Marine Biology (1998) 132: 435±444 Ó Springer-Verlag 1998

P. G. Beninger á R. Larocque Gonopod tegumental glands: a new accessory sex gland in the Brachyura

Received: 26 January 1998 / Accepted: 11 June 1998

Abstract It is not yet known whether gonopod tegu- neutral mucopolysaccharide (NMPS) layers in the three mental glands (GTG) previously described in one species Portunidae, AMPS only in Cancer irroratus, and NMPS of brachyuran crab (Chionoecetes opilio) are a general only in Grapsus grapsus and Petrolisthes armatus. When feature in this large taxon. In order to determine the combined with data on gonopod morphology and oc- prevalence and role of GTG in the Brachyura, the ®rst currence of spermatophore-less sealant in the ejaculate gonopods of six species of boreo-temperate and tropical of various brachyurans, two plausible functions of the brachyurans belonging to four families were examined AMPS GTG secretions emerge: protection of the male's morphologically and histologically, using the PAS±Alc- genetic investment (stored spermatophores) from op- ian-blue staining protocol: Carcinus maenas, Portunus portunistic microbes following copulation, and the re- sebae, and Ovalipes ocellatus (Portunidae), Cancer ir- ciprocal processes of sperm competition and paternity roratus (Cancridae), Grapsus grapsus (Grapsidae), and assurance. The NMPS secretions may function as a lu- Petrolisthes armatus (Porcellanidae). Discrete rosette- bricant to reduce mechanical wear of the ejaculatory type GTG were found in all species examined, although canal by the second gonopod during copulation, and to the longitudinal extent and location di€ered somewhat reduce the viscosity of the ejaculate from the vas de- between taxa. The GTG were invariably grouped about ferens as it enters the narrow ejaculatory canal. the ejaculatory canal, and communicated with the lumen of the ejaculatory canal via ducts which traversed pores in the cuticle; staining properties of secretions at the duct openings to the ejaculatory canal matched those of the Introduction GTG. Neither GTG, ducts, nor pores were observed in regions distal to the ejaculatory canal. These data indi- Although crabs currently represent the greatest pro- cate that the prime, if not exclusive, role of the GTG is portion of crustacean captures in the world's ®sheries, at in reproduction, and that GTG may therefore be con- 1 738 407 tons declared in 1993 (Anonymous 1995), sidered accessory sex glands. Together with previous and ®sheries management has been limited by the availability current investigations such GTG have been observed in of biological data concerning the extremely diverse all eight brachyuran species examined from ®ve families, assemblage of crab species (Haefner 1985; Bailey and and are thus probably ubiquitous within the Brachyura. Elner 1989). In particular, investigations of crab repro- The organization and nature of the gland secretions duction are thinly scattered both in level of approach di€ered between taxa: alternating acid (AMPS) and and taxa studied. The information which has emerged over the past 20 years indicates that in the largest group, the infraorder Brachyura, reproduction is much more complex than was previously believed (see Adiyodi 1983; Sastry 1983; Bauer and Martin 1991; Wenner and Kuris Communicated by O. Kinne, Oldendorf/Luhe 1991; Elner and Beninger 1995 for reviews and refer- P.G. Beninger (&) ences). Multi-level data exist for very few species (Sastry Laboratoire de Biologie Marine, 1983), the foremost being the circumboreal majid Faculte des Sciences, Universite de Nantes, F-44322 Nantes Ce dex 03, France Chionoecetes opilio. Its status as a major ®shery species of Canada, the United States, Japan, and Russia has R. Larocque De partment de Biologie, Faculte des Sciences, doubtless contributed to the impetus of the research Universite de Moncton, Moncton, New Brunswick, e€ort. E1A 3E9, Canada 436

An understanding of the functions of the reproduc- tive system components is fundamental to the elucida- Materials and methods tion of reproductive processes in these organisms. Recent studies of the male and female systems have Sampling shown that events surrounding copulation, sperm Boreo-temperate species transfer, storage, and fertilization in the Brachyura now appear to be quite complex, with possibilities of sperm Mature male specimens of Cancer irroratus (carapace width, CW, competition and paternity assurance (Christy 1987; Be- 91.5 to 101.1 mm) were collected via SCUBA in the Northumber- land Strait (46°14¢N; 64°12¢W), 4 July 1995, 25 July 1995, and ninger et al. 1991, 1993; Diesel 1990, 1991; Sevigny and 15 August 1995. The gonopods of these individuals were removed in Sainte-Marie 1996), self-fertilisation by females whose the ®eld and ®xed in aqueous Bouin's solution for later histological broods are compromised (Beninger et al. 1993), di€er- processing. In order to detect any di€erences in pre- and post-cop- ential spermatophore dehiscence (Beninger et al. 1988, ulatory gland characteristics, three couples in pre-copulatory em- brace were collected via SCUBA on 2 July 1996 at the same sampling 1993), and enhancement of oocyte and stored sperm site and returned to the laboratory, where they were placed in a viability through protection from opportunistic recirculating seawater system at the same temperature, salinity and microbes (Beninger et al. 1993; Elner and Beninger 1995; approximate ambient light conditions as the sampling site (20 °C, Lanteigne et al. 1996). 24&, natural shaded daylight). The aquarium bottom contained large rocks as refuges. One male and one female were placed together The copulatory appendages, or gonopods, are of in three separated portions of the aquarium. All couples formed pre- particular interest in the brachyuran male reproductive copulatory embraces of variable duration; the left ®rst gonopods system, because they are morphologically diverse yet were removed and ®xed as above upon observing pair formation, taxonomically very conservative, and hence provide and prior to female moulting which precedes copulation in this species (Hartnoll 1969). Although removal of the remaining ®rst valuable taxonomic and phylogenetic information (Ste- gonopod was intended following copulation, males were unable to phensen 1946; Garth 1958; Williams 1965; Nishimura successfully copulate after removal of the left ®rst gonopod. 1967; Abele 1971; Hartnoll 1975; Chambers et al. 1980; Mature male Ovalipes ocellatus and additional mature male Bauer 1986; Martin and Abele 1986). These structures Cancer irroratus were subsequently collected at the sampling site may be of even greater importance in the Brachyura, as 11 July 1996. The gonopods were removed and placed in aqueous Bouin's ®xative immediately upon surfacing. a glandular function has recently been attributed to the Mature male specimens of Carcinus maenas and an embracing ®rst gonopods of Chionoecetes opilio (Beninger et al. pair (hard-shelled male, soft-shelled female) were sampled from the 1991, 1995). Although considerable progress has been intertidal in Passamaquoddy Bay (45°7¢N; 67°4¢W) on 25 July 1995 made in the study of the glandular functions of the vas and placed in the recirculating seawater system. The embracing pair were observed to copulate upon being placed in the aquarium. The deferens and spermatheca (Spalding 1942; George 1963; ®rst gonopods were removed and ®xed as above (after completion Ryan 1967; Johnson 1980; Beninger et al. 1988, 1993; of copulation in the case of the copulating male). Lanteigne et al. 1996) , the gonopod tegumental glands All crabs in the aquarium were fed daily with pollock and oc- (GTG) have received scant attention. Emerging from the casionally mussels, scallops, or oysters. Excess food was removed debate concerning the functions of such glands in the 3 h after presentation, and feces were removed daily. The aquarium ®lters were cleaned daily with boiling water in order to prevent crustacean exoskeleton in general is the overarching microbial proliferation. need for more data (see Stevenson 1985; Felgenhauer 1992; Fingerman 1992; Subramoniam 1993; Talbot and Demers 1993 for reviews). That the GTG are related to Tropical species reproductive activity in the male was ®rst suggested by Mature male Portunus sebae were collected from Discovery Bay, Beninger et al. (1991) for C. opilio; subsequent investi- Jamaica (18°24¢N; 77°27¢W) via SCUBA on 20 and 29 December gations con®rmed their role as male accessory sex glands 1995. Mature specimens of Grapsus grapsus were also collected in in this species (Beninger et al. 1995). No further studies the intertidal in Discovery Bay on 26 December 1995. Mature have been published to date concerning this aspect in specimens of Petrolisthes armatus were collected via SCUBA from other brachyurans, or indeed in other decapods, so no Runaway Bay (18°24¢N; 77°20¢W) on 27 December 1995. evaluation of its occurrence or role within these taxa has been possible. The present study reports on investiga- Histological processing tions into the distribution, histology, and secretion types of GTG discovered in six additional brachyuran species In order to improve paran penetration, the gonopod cuticles were perforated using entomological needles in the region opposite the from four additional families, encompassing both boreo- ejaculatory canal. The gonopods were stored in aqueous Bouin's temperate and tropical habitats, as well as both aquatic ®xative for at least 3 months, in order to achieve sucient softening and semi-terrestrial species. The results are related to of the cuticle (Beninger et al. 1995). Scienti®c drawings were made gonopod morphology, possible secretion roles, and under stereoscopic microscope of the ®rst gonopods for all species, from both the anterior and posterior aspects. The gonopods were evolution of the functions of the ventralmost ejaculate bisectioned transversally and processed for histology as described within the brachyuran spermatheca. in Beninger et al. (1995). The two gonopod lengths were embedded side-by-side in order to facilitate serial sections. Transverse serial sections were cut at 10 lm (optimum thickness for intact sections) in order to determine gland distribution within the ®rst gonopod of each species. Once this distribution was ascertained, subse- quent sections were performed directly in the gland region of the gonopods. 437

Preliminary tests using the PAS±Alcian-blue staining protocol (Beninger et al. 1995) resulted in the use of staining times as indi- cated in Table 1. These protocols allowed visualization of both cuticle and gonopod tissues, although in some cases staining of the glands and hypodermis was unavoidably intense due to the weak staining anities of the cuticle. Diastase-digested negative controls were run to ensure that the stained substances were mucopolysaccharides and not glycogen deposits. Photomicrographs of gonopod internal structure, gland presence, position, and secretion type were obtained using a Leitz DMRB microscope. The secretion colours were standardized using the Pantone standard colour scale (Beninger et al. 1993).

Results

Gland type and cuticle pores

Glands were observed within the ®rst gonopods of all specimens studied (Figs. 1 to 4). Their histology corre- sponded to the rosette-type tegumental glands (Talbot and Demers 1993).The longitudinal positions of the glands varied somewhat between species, as did the type of secretion (see below). However, in all species, glands were grouped about the ejaculatory canal, which in- variably presented large pores (diameter 2.5 to 3.0 lm), lined with ducts often containing the same secretions of the glands (Figs. 3, 4). Continuity between the ducts of the gland system and the pores was often apparent in sections (e.g. Fig. 4.6, 4.7, 4.9). No pores or ducts were observed elsewhere than in the cuticle of the ejaculatory duct.

Secretion types

Table 1 shows the visual colours of the gland secretions, their corresponding Pantone equivalents, and muco- polysaccharide (MPS) types. Both acid and neutral mucopolysaccharides were observed, depending on the species (see below).

Gonopod morphology, gland distribution and secretion types by species

Carcinus maenas

The ®rst gonopod tapered quickly to a thin, curved process terminating in a point. Tegumental glands were situated much further distally than in the remaining Fig. 1 Schematic representation of ®rst gonopods of boreo-temperate species, between approximately 3/5 and 4/5 of the go- brachyurans studied. 1.1, 1.3, 1.5, anterior aspect; 1.2, 1.4, 1.6, posterior aspect. Hatched areas indicate segments of gonopod in nopod length (Fig. 1.1, 1.2). In the single males the which tegumental glands are localized glands appeared to be paired, with an intensely staining neutral mucopolysaccharide (NMPS) layer beneath an Portunus sebae intensely staining acid mucopolysaccharide (AMPS) layer (Fig. 4.2, 4.3; Table 2). In the male which had The ®rst gonopod tapered abruptly to a thin, curved copulated, only NMPS secretions were visible, indicat- process. The tegumental glands were located at approxi- ing that all of the AMPS had been transferred during mately 2/5 of the gonopod length (Fig. 1.3, 1.4), and copulation (Fig. 4.4, 4.5). The remaining NMPS entirely showed alternating layers of NMPS and AMPS glands ®lled the glands, suggesting that none had been trans- tightly appressed to one another, as in Carcinus maenas ferred (Fig. 4.5). (Table 2). 438

gonopod (Fig. 1.5, 1.6). The glands were organized in bilayers of intensely staining NMPS-containing glands beneath intensely staining AMPS-containing glands (Fig. 4.6; Table 2). The contours of the two gland types were almost exactly identical.

Cancer irroratus

The ®rst gonopods were quite robust, very slightly curved at the distal extremity, tapering to a point. The tegumental glands were con®ned to the proximal 2/5 of the gonopod (Fig. 2.1, 2.2). Only AMPS was present in these glands (Fig. 4.1; Table 2), both in the single males and in the males which had formed precopulatory embraces.

Grapsus grapsus

The ®rst gonopod was squat, straight, and presented a ¯at distal extremity. The tegumental glands were situ- ated in the proximal 2/5 of the gonopod length (Fig. 2.3, 2.4). Only NMPS secretions were observed in these glands, and the intense fuschia colour (Table 1) indi- cated that the secretions were fully constituted. The mating status was unknown.

Petrolisthes armatus

The ®rst gonopod morphology was similar to that of Grapsus grapsus: straight, somewhat narrower, termi- nating in a ¯at tip. The tegumental glands were con®ned to a very restricted region (50 lm) at the base of the gonopod (Fig. 2.5, 2.6). Although in all other species examined, the cuticle pores were grouped around the extent of the ejaculatory canal, in P. armatus the pores were clustered around the deepest part of the ejaculatory canal (Fig. 3.5). The shrunken duct walls lining the pores made their identi®cation somewhat more dicult. The gland secretions consisted of NMPS only, and their

c Fig. 3 Transverse histological sections of the ®rst gonopod in the brachyurans studied (periodic acid±Schi€±Alcian-blue protocol). Transverse section of whole gonopod in: 3.1 Grapsus grapsus, 3.2 Carcinus maenas, 3.3 Cancer irroratus. Note the position of the glandular region (GR ) proximal to the ejaculatory canal (EC ), typical Fig. 2 Schematic representation of ®rst gonopods of tropical bra- of all species studied. 3.4 Detail of glands (G) in Petrolisthes armatus, chyurans studied. 2.1, 2.3, 2.5, anterior aspect; 2.2, 2.4, 2.6, posterior showing rosette structure typical of all species studied. 3.5 Detail of aspect. Hatched areas indicate segments of gonopod in which ejaculatory canal (EC ) region in Petrolisthes armatus. Note pores tegumental glands are localized (arrows ) in cuticle (CU ) at base of ejaculatory canal (GR gland region; H hypodermis, mechanically detached from cuticle during histological processing). 3.6 Detail of ejaculatory canal (EC ) in Ovalipes ocellatus Cancer irroratus. Note pores (arrows ) traversing cuticle (CU ). Subjacent hypodermis (H ) and gland region (GR ) intact. 3.7 Petrolisthes armatus, detail of cuticle (CU ) distal to ejaculatory canal. Like the other portunids Carcinus maenas and Portunus Note absence of pores in cuticle (H hypodermis). 3.8 Cancer irroratus, sebae, the gonopods were tapered and curved, with detail of cuticle (CU ) in region distal to ejaculatory canal. Note tegumental glands located in the proximal 2/5 of the absence of pores in cuticle 439 440

Table 1 Visual and Pantone standard colours of gonopod tegumental glands (GTG) following staining with PAS±Alcian-blue protocol and corresponding mucopolysaccharide type (AMPS acid mucopolysaccharide; NMPS neutral mucopolysaccharide)

Species Staining times (min) Visual colours Pantone Secretion standard colours type Alcian-blue PAS

Carcinus maenas 1 1 Before copulation Alternating layers 2927 c AMPS + NMPS of blue and pink After copulation Light pink 251 c NMPS Portunus sebae 6 6 Alternating layers 2745 c, 2727 c AMPS + NMPS of dark blue and pink 239 c Ovalipes ocellatus 3 4 Alternating layers of blue 293 c AMPS + NMPS and pink 251 c Cancer irroratus 6 6 Dark blue 293 c AMPS Grapsus grapsus 7 5 Fuschia pink 225 c NMPS Petrolisthes armatus 7 3 Very light purple-pink 677 c NMPS pale staining indicated that the secretions were not fully between families. While the glands were usually located constituted (Table 1). somewhere in the proximal region of the endopod, they were found in the distal region in Carcinus maenas. Similarly, the longitudinal extent of the glandular region Discussion di€ered between taxa, ranging from 50 lm in Petrolis- thes armatus to 2±2.5 mm in the remaining species, for Gland distribution similar gonopod sectional diameters. While it is impos- sible at this time to conclude whether the di€erences in Gonopod tegumental glands were found in all six species longitudinal position correspond to functional attri- of the present study; when combined with published butes, it is clear that the volume of secretion will be reports on the circumboreal majid Chionoecetes opilio proportional to the gland volume, and hence much re- (Beninger et al. 1991, 1995) and current investigations duced in P. armatus. The signi®cance of these di€eren- on the tropical majid Mithrax spinosissimus (author's ces, in turn, must await further studies on the exact roles personal observations), such glands have been revealed of the GTG in reproduction in the various brachyuran in all eight of the species studied, comprising ®ve bra- families. In the meantime, it must be emphasized that chyuran families. Their presence in the Brachyura thus the site of the GTG must be determined by serial section appears to be general, and several salient features are for each new species studied. common. (1) The glands are invariably grouped around and in proximity to the ejaculatory canal; no glands are observed distal to the canal. (2) The glands communicate with the lumen of the ejaculatory canal via ducts which pass through large (2.5 to 3.0 lm) cuticular pores. No c such ducts or pores are observed elsewhere in the Fig. 4 Color photomicrographs of histological sections of ®rst gonopod. (3) Staining anities of secretions at the pore gonopods in the brachyurans studied, illustrating gland secretion openings to the ejaculatory canal lumen match those of types (periodic acid±Schi€±Alcian-blue protocol). 4.1 Detail of two the glands. rosette glands in Cancer irroratus (CL common locus; N nucleus; S secretions; dashed lines indicate cell contours). 4.2, 4.3 Carcinus These three common features con®rm that the maenas rosette glands (G) proximal to the ejaculatory canal (EC ). The gonopod tegumental glands of brachyurans are involved acid mucopolysaccharide secretions (AMPS, Alcian-blue positive, chie¯y, if not exclusively, in reproduction. Experimental Fig. 4.2) are situated in a plane of focus above the neutral con®rmation of this function can be found in the dif- mucopolysaccharides (NMPS, periodic acid±Schi€ positive, Fig. 4.3), indicating that the two secretion types are typically strati®ed ference between pre- and post-copulatory secretion types in this manner (CU cuticle; H hypodermis). 4.4, 4.5 AMPS of of the Carcinus maenas glands, where it is clear that Carcinus maenas present prior to copulation (Fig. 4.4) are absent in a AMPS were transferred to the female during copulation. male known to have copulated recently, which shows only NMPS The major di€erences between taxa were in the (Fig. 4.5) (GR gland region). 4.6±4.9 Details of pores and gland ducts in ejaculatory canal cuticle. 4.6 Ovalipes ocellatus, gland region (GR) longitudinal position of the glands within the gono- containing Alcian-blue positive secretions, and a cuticular duct (D) pods and in the gland secretion types, as highlighted containing Alcian-blue positive secretions (H hypodermis). 4.7 below. Portunus sebae, detail of cuticular pore (P) , and duct (D), with secretions (S) on the pore opening to the ejaculatory canal (EC ) (CU cuticle; GR gland region; H hypodermis). 4.8 Ovalipes ocellatus, detail Longitudinal position of glands of cuticlar pores (P) and ducts (D), with secretions (S) on the pore opening to the ejaculatory canal (EC ) (GR gland region; H hypodermis). 4.9 Grapsus grapsus, detail of duct (arrowheads) from The precise longitudinal position of the glands within gland region penetrating pore (P) in cuticle (CU ) of ejaculatory canal the ®rst gonopods showed considerable variation (H hypodermis) 441 442

Table 2 Characteristics of gonopods and GTG secretions for Brachyura investigated to date

Family Species Gonopod morphology GTG secretions

Tapered Curved AMPS AMPS + NMPS NMPS alternating layers

Portunidae Carcinus maenas + + Before copulation + After copulation + Portunus sebae + + + Ovalipes ocellatus + + + Cancridae Cancer irroratus + ± + Majidae Chionoecetes opilio + + + + (Beninger et al. 1991, 1995) Mithrax spinosissimus + + + + (Beninger unpublished) Grapsidae Grapsus grapsus ± ± + Porcellanidae Petrolisthes armatus ± ± +

Cuticular pores Spalding 1942; Ryan 1967; Hartnoll 1969; Bawab and El-Sherief 1989). The hypothesis that the sealant is de- The cuticular pores associated with the GTG system rived at least in part from the gonopod tegumental appear to be speci®c to this context. The normal ubiq- glands was proposed by Spalding (1942) for Carcinus uitous cuticular pores of the crustacean exoskeleton are maenas, although he did not perform the most appro- much smaller than those observed in the present study priate histochemical tests (PAS and Alcian blue) and and in Beninger et al. 1995 [0.06 to 0.2 lm, Halcrow therefore was unable to determine the dominant com- (1993) vs 2.5 to 3 lm]. The size of the GTG cuticular position of the gland secretions and the sealant itself. pores probably constitutes an adaptation for the secre- Nevertheless, his hypothesis has been virtually ignored tion of relatively large quantities of MPS, as would be ever since. Beninger et al. (1995) reasoned that the origin necessary during copulation. of this substance must be found outside the vas deferens, which contains spermatophores, whereas the sealant does not; hence the most likely origin must be the GTG. Gland structure, secretion types, and possible roles The sealant in the vagina of Chionoecetes opilio is known to consist entirely of AMPS (Beninger et al. 1993). The GTG in all of the species examined to date present Phenolic tanning of the sealant is also generally sup- the same histological features, belonging to the class of posed (Bawab and El-Sherief 1989), although hardening crustacean tegumental glands termed rosette glands could also be accomplished by speci®c AMPS compo- (Talbot and Demers 1993). The homogeneous distribu- nents such as chondroitin sulphate (Radha and tion of secretions within the secretory cells corresponds Subramoniam 1985). to the pro®le of the common Type A subclass (Talbot The sealant may participate in several complex and Demers 1993). In contrast to their histological ho- functions due to its mechanical and chemical properties. mogeneity, the GTG present inter-familial di€erences in AMPS are highly sulphated, and components such as the types and distributions of secretions. The Portunidae hyaluronic acid and chondroitin sulphate are anti-mi- are characterized by glands which possess distinct su- crobial (Sasikala and Subramoniam 1987; Subramoniam perimposed layers of AMPS and NMPS, and the result 1991); these characteristics may therefore function to of the Carcinus maenas copulation indicate that, whereas protect the female gonoduct and thus both directly and NMPS remains following ejaculation, all of the AMPS is indirectly the male's genetic investment following transferred. These observations suggest that indepen- transfer of spermatophores to the spermatheca (Benin- dent control of the two secretion types is possible in the ger et al. 1993). The most complex and controversial role Portunidae, rendering previous hypotheses on their may, however, be in the interplay between sperm com- function plausible (Beninger et al. 1995). The low-vis- petition and paternity assurance. cosity NMPS could thus be used to facilitate the passage First developed in the ®eld of ecological entomology of the more viscous vas deferens ejaculate in the narrow (Parker 1970), the concept of sperm competition was ejaculatory canal, whereas the more viscous AMPS evoked in brachyurans independently by Diesel (1990, could constitute the layers of sealant (often unsuitably 1991) and Beninger et al. (1991). Brie¯y, since females referred to as ``sperm plugs'') observed in the sperm- can store large quantities of viable sperm for protracted atheca and vagina following copulation in many species, periods, the sperm of several males could thus complete including Cancer irroratus, Carcinus maenas, and Oval- for fertilization of oocytes at ovulation. Since decapod ipes ocellatus of the present study (Chidester 1911; sperm are non-motile, such competition essentially cen- 443 tres around the strategic positioning of sperm by the may have appeared with the hardening of this AMPS male within the spermatheca, i.e. closest to the oviduct. mass in the more advanced families such as the Males may increase the probability of representation of Portunidae, within which the processes of paternity as- their genome either by o€ensively displacing and sealing surance have putatively been evolving synergistically o€ rival sperm and substituting their own, or by defen- ever since. sively ``locking'' their sperm into place (see Diesel 1991 Although the potential roles of the mucopolysac- for review). Ventral sealing of the vagina (``sperm plug'') charide secretions in the GTG are considerable, it has thus been ascribed a defensive role (e.g. Bauer and should be stressed that a complete chemical character- Min 1993), whereas displacement and sealing of a pre- ization of the GTG secretions will be necessary for a full vious male's spermatophoric mass accomplished by the understanding of their roles in reproduction. Research gonopod and its ejaculate would ful®ll an o€ensive role on insects, which is incomparably more advanced than (e.g. Diesel 1990; Beninger et al. 1991). If indeed the that on Crustacea, has demonstrated biological activity origin of the sealant lies with the GTG, it is of interest to in the secretions of male accessory sex glands, such as note that both o€ensive and defensive roles would elevation of oviposition and repression of subsequent therefore have evolved in the same structure (the ®rst sexual receptivity (Chen 1984) ± again increasing the gonopod), as is the case for the insects in which sperm probability of success for the copulating male's genome. competition has been observed (Waage 1979). Having demonstated the ubiquity and reproductive The evolution of the reciprocal phenomena of sperm nature of GTG in the Branchyura, these multiple competition and paternity assurance in the Brachyura perspectives are open for exploration. thus centres around the ®rst gonopod. As any im- provement in the defensive mechanisms automatically Acknowledgements We thank the sta€ of the Discovery Bay selects for improvements in o€ensive mechanisms, this Marine Laboratory, Jamaica, for use of facilities and assistance with sampling the tropical species. This work was supported by evolutionary interplay would be synergistic. For exam- operating grants from the Natural Science and Engineering ple, Diesel (1991) has proposed that in the time required Research Council and the Faculte d'etudes supe rieures et de la for hardening of the ventral sealant in Portunus sang- recherche de l'Universite de Moncton. uinolentus, the pointed extremity of a rival male's ®rst gonopod could pierce the sealant and deposit its sperm somewhat more dorsally in the spermatheca, displacing References the previous male's sperm ventrally and placing its own in a more favorable position with respect to the oviduct Abele LG (1971) Scanning electron micrographs of brachyuran gonopods. Crustaceana 21: 218±219 in this species. This is an intriguing area of biology, both Adiyodi RG (1983) Reproduction and its control. In: Bliss DE, from the practical and theoretical points of view. Un- Mantel LH (eds) The biology of Crustacea. Vol. 9. Integument, fortunately, although much indirect evidence points to pigments, and hormonal processes. Academic Press, New York, the existence of sperm competition and paternity as- pp 147±215 surance in the Brachyura, very little experimental con- Anonymous (1995) Fishery statistics ± captures and landings. FAO, Rome ®rmation has been provided ± in fact the hypotheses and Bailey RF, Elner RW (1989) Northwest Atlantic snow crab ®sh- concepts have far outstripped the available data base of eries: lessons in research and management. In: Caddy JF (ed) two majid species, Inachus phalangium (see Diesel 1990, Marine invertebrate ®sheries: their assessment and manage- 1991) and Chionoecetes opilio (Se vigny and Sainte-Marie ment. John Wiley and Sons, New York, pp 261±280 Bauer RT (1986) Phylogenetic trends in sperm transfer and storage 1996). Adequate understanding of these processes re- complexity in decapod crustaceans. J Crustacean Biol 6: 313± quires nearly simultaneous studies of multiple facets of 325 the reproductive biology of each species, and a pan- Bauer RT, Martin JW (eds) (1991) Crustacean sexual biology. orama of such research to provide the taxonomic com- Columbia University Press, New York Bauer RT, Min LJ (1993) Spermatophores and plug substance of parisons necessary for evolutionary insight. In the marine shrimp Trachypenaeus similis (Crustacea: Decapoda: particular, such studies must carefully relate (1) gono- Peneidae): formation in the male reproductive tract and dis- pod morphology, (2) spermatheca morphology and position in the inseminated female. Biol Bull mar biol Lab, oviduct position, (3) composition of GTG secretion Woods Hole 185: 174±185 transferred, (4) composition of sealant, and (5) behav- Bawab FM, El-Sherief SS (1989) Contributions to the study of the origin, nature, and formation of the plug in the spermatheca of ioral elements such as mate guarding. the female crab Portunus pelagicus (Linneus, 1766) (Decapoda, An interesting perspective opened by the studies to Brachyura). Crutaceana 57: 9±24 date on the GTG is that the primitive function of their Beninger PG, Elner RW, Foyle T, Odense P (1988) Functional secretions may have been simply to seal the vagina from anatomy of the male reproductive system and the female spermatheca in the snow crab Chionoecetes opilio (O. Fabricius) entry of either seawater (which could cause premature (Decapoda: Majidae) and a hypothesis for fertilization. spermatophore dehiscence and spermatozoan acrosome J Crustacean Biol 8: 322±332 reaction; Beninger et al. 1988, 1993) or opportunistic Beninger PG, Elner RW, Poussart Y (1991) Gonopods of the majid microbes ± both accomplished by the viscous AMPS crab Chionoecetes opilio (O. Fabricius). J Crustacean Biol 11: 217±228 observed in the ventral region of the vagina in the Beninger PG, Ferguson A, Lanteigne C (1995) The gonopod primitive majid Chionoecetes opilio (Beninger et al. tegumental glands of snow crab (Chionoecetes opilio) are ac- 1993). The additional advantage of paternity assurance cessory reproductive glands. J Shell®sh Res 14: 365±370 444

Beninger PG, Lanteigne C, Elner RW (1993) Reproductive pro- Martin JW, Abele LG (1986) Notes on male pleopod morphol- cesses revealed by spermatophore dehiscence experiments and ogy in the brachyuran crab family Panopeidae Ortmann, by histology, ultrastructure, and histochemistry of the repro- 1893, sensu Guinot (1978) (Decapoda). Crustaceanea 50: 182± ductive system in the snow crab Chionoecetes opilio (O. Fab- 198 ricius). J Crustacean Biol 13: 1±16 Nishimura S (1967) Male ®rst pleopods of the majid brachyurans Chambers CL, Payne JF, Kennedy ML (1980) Geographic varia- Chionoecetes opilio (O. Fabricius) and C. japonicus Rathbun tion in the ®rst pleopod of the form I male dwarf cray®sh, from the Japan Sea. Publ Seto mar biol Lab 15: 165±171 Cambarellus puer Hobbs (Decapoda, Cambaridae). Crusta- Parker GA (1970) Sperm competition and its evolutionary conse- ceanea 38: 169±177 quences in the insects. Biol Rev 45: 525± 568 Chen PS (1984) The functional morphology and biochemistry of Radha T, Subramoniam T (1985) Origin and nature of the insect male accessory glands and their secretions. A Rev Ent 29: spermatophoric mass of the spiny lobster Panulirus homarus. 233±255 Mar Biol 86: 13±19 Chidester FE (1911) The mating habits of four species of the Ryan EP (1967) Structure and function of the reproductive system Brachyura. Biol Bull mar biol Lab, Woods Hole 21: 235±248 of the crab Portunus sanguinolentus (Herbst) (Brachyura: Christy JH (1987) Competitive mating, mate choice, and mating Portunidae). The female system. In: Proceedings of the sym- associations of brachyuran crabs. Bull mar Sci 41: 177±191 posium on Crustacea, Ernakulam. Part II. Marine Biological Diesel R (1990) Sperm competition and reproductive success in a Association of India, Bangalore Press, Bