synchronized. The shell ducts arepaired in Gaussia and Metridia, unpaired in Pleuro-mamma; these arise from glands situated in the lateralexpansions of the last prosomite and lead into the distalpart of each egg-laying duct in Gaussia andMetridia andof the egg-laying duct receiving the seminal duct in

Pleuromamma. The position and anatomy of thesestructures are compared to those of other families andgenera, and a functional interpretation of their mor-phology is proposed. The main evolutionary trends ofthe di�erent structural patterns of female calanoid gen-italia are presented in tabular form.

Introduction

The genital structures of female calanoid copepodsfunction in the storage of seminal products, the fertil-ization and laying of oocytes, the formation of the egg-sac and the release of egg-shell material. They are locatedon the ventral face of the genital double-somite and to-gether constitute the female genitalia, the organizationand functioning of which are very poorly known.

Most of the previous morphological data have comefrom studies of specimens ``in toto'' and do not allow aprecise understanding of the anatomy of the internalstructures, despite the high quality of some descriptionsbased on cleared specimens, such as those of Frost andFleminger (1968) for the genus Clausocalanus. Genitalanatomy has been carefully studied in the genus Acartia(Acartiidae) (Steuer 1923), Calanus ®nmarchicus(Calanidae) (Lowe 1935; Marshall and Orr 1955) andEpilabidocera amphitrites (Pontellidae) (Park 1966).Geptner (1968) was the ®rst to propose an anatomicalreconstruction of the genital double-somite of females ofPareuchaeta (Euchaetidae), after studying 18 species.These studies revealed di�erences in the organization ofthe genital structures and do not allow any generaliza-tion. Vaupel-Klein (1982) noted the insu�ciency of datain this ®eld and gave a precise, although incomplete,description for Euchirella messinensis (Aetideidae) basedon SEM observations of the external genital area. Otherinteresting illustrations were published by Blades (1977),Hammer (1978), Blades and Youngbluth (1979) andBlades-Eckelbarger (1991), mainly in investigations ofthe mating processes. More data concerning femalegenital structures in copepods have appeared in recent

Marine Biology (1997) 129: 651±665 Ó Springer-Verlag 1997

Communicated by A. RodrõÂ guez, Puerto Real

C. Cuoc á M. Brunet á J. Mazza (&)UPRES Biodiversite , Laboratoire de Biologie Animale (Plancton),Universite de Provence, Place Victor Hugo,F-13331 Marseille cedex 3, France

D. DefayeMuse um National d'Histoire Naturelle,Laboratoire de Zoologie (Arthropodes),61 Rue de Bu�on, F-75231 Paris cedex 05, France

R. NotonierService Commun de Microscopie Electronique,Universite de Provence, 3 Place Victor Hugo,F-13331 Marseille cedex 3, France

works such as Ferrari and Dojiri (1987) and, particu-larly, Huys and Boxshall (1991) and Ohtsuka et al.(1994). In addition to SEM data, the latter authorsprovided a detailed diagram of external and internalstructures observed in whole specimens after clearing.Finally, a comprehensive approach to the anatomicalrelationships of the di�erent genital elements was de-voted to Hemidiaptomus ingens (Cuoc et al. 1989) fromSEM observations of internal and external structuresand light microscopy observations of semi-thin sections.Such investigations of inseminated and non-inseminatedfemales provide the elements necessary for a correctfunctional interpretation of the genital structures. Thusfar, they have been carried out for 20 species of cala-noids. We present here the results obtained from in-vestigations of the family Metridinidae, of thesuperfamily Arietelloidea (previously Augaptiloidea:Andronov 1974, 1991; Park 1986), which contains someof the most primitive calanoid families. The Me-tridinidae are pelagic copepods with a large bathymetricdistribution, and are represented here by the generaGaussia, Metridia and Pleuromamma. The genitalstructures of Metridinidae are only incompletely knownfor G. asymmetrica (BjoÈ rnberg and Campaner 1988) andthe genus Pleuromamma (Blades-Eckelbarger 1991), buthave not, to our knowledge, been previously describedfor Metridia species.

Materials and methods

Material examined

Material of the six species examined came from three institutions:Muse um National d'Histoire Naturelle, Paris (MNHN); Labor-atoire de Biologie Animale, Plancton, Universite de Provence,Marseille (UP); and US National Museum of Natural History,Washington (USNM).

Gaussia n. sp. (description in preparation by Defaye). North Paci®c:9 November 1962, Station 8295 (33°16¢ to 33°25¢N; 118°36¢ to118°50¢W), depth 1262 to 1271 m (USNM).

Metridia princeps (Giesbrecht, 1889 ). South Atlantic: 17 October1962, Station 259 (62°00¢ to 62°14¢S; 68°01¢ to 68°14¢W), 2615 m;North Atlantic: June 1955 and 1956, Station 187 (41°08¢N;19°45¢W) and Station 195 (41°08¢N; 16°27¢W), depth 0 to 1000 m(UP and USNM).

Metridia lucens (Boeck, 1864 ). Razouls collection, MediterraneanSea, o� Banyuls (MNHN).

Pleuromamma abdominalis (Lubbock, 1856 ). North Atlantic, o�coast of Portugal: June 1955 and 1956, Station 216 (39°48¢N;11°12¢W), depth 0 to 1000 m (MNHN and UP).

Pleuromamma xiphias (Giesbrecht, 1889 ). North Atlantic, Bermu-das: 15 July 1992 (32°09¢N; 64°34¢W), depth 0 to 800 m; Azores:June 1955 and 1956, Station 187 (41°08¢N; 19°45¢W), depth 0 to1000 m (MNHN, UP and USNM).

Pleuromamma gracilis (Claus, 1863 ). Razouls collection, Mediter-ranean Sea, o� Banyuls (MNHN).

Methods

Scanning electron microscopy. For studies of external morphology,female specimens were rinsed in distilled water, treated with 2%sodium hypochloride to eliminate the organic remains on the sur-face of the genital double-somite, rinsed again, dehydrated in ac-etone, and critical-point dried. For studies of internal morphology,a dorsal cut (as broad as possible) was made into the genitaldouble-somite. The urosome was then isolated and treated with 2%sodium hypochloride in order to eliminate all the soft parts, otherthan the muscles associated with the genital area. After rinsing indistilled water, samples were stained in an aqueous solution ofchlorazol black, and then dried in the same way as mentionedabove. Samples were then mounted on a stub and coated with goldor gold-palladium. Observations were carried out using a JEOLJSM 35C or a Hitachi 5 570 scanning electron microscope.

Light microscopy. Specimens for light microscopy were dehydratedand embedded in Epon. Semi-thin sections of the genital double-somite were cut using an LKB ultramicrotome and stained withUnna Blue.

Results

We use the term genital area (Cuoc et al. 1989) to des-ignate all internal and external regions of the genitaldouble-somite, including the female genitalia and theirassociated muscles. For the various structures of thegenital area, we follow the nomenclature of Huys andBoxshall (1991) and Ohtsuka et al. (1994), with somechanges which will be discussed later.

Fig. 1 Gaussia sp. 1±7 Scanning electron micrographs of femalegenital area: 1 outer ventral face of urosome, note position of closedgonopores (cg) and seminal receptacles (sr), right receptacle beingempty and left containing spermatophoral mass (sm), thoracicexpansions (stars) of last prosomite segment, and strong hook (h)and lateral protuberances (p) of genital double-somite; 2±3 compar-ative outer ventral (2) and inner dorsal (3) views of anterior part ofgenital double-somite; 4 insemination site or copulatory pore of non-inseminated right receptacle, note copulatory pore closed by a ®nemembrane, cracked on its external side (arrowheads); 5 inner dorsalview of right part of genital area; 6 detail of inner-right gonoporalzone, note semicircular shape of egg-laying duct (d3) with double wall(arrows), and insertion (arrowhead ) of muscles (m) of egg-laying duct;7 inner view of right seminal receptacle (sr) and right seminal duct(d1), note multilobed proximal end (arrow) of seminal duct. 8±13Photomicrographs of transverse sections through last prosomite (8)and genital double-somite (9±13): 8 left oviduct (od ) and thoracicglands (tg), with proximal end of shell duct (d2); 9 shell duct (d2)opening into right egg-laying duct; 10 left egg-laying duct and shellduct (d2), (stippling delimits muscles of egg-laying duct); 11inseminated left receptacle, note seminal contents (*), layered envelope(arrows) of spermatophoral mass (sm), and shallow tegumental cavity,corresponding to the seminal receptacle (sr); 12 inseminated rightreceptacle (sr), with proximal end of seminal duct (arrow); 13 junctionbetween seminal duct (d1) and right egg-laying duct (cg closedgonopores; d1 seminal duct; d2 shell duct; d3 egg-laying duct; gpgonoporal plates; gs gonoporal slits; h hook of genital double-somite;m muscles of egg-laying duct; oe epithelial wall of oviduct; p lateralprotuberances of genital double-somite; sr seminal receptacle; tgthoracic glands; w wall of egg-laying duct)

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Gaussia n. sp. (3 females)

Observations

SEM observations of the external genital area of aspecimen with a single inseminated seminal receptacleand of the internal genital area of an another specimenwith both receptacles inseminated; light-microscopeobservations of semi-thin sections of the genital double-somite of the third specimen with two inseminated re-ceptacles.

Description

External genital area. The genital double-somite(Fig. 1: 1) is trapezoidal, 1.4 to 1.5 mm long, with awidth varying from 1.0 mm posteriorly to 1.6 mm an-teriorly. The anterior part has a strong, hook-shapedspine and two, rounded, lateral prominences. The gono-pores are situated lateroventrally, near the anterior edgeof the somite. They are closed, but are visible as asemicircular gonoporal slit, which delimits a gonoporalplate, 110 to 120 lm in diameter (Fig. 1: 2). In theposterior part of the genital double-somite, two shallow,oval, lateroventral cavities function as seminal recepta-cles, one or both of which may be inseminated (Fig. 1:1). Non-inseminated receptacles are covered by a mem-brane with a cuticular appearance, which indicates theinsemination site or copulatory pore (Fig. 1: 4). Insem-inated receptacles are ®lled with a prominent, volumi-nous, spherical, spermatophoral mass (Fig. 1: 1) that isdark brown in colour.

Internal genital area. Two short, cuticular, egg-layingducts are present at the level of the gonopores. Eachduct is crescent-shaped with a double wall which leavesonly a narrow lumen corresponding to the external gono-poral slit (Fig. 1: 3, 5, 6). Paired muscles insert on theinternal side of each gonoporal plate and the latero-dorsal wall of the genital double-somite (Fig. 1: 6, 10).Two cuticular ducts arrive at the level of each egg-layingduct. That arriving posteriorly is a seminal duct, only 3to 4 lm in diameter, which leaves the seminal receptaclevia a multilobed bulb (Fig. 1: 5±7). The other is a shell-duct, 8 to 9 lm in diameter, which arises from glandslocated in the lateral expansions of the last prosomite(Fig. 1: 8). Transverse sections at the level of the seminalreceptacle clarify the structure of the egg-laying ductsand their relationship with the shell and seminal ducts.The wall of each duct is formed of a short invaginationof the ventral cuticle, connected to secretory epitheliumof the oviduct (Fig. 1: 10). The shell duct and the sem-inal duct open approximately mid-way along the egg-laying duct (Fig. 1: 9, 13). The seminal ducts start fromthe central region of the integumentary depressions,which constitute the openings of the receptacles (Fig. 1:11, 12). The seminal products are barely recognizableamongst the spermatophoral mass. They are protected

by a thick envelope of at least four layers, which adheresstrongly to the ventral cuticle. The internal layer pre-sents a cuticular structure, while the external layers havethe form of a solidi®ed gel (Fig. 1: 11).

Metridia spp. (M. princeps, 10 females,M. lucens, 1 female)

Observations

SEM observations of the external genital area of severalspecimens of Metridia princeps and the single specimenof M. lucens, and of the internal genital area of severalspecimens of M. princeps. Light-microscope observa-tions of a series of semi-thin transverse sections throughthe genital double-somite of M. princeps.

Description

External genital area. In these species, the genital double-somite has the shape of a laterally compressed cylinder,with the anteroventral part showing a marked depres-sion (Fig. 2: 14, 16, 19). The genital area occupies theanterior half of the genital double-somite, including thedepressed zone. The closed gonopores are located on theanterior edge of this zone and are clearly separate. Eachappears as a semicircular slit, the convexity of which isdirected anteriorly and exteriorly, bounding a gonoporalplate, which is 75 to 80 lm in diameter in Metridiaprinceps and �25 lm in M. lucens (Fig. 2: 15, 19). Twoslight furrows are visible on the median part of thegenital area. These begin at the internal edge of eachgonoporal slit, pass posteriorly in parallel, and end justbefore the medioventral region of the genital double-somite. The slightly divergent extremities of the twofurrows are enlarged posteriorly as they reach the cop-ulatory pores (Fig. 2: 14±19). These pores are only vis-ible in inseminated females (Fig. 2: 18); in virginfemales, they are covered by a cuticular membrane. Insome cases only one may be visible, indicating a uni-lateral insemination (Fig. 2: 17). Behind the copulatorypores of one female M. princeps, the ventral region ofthe genital double-somite bears ®xation discs from sev-eral spermatophores (Fig. 2: 20), behind the genital areaitself. Other females were observed with scattered re-mains on the surface, marking the ®xation zones ofeliminated spermatophores (Fig. 2: 21). This zone seemsto have at least three pairs of small glandular pores in itsposterior part and a sensory pore at the level of thecopulatory pores (Fig. 2: 17, 21).

Internal genital area. Two, spindle-shaped, seminal re-ceptacles can be seen on the internal side (Fig. 2: 22).These have one end thin, linked to the copulatory poreby a short copulatory duct (Fig. 2: 24), while the other isclub-shaped, extended by a long, thin seminal ductwhich leads to the base of the associated egg-laying duct

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(Fig. 2: 22, 23). Each egg-laying duct also receives a thinshell-duct and is provided with a muscle inserting on thegonoporal plate (Fig. 2: 23; 4: 37, 38). Two ridges, cor-responding to the external furrows, link the egg-layingducts to the copulatory pores (Fig. 2: 22). Sections of thegenital double-somite indicate that the seminal ducts areattached to the ridges for most of their length. The di-ameter of the seminal ducts does not exceed 4 lm. Theirclosed extremities are located on the internolateral edgeof the egg-laying ducts (Fig. 4: 41). These ducts receivethe oviducts and have a rather thin cuticular wall, exceptnear the gonoporal plates where it is thickened. Strong,paired muscles insert on the thickened region, the other

end being attached to the laterodorsal wall of the genitaldouble-somite (Fig. 4: 37). At the level of the insemi-nated receptacles, the copulatory duct is always ®lledwith strongly-staining spermatophoral material, forminga plug, which is sometimes visible at the level of thecopulatory pores (Fig. 2: 17, 18; 4: 40). In the case ofnon-inseminated receptacles, the copulatory duct isempty and the associated pore is closed by a thin epi-cuticular membrane (Fig. 4: 40, 42).

Fig. 2 Metridia spp. Scanning electron micrographs of genital area offemales. 14±18 M. princeps: 14±16 ventral face of genital double-somite of two specimens (14, 16) and detail of genital area (15); 17, 18copulatory pores (cp) of females with single insemination on right side(17) and with insemination on both sides (18), note spermatophoralplugs (black arrowheads ), closed copulatory pore (arrow), and sensorypore (?) (white arrowhead ). 19 M. lucens, genital area of non-inseminated female; note closed gonopores (black arrowheads),shallow super®cial furrows (white arrows) and closed, obscurecopulatory pores (white arrowheads). 20±24 M. princeps: 20 ®xation

sites of three spermatophores, note ®xation disk level with rightcopulatory pore (large arrow), two posterior disks (triangles), andsuper®cial furrows (small arrow); 21 posterior part of genital double-somite, with spermatophoral remnants and some glandular pores(arrows); 22±24 inner dorsal view of genital area (22), with details ofright egg-laying duct (d3) (23) and copulatory ducts (arrows) (24) (cgclosed gonopores; cp copulatory pores; d1 seminal duct; d2 shell duct;gp gonoporal plates; gs gonoporal slits; f folds of super®cial furrows;m muscle of egg-laying duct; sg super®cial furrows betweencopulatory pores and gonopores; sr seminal receptacles)

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Pleuromamma spp. (P. abdominalis, P. xiphiasnumerous specimens, P. gracilis, 1 female)

Observations

SEM observations of the external genital area of severalinseminated females and a virgin female of bothPleuromamma abdominalis and P. xiphias, and one in-seminated female of P. gracilis. SEM observations of theinternal genital area of P. abdominalis (4 inseminatedfemales) and P. xiphias (6 inseminated females). Light-microscope observations of transverse and longitudinalserial sections of the genital double-somite of two in-seminated females of P. abdominalis.

Description

External genital area. The genital area is located in theanterior ventral half of the genital double-somite in thethree species (Fig. 3: 25). The ventral face of the somiteis strongly rounded, except towards the anterior edgewhere the two gonopores are situated close together. Asin Gaussia and Metridia, the gonopores are closed andappear as two semicircular slits which delimit an almostspherical gonoporal plate, 30 to 40 lm in diameter.There is a single, medioventral copulatory pore, which isalways sealed (Fig. 3: 25±28). In virgin females, thispore is closed by a thin, fragile cuticular membrane.When broken, this membrane reveals a wide openingonto the empty seminal receptacle (Fig. 3: 29, 30). Ininseminated females, the pore is ®lled with an oval(Pleuromamma abdominalis, P. gracilis) or spherical (P.xiphias) spermatophoral plug, which has an eccentric,pediculate bulge that is the insemination point of aspermatophore (Fig. 3: 25±28). The removal of this plugand an appropriate treatment allow the receptacle full ofspherical or oval sperm (diam 8 to 10 lm) to be seen(Fig. 3: 31).

Internal genital area. The two egg-laying ducts, whichhave the same structure as that observed in the othergenera, open onto the internal face, at the level of thegonopores. A single, strong muscle links the anteriorinternal face of each gonoporal plate to the laterodorsalwall of the genital double-somite (Fig. 3: 32±36). Pos-teriorly, a single seminal receptacle forms a wide, bi-lobed structure, with a sub-rectangular shape(�330 ´ 230 lm) in Pleuromamma abdominalis; 200 to250 ´ 150 to 200 lm in P. xiphias. The seminal duct (180to 200 lm long) separates from the receptacle sagittallyand extends to the internal edge of the left gonopore inP. abdominalis, or to the right or left gonopore inP. xiphias. A short blind duct (�60 lm long) opens atthe internal edge of the other gonopore (Fig. 3: 32±36).A single, thin shell-duct passes anteriorly into the samegonopore as the seminal duct (Fig. 3: 35, 36).

Sections of the genital double-somite show that thecuticular wall of the seminal receptacle is continuouswith the ventral cuticle of the genital double-somite. The

seminal products are packed into a homogeneous en-velope within the receptacle, forming a plug near thecopulatory pore (Fig. 4: 43, 44). The seminal duct andthe blind duct have a cuticular wall, the latter duct dis-playing an empty, narrow lumen. These ducts open atthe laterodistal end of the egg-laying ducts and theirapertures are normally closed by a constriction of thewall (Fig. 4: 45b, c). The egg-laying ducts, which receivethe oviducts, are short and similar to those of Gaussiaand Metridia. The cuticle only seems to be thickened atthe level of the wall of the egg-laying ducts and the gono-poral plates (Fig.4: 45a, b).

Discussion

Comparative organization of genital area

The genital area of the three genera is characterized bythe presence of paired gonopores, clearly separatedfrom the copulatory pores (paired in Gaussia andMetridia, unpaired in Pleuromamma). It is delimited bythe position of the pores and occupies almost the wholeventral face of the genital double-somite in Gaussia androughly the anterior ventral half in Metridia andPleuromamma.

Copulatory pores and seminal receptacles

Our results for Gaussia sp. con®rm and complementthose of BjoÈ rnberg and Campaner (1988) for G. princepsand G. asymmetrica. The present study reveals that thestorage sites for seminal products are reduced to simpleintegumentary depressions, provided with long ductsconnected to the gonopores, as in the other two genera.These function as seminal receptacles, with broad ex-ternal openings, into which the spermatophoral prod-ucts are discharged. We therefore retain the terms``copulatory pores'' and ``seminal receptacles'' to desig-nate the insemination and storage sites, respectively.

Fig. 3 Pleuromamma spp. Scanning electron micrographs of femalegenital area. 25 P. abdominalis, lateroventral view of the genitaldouble-somite; 26±28 comparative outer genital areas of inseminatedfemales of P. abdominalis (26), P. xiphias (27) and P. gracilis (28), notepedunculate structure (arrow) on spermatophoral plug (sp); 29P. xiphias, epicuticular membrane (star) masking the copulatory pore(cp) of virgin female; 30 P. abdominalis, copulatory pore (cp) andseminal receptacle (sr) of virgin female after partial elimination ofepicuticular membrane (star), note opening of seminal duct (arrow);31 P. abdominalis, seminal contents (arrow) of inseminated femaleafter removal of spermatophoral plug; 32 P. xiphias, inner dorsal viewof left egg-laying duct (d3); 33±34 comparative inner dorsal views ofgenital area of P. abdominalis (33) and P. xiphias (34), note position ofseminal (d1) and blind (bd) ducts; 35±36 inner dorsal views of parts ofgenital areas of P. abdominalis (35) and P. xiphias (36), note singleshell duct (d2), (bd blind duct; cg closed gonopores; cp copulatorypore; d1 seminal duct; d2 shell duct; d3 egg-laying duct; gp gonoporalplate; gs gonoporal slits; m muscle of egg-laying duct; sp spermato-phoral plug; sr seminal receptacle)

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These sites are only accessible after the rupture of anobturating membrane, which is probably homologouswith the epicuticular layer which closes the copulatorypores of virgin females of Metridia and Pleuromamma.The broad receptacular openings observed in Gaussiaare not known in any other calanoid. BjoÈ rnberg andCampaner considered the material ®xed to each recep-tacle of G. princeps and G. asymmetrica to correspond toa spermatophore. The spermatophore is a structuresynthesized in the male genital system, containing theseminal products and various other secretions. Accord-

ing to results for several species (see review by Blades-Eckelbarger 1991), the spermatophore generally subsistsfor only a brief period after the discharge of its contentsinto the seminal receptacles. The morphology of thespermatophore does not seem to have been describedpreviously in Gaussia. Our observations of a malespecimen of Gaussia sp. show that it has the shape of anelongated bulb, as in many calanoids. It appears that thematerial stored in the receptacles actually corresponds tothe extrusion products of the spermatophore, whichconstitute an external ``spermatophoric mass'', a term

Fig. 4 Metridia princeps and Pleuromamma abdominalis. Photomi-crographs of sections of genital double-somites of females. 37±42M. princeps, transverse sections of a left-inseminated female: 37section through left egg-laying duct (d3), note muscle insertion onlaterodorsal wall of genital double-somite (arrow); 38 detail of egg-laying duct, note narrow lumen (arrows) and thick wall of duct(arrowhead ) at level of muscle insertion (m); 39 section through leftinseminated receptacle; 40 section through copulatory ducts (cd ), notespermatophoral plug in left duct; 41 distal end of right seminal duct

(d1), close to right egg-laying duct (d3); 42 section at level of rightcopulatory pore, note epicuticular membrane (arrowhead ) closingpore. 43±45 P. abdominalis, sections of inseminated females: 43transverse section of seminal receptacle (sr), note spermatophoral plug(sp) closing copulatory pore; 44 sagittal section of seminal receptacleof another female; 45 a±c transverse sections at level of egg-layingducts (cw cuticular wall; bd blind duct; d1 seminal duct; d3 egg-layingduct; gp gonoporal plate; sc seminal contents; sp spermatophoral plug;sr seminal receptacle)

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employed by Bauer (1986) for analogous structures inDecapoda. Although a similar form of storage is prob-ably present in Lucicutiidae ± judging from the illus-trations provided for females of Lucicutia species(Hulsemann 1966; Razouls 1994) ± this process is onlywell known in Hemidiaptomus ingens and Mix-odiaptomus kupelwieseri (Diaptomidae) (Cuoc et al.1989). In the Diaptomidae, which lack seminal recep-tacles and ducts, the genital area is reduced to a smallregion, where the paired, closed gonopores are located.It serves as storage site and, in inseminated females, isalmost completely obscured by the spermatophoralmass. The latter term is preferred to that of externalspermatheca (Cuoc et al. 1989), since the term sperma-theca, as employed in higher Crustacea, refers to astorage structure in females, synonymous with seminalreceptacle (Bauer 1986).

The membranes which close the copulatory poresprobably play a role during the mating process (cf.subsection ``Functional interpretation'' below). Accessto the receptacles is gained with the emission of thespermatophore content during mating. The accessorysecretions of the spermatophore form a multi-layeredenvelope, as in Gaussia sp. and G. princeps (BjoÈ rnbergand Campaner 1988). One of these layers has the ap-pearance of chitinous material, the presence of whichhas been suggested in the seminal secretions of Crusta-cea (Subramoniam 1991). The envelope protects theseminal products and prevents subsequent access to thereceptacles. In Metridia and Pleuromamma, the secre-tions form a spermatophoral plug, which closes thecopulatory pores; such structures are also present inCrassarietellus (Ohtsuka et al. 1994). In females ofCentropages typicus and Temora stylifera, this type ofmaterial occurs in the genital cavity and could play thesame role (Cuoc et al. 1996). It is likely that all calanoidshave analogous mechanisms of pre- and post-copulatoryclosing of the copulatory pores, which isolate the inter-nal structures from the outside.

In the three genera studied, the seminal receptaclesare connected to the gonopores by seminal ducts. Thecopulatory pores, receptacles and seminal ducts arepaired in Gaussia and Metridia. In Pleuromamma, thesestructures are unpaired, but the bilobed morphology ofthe receptacle, and the persistence of a blind, vestigialduct associated to one gonopore, suggest that this is aderived character. This organization has functionalimplications (cf. subsection ``Functional interpretation''below). The tendency towards an unpaired, asymmet-rical organization is expressed in other calanoids. Thus,in Arietellidae, paired receptacles are generally the rule,but several genera have an unpaired copulatory pore. Inthe genus Paramisophria, the position of this pore caneven vary from median, left or right, depending on thespecies (Ohtsuka et al. 1994). In the closely-relatedfamily Hyperbionychidae, represented only by Hype-rbionyx pluto, a single, functional, seminal receptacle ispresent on the left side (Ohtsuka et al. 1993). The samepattern is observed in Stephos lucayensis of the family

Stephidae (Huys and Boxshall 1991). In Pleuromamma,the right or left position of the seminal duct observed inabout ten specimens each of the species P. abdominalisand P. xiphias cannot be considered as a speci®ccharacter. Ferrari (1984) showed that this genus pro-vides an example of ``looking-glass copepods'', whichexhibit an asymmetric dimorphism in position of boththe pigment knob1 and the aperture of the seminal ductin females of the P. xiphias species-group (P. abdomi-nalis and P. indica): the left lateral aperture of theseminal duct (``anterior tube of the genital segment'',Ferrari 1984) is associated with the right lateral positionof the pigment knob, and vice versa. The shell duct isalso involved in this asymmetry, following the positionof the seminal duct. According to Ferrari, a pleiotropicprocess could explain this unique group of combina-tions. A second group, comprising the other species ofthe genus (particularly P. gracilis), presents only onetype of asymmetry. In P. gracilis, this concerns thepigment knob (right side), the seminal duct (left side)and the asymmetrical position of the copulatory pore(``genital opening'', Ferrari 1984); this asymmetry is notclear on the single specimen studied here (Fig. 3: 28).

The seminal ducts open close to the gonopores inMetridia and Pleuromamma, and at the sub-distal end ofthe egg-laying ducts in Gaussia sp. Like the receptacles,the ducts possess a cuticular wall. InMetridia, their sub-integumentary location, coinciding with the thin furrowsvisible on the external area, suggests that they may beformed from integumentary invaginations which close toisolate them from the external cuticle. Adopting thishypothesis, we can suppose that the initial state was ofdiaptomid-type (external storage sites for seminalproducts) and that the distal migration of the gonoporeslead to the formation of true ducts from theintegumentary invaginations and the internalization ofreceptacles. This internalization is incomplete in Gauss-ia, which may constitute an intermediary step in thesechanges. In Metridia and Gaussia, the seminal ductsshare a remarkable character: they are very long andthin, not exceeding 3 to 4 lm in diameter. Spermatozoapassing through these ducts to fertilize oocytes musttherefore have an appropriate, ®liform morphology, asis suggested by the content of inseminated receptacles inMetridia (Fig. 4: 39). They should be very di�erent, inthis respect from the spherical or oval spermatozoa,several micrometres in diameter, of Pleuromamma xi-phias (Fig. 3: 31) and calanoids in general (Brown 1970;Blades-Eckelbarger and Youngbluth 1982; Pochon-Masson 1994). They would be more similar to the long,thin spermatozoa of harpacticoids, which do not exceed1 lm in diameter (Pochon-Masson and Gharagozlou-van Ginneken 1977; Hosfeld 1994).

1Term used by Herring (1988); synonymous with the ``black-organ'' of Ferrari (1984)

659

Gland ducts of lateral expansions of the last prosomite

In the three genera studied, ducts coming from glandslocated in the lateral expansions of the last prosomiteemerge at the level of the gonopores, at the distal end ofthe egg-laying ducts. These ducts have only been men-tioned by Park (1966) in Epilabidocera amphitrites andby Cuoc et al. (1989, 1994) in Hemidiaptomus ingens.The latter authors called them shell ducts, in reference tothe role of the secretions of the thoracic glands in thesynthesis of the egg-shell in this species. They are pairedin Metridia and Gaussia sp., and we have observedsimilar ducts in other calanoids, particularly in Arietellussimplex. In Pleuromamma, however, a single shell duct ispresent and opens into the gonopore receiving thefunctional seminal duct.

Gonopores and egg-laying ducts

The paired gonopores are located near the anterioredge of the genital double-somite and correspond to theapertures of the egg-laying ducts. While lateroventrallysituated and clearly separated from each other inGaussia sp., they are closer together in Metridia, andadjacent and medioventral in Pleuromamma. They havethe form of a hemispherical slit, corresponding to theclosed state of the egg-laying ducts into which theoviducts lead. The gonoporal plates, or genital operculaof previous authors (Cuoc et al. 1989; Huys and Boxs-hall 1991, Ohtsuka et al. 1994), are part of the ductwalls and form the closing apparatus. Gonopores andgonoporal plates are easily observed in the familyDiaptomidae, in which they are approximately medio-ventrally situated, in a well-delimited genital area (Cuocet al. 1989), as well as in families considered to beprimitive ± Pseudocyclopidae, Boholinidae and Ari-etellidae. In the latter family, in several genera, they arepositioned very anteriorly as in Metridinidae (Huys andBoxshall 1991; Ohtsuka et al. 1994). However, the gono-pores and gonoporal plates of most calanoids aremedioventrally situated and hidden by an external cu-ticular ¯ap, the anterior edge of which is fused to thegenital double-somite. A variety of terms has been usedto designate this ¯ap, the space just below it, and thegenital structures it protects. Vaupel-Klein (1982)named the external ¯ap the genital operculum2, a termalso used in recent papers by Blades-Eckelbarger (1991)and Huys and Boxshall (1991). Huys and Boxshall, inanalyzing the modi®cations of the female genital systemof copepods, considered that this single genital oper-culum resulted from the fusion of the genital operculaor gonoporal plates of more primitive calanoids. Con-

sequently, the external median opening was called thegonopore or common genital pore, which would haveappeared in most female calanoids after fusion ofprimitively paired gonopores. This genital pore givesaccess to a sub-opercular space ± the genital atrium3

(Blades-Eckelbarger 1991) ± into which the oviductsopen through paired openings, according to Blades-Eckelbarger, or paired or unpaired openings, accordingto Huys and Boxshall (1991). Vaupel-Klein (1982)noted the disagreements between authors concerningthe paired or unpaired nature of these openings, alsonamed genital openings or gonopores. These termscreate confusion with the genital pore or gonopore usedto designate the external aperture of the genital atrium.The characteristics observed in Diaptomidae and rep-resentatives of other calanoid families allow some ofthese ambiguities to be resolved. In Diaptomidae, thegenital area is usually delimited and partially protectedby a pad, which Huys and Boxshall consider to be thecommon genital operculum. Yet, the gonopores andegg-laying ducts of this family, studied in Hem-idiaptomus ingens and Mixodiaptomus kupelwieseri(Cuoc et al. 1989), present exactly the same organiza-tion as in Metridinidae. This was con®rmed in threeother diaptomids of the sub-family Diaptominae: Arc-todiaptomus wierzejskii, Diaptomus cyaneus and Hem-idiaptomus maroccanus (Cuoc et al. unpublishedobservations). No study has yet elucidated the exactorganization of the egg-laying ducts and their rela-tionship to the genital atrium for calanoids with agenital area closed by an operculum. However, SEMobservations of the internal genital structures ofTemora stylifera, Eurytemora velox, Candacia simplex,Centropages typicus, Gaetanus pileatus, Euchirella in-termedia and Eucalanus elongatus revealed the presenceof paired egg-laying ducts under the genital operculumin all these species. The ducts are hemispherical-shaped,with a pair of muscles and paired shell ducts, thushaving a pattern comparable to that of Metridinidae.Cuoc et al. (1989) have already mentioned the simi-larity in the structure of C. typicus, H. ingens andM. kupelwieseri. In our opinion, the anterior thickeningof the genital area of Diaptomidae and the genitaloperculum of numerous other calanoids are homolo-gous structures which probably have a protectivefunction. The gonoporal plates, often termed genitalopercula in the literature, have the same origin as theegg-laying ducts, on which they are dependent, andthey serve to close the egg-laying ducts (cf. followingsubsection). Hence, the term gonopore should be re-served for the openings of the egg-laying ducts, whilethe external aperture of atrium, partially covered by thegenital operculum, will be called opercular or atrial slit,synonymous with the ``genital valve'' used by Geptner(1968) for Euchaetidae.

2 This term is synonymous with ``genital ¯ap'' (Steuer 1923 andHammer 1978, in Acartia spp.), ``operculum'' (Brodskii 1967,in Calanus spp.), ``genital plate'' (Frost and Fleminger 1968, inClausocalanus spp.) and ``plaque genitale'' (Cuoc et al. 1989,in Centropages typicus).

3 Term previously used by Fleminger (1967) and Boxshall (1982);synonymous with ``vulval cavity'' (Vaupel-Klein 1982) and ``genitalcavity'' (Geptner 1968; Cuoc et al.1989).

660

Functional interpretation

The genital area of female copepods is involved in twokey phases of reproduction: insemination, comprisingthe ®xation of the spermatophore during mating and thetransfer of seminal products to the female storagestructures, followed by fertilization and oviposition. Theanatomical organization of this area in Metridinidae hasimportant functional implications during these twosteps.

Modes of insemination

One of the problems to be solved by the male duringmating is the correct positioning of the spermatophore.The morphology of the genital double-somite, and thepresence of sensory organs and pheromone-secretingglands, play an essential role in the recognition of thespermatophore ®xation site, close to or on the copula-tory pores (Vaupel-Klein 1982; Hulsemann and Flem-inger 1990; Blades-Eckelbarger 1991). In Metridinidae,the thin epicuticular membrane closing the copulatorypores of virgin females may constitute an identi®ablestructure for the male during mating. This form of rec-ognition is suggested by the unilateral inseminationsobserved in Gaussia and Metridia and by the remains of®xed spermatophores near the copulatory pores seen inMetridia (Fig. 2: 20). The breaking open of the copula-tory pore is induced either mechanically by the male, orby the pressure exerted by the insemination tube of thespermatophore, or the spermatophore itself, during thedischarge of the seminal products and associated secre-tions. These secretions make up the greater part of thespermatophoral mass in Gaussia, and the plugs whichblock the copulatory ducts of Metridia and the copula-tory pore of Pleuromamma. It is probably one of theseexternal secretions which takes on the brown colourobserved, particularly in Gaussia, perhaps owing to thepresence of a tanned chitinous material (Subramoniam1991). These modes of insemination suggest that onlyone spermatophore is discharged into a seminal recep-tacle. The bilateral inseminations associated with thepaired receptacles of Gaussia and Metridia imply twomatings, which are unlikely to be due to the same male,since each male only produce one spermatophore at atime (Katona 1975; Blades and Youngbluth 1981). InPleuromamma, the role played by the membrane inclosing the copulatory pore seems evident at thespermatophore-®xing phase. All the inseminated femalesobserved show only a single trace of a spermatophore, inthe form of a small protuberance on the spermatophoralplug. No sign of attachment is visible around the pe-riphery of the ventral face of the genital double-somite.In all these cases, a single male would be responsible forthe insemination of a female. Thus, Pleuromamma fe-males would only receive one spermatophore, whilethose of Gaussia and Metridia could receive severalspermatophores for each pore, even if only one e�ec-tively inseminates each receptacle. These conclusions

support those of Hopkins and Machin (1977) forEuchaeta norvegica, in which the ®xing of the ®rstspermatophore is precise and e�cient, while the ®xing ofother spermatophores has no functional importance andis only linked to a favourable sex-ratio.

Functioning of egg-laying ducts

The seminal products stored in the receptacles are usedto fertilize the oocytes during oviposition. The openingand closing of the egg-laying ducts of Metridinidae arepresumed to occur according to the pattern described inHemidiaptomus ingens (Cuoc et al. 1989), through theaction of duct muscles or the dilator muscles inserted atthe level of the gonoporal plates, which are the onlymuscles associated with the genital area in Gaussia,Metridia and Pleuromamma. Fig. 5 outlines their func-tioning and shows that: (1) the closed state induces agutter-like con®guration of the ducts, as seen at timesother than during oviposition; (2) the open, cylindricalcon®guration is active, due to the contraction of dilatormuscles which widely open the gonopores by completelyinvaginating the gonoporal plates. Relaxation of themuscles allows a passive return to the closed state

Fig. 5 Metridinidae. Schematic three-dimensional interpretation ofstructure and function of egg-laying ducts. A1, A2 Dorsal represen-tations of egg-laying duct when closed (A1) and open (A2); B1, B2details of part of duct when closed (B1) and open (B2), showing howcontraction and relaxation of muscles of egg-laying duct result in co-ordinated opening and closing of the gonopores and seminal ducts(black arrows), allowing release of gametes (open arrows) (the shellducts, which open into the egg-laying ducts, are not shown ondiagram). Functioning of egg-laying ducts of Diaptominae corre-sponds to this model, but the seminal ducts are absent

661

through the ¯exibility of the cuticular wall of each duct,particularly the gonoporal plates. These plates are not,therefore, independent, opercular-type closing structuresin the zoological sense, but rather mobile sternal plates,belonging to the walls of the self-closing egg-layingducts. The organization and functioning of these ductsare probably very similar in all calanoids. In most casesthese ducts open into the genital atrium, which is closedby the genital operculum such that the gonopores arenot immediately visible externally. The operculum iscompletely absent only in Hyperbionychidae, Boholin-idae, Arietellidae and Metridinidae. In Diaptomidae,however, the anterior edge of the genital area forms amore or less protruding thickening, which partiallycovers the gonopores (Diaptominae) or is extended toform an operculum (Paradiaptominae) (Defaye et al.1996). The homology of these structures is con®rmed bythe arrangement of the associated musculature: threepairs of muscles are associated with the genital area inH. ingens (Diaptominae) (Cuoc et al. 1989), of whichone pair is inserted on the anterior thickening. A similarmusculature is present in Paradiaptomus (Lovenula)falcifera (Paradiaptominae), Centropages typicus (Cen-tropagidae), Temora stylifera and Eurytemora velox(Temoridae), Candacia simplex (Candaciidae) (Cuoc etal. unpublished observations), which all belong to Cen-tropagoidea.

Modes of fertilization and oviposition

The muscles of the egg-laying ducts act synchronouslyand the release of oocytes occurs simultaneouslythrough both gonopores, as has been observed inHemidiaptomus ingens (Cuoc et al. 1989). In Metri-dinidae, the fertilization of oocytes can only occur at thistime, at the distal end of the egg-laying ducts, since theseminal ducts open into this part and are closed at othertimes. This closure prevents any spermatozoa enteringthe oviducts before oviposition; the topographic ar-rangement of the di�erent ducts suggests that theiropening is synchronized with the contraction of the di-lator muscles (Fig. 5).

In Gaussia and Metridia, which have clearly sepa-rated gonopores, the oocytes are fertilized at each gono-pore by sperm coming from the associated receptacle.The relative positions of the gonopores and seminalducts support the hypothesis of two successive matings.The unilateral insemination observed in some femalessimply indicates that the mating phase is not ®nished.Although Pleuromamma only has a single functionalseminal duct, its diameter is clearly larger than in theother genera, which would facilitate a greater emissionof sperm, allowing the successful fertilization of all oo-cytes discharged from the adjacent gonopores. The sameprobably applies to the secretions of the single shell duct.Indeed, it is a remarkable anatomical adaptation. Moregenerally, paired gonopores appear to be a common, ifnot general, trait of calanoids. This arrangement should

o�er a selective advantage over an unpaired systemformed by the terminal fusion of the oviducts and/oregg-laying ducts. In the latter case, it is probable thatoocytes would have to be released one by one, whichwould double the oviposition time and consequently theassociated energetic cost.

Seminal products stored in the receptacles allow thefertilization of oocytes during oviposition. If insemina-tion of the receptacles occurred only once in the life of afemale, the stored sperm would have to be su�cient tofertilize all subsequent ovipositions. Corkett andMcLaren (1978) estimated that the number of fertilizedovipositions in Pseudocalanus females could reach 8 to10 during the reproductive period. However, an earlydepletion of the stored seminal products, resulting fromthe ``poor quality'' of the male, will lead to non-viableeggs. This was suggested by Corkett and McLaren as anexplanation for the laying of non-viable eggs during thepost-reproduction period. Finally, a consequence of thesingle insemination in Metridinidae and most other ca-lanoids is that the ``useful life'' of males, in terms ofreproduction, is much shorter than that of females. Atthe population level, these modes probably in¯uence theevolution of the sex-ratio which, despite varying ac-cording to environmental conditions, usually favoursmales initially and then females (Kouwenberg 1993).However, a stable sex-ratio, of �1:1 was observed insome diaptomid populations which are not subject topredation (Hairston et al. 1983), the females of whichmust be fertilized before each oviposition (Cuoc et al.1989).

Evolutionary trends in organizationof genital area in calanoids

Although limited to some species of the family Metri-dinidae, the present study of the genital area con®rms thepattern of separate gonopores and copulatory pores inthe Arietelloidea, despite the wide variety of evolutionarytrends present, particularly in the Arietellidae (Ohtsukaet al. 1994). Based on the genital structures of the hy-pothetical calanoid ancestor constructed by Huys andBoxshall (1991) and adopting Ohtsuka et al.'s models fortheir representation, we propose a diagram (Fig. 6)which takes into account the present data for metridinidsand main forms presently known in calanoids. The an-cestral state, de®ned by Huys and Boxshall, is charac-terized by a paired organization, with a medioventralposition of the genitalia, in which the gonopores andcopulatory pores are adjacent. The latter lead to seminalreceptacles via a copulatory duct. Seminal ducts connectthese receptacles to the gonopores and allow the fertil-ization of oocytes at oviposition. Studies to date indicatethat two main trends in the evolution of the calanoidfemale genital system can be recognized. In the ®rst,which particularly characterizes the superfamily Arie-telloidea, the gonopores and copulatory pores are notcovered, being directly accessible as in the calanoid

662

ancestor, but their relative arrangement has been mod-i®ed according to numerous trends. The main changeswhich have occurred in this superfamily (Fig. 6) involve:(1) the anterior migration of gonopores and copulatorypores, observed only in Boholinidae (Ohtsuka et al.1994); (2) the lateral migration of gonopores in thefamilies Hyperbionychidae (Hyperbionyx), in which thislateral migration is accompanied by a quasi-total re-gression of structures located on the left side (Ohtsuka etal. 1993), and in Arietellidae, in which the genera Scu-togerulus and Crassarietellus have lateral gonopores,with the copulatory pores hidden by a ventral ¯ap in thelatter (Ohtsuka et al. 1994); (3) the anterior migration ofgonopores, as shown by two families: Arietellidae (Pa-ramisophria and the derived genera Arietellus and Sar-sarietellus) (Ohtsuka et al. 1994), and Metridinidae(Metridia); (4) the anterior migration of gonopores andthe posterior migration of copulatory pores in Arietell-idae (Paraugaptilus) (Ohtsuka et al. 1994) and Metri-dinidae (Gaussia); (5) the posterior migration ofgonopores and copulatory pores, known only in Arie-tellidae (Metacalanus) (Ohtsuka et al. 1994); (6) the an-terior migration of the gonopores and transformation ofthe remaining genitalia into unpaired structures inPleuromamma. This scheme of evolutionary trends thusprovides several examples of parallel evolution. More-over, in the family Metridinidae, it indicates that themost derived characters of the genital area are found inGaussia (arrangement of gonopores and copulatorypores) and Pleuromamma (unpaired genitalia, excludingegg-laying ducts). From the ancestral state, the evolutionof the arrangement of the seminal receptacles may be

interpreted in the same way, with Metridia illustrating a``primitive type'', while Gaussia and Pleuromamma eachshow unique characters for calanoids. Pleuromamma,with an unpaired copulatory pore, provides the ®rst ex-ample of a bilobed seminal receptacle, resulting from thefusion of two primitive receptacles. The unpaired re-ceptacles of Hyperbionychidae (Ohtsuka et al. 1993) anda species of Metacalanus (Arietellidae) (Ohtsuka et al.1994) are not the result of fusion, but of a regression ofthe genital structures on one side of the body. Gaussiademonstrates a remarkable evolutionary trend in havingboth receptacles quasi-external, despite possessing longinternal seminal ducts. Finally, shell ducts are present inMetridinidae and Arietellus simplex, and represent ele-ments of the genital area liable to be modi®ed, as is thecase in Pleuromamma which has an unpaired duct.

In the second evolutionary trend, which occurs inmost calanoids, the gonopores and copulatory poresremain adjacent, but are covered by a cuticular fold oroperculum, which delimits the genital atrium (Cuoc et al.1996). Within this trend, the family Diaptomidae mayrepresent a transition group, since its members have thegonopores partially covered by a cuticular thickening(modi®ed or rudimentary operculum), or uncovered(Diaptominae), or hidden by an operculum (Paradi-aptominae: Defaye et al. 1996). This organization isassociated with the loss of the copulatory pores andseminal receptacles. Their replacement by an external,pseudo-receptacle has the physiological consequencethat seminal products can only be stored for a briefperiod, which necessitates a new insemination for eachoviposition, as occurs in Hemidiaptomus (Cuoc et al.

Fig. 6 Diagrammatic represen-tation of main evolutionarytrends in organization of femalegenital area in calanoids(modi®ed after Huys andBoxshall 1991 and Ohtsukaet al.1994) (* seminal duct ofPleuromamma may be situatedon left or right, according toindividual)

663

1989). Shell ducts have been observed in Pontellidae(Park 1966), Diaptomidae (Cuoc et al. 1989, 1994) andsome species of other calanoid families which have agenital operculum (Cuoc et al. 1996). The presence ofsuch ducts in the latter remains to be con®rmed, as doesthe presence and arrangement of the copulatory poresand seminal receptacles.

Acknowledgements We wish to thank the following who kindlysupplied us with di�erent samples: Dr F. Ferrari, US NationalMuseum of Natural History (Washington), and Dr G.A. Boxshall,Natural History Museum (London). We are also grateful toM. Judson (Muse um national d'Histoire naturelle, Paris) for helpwith the English translation.

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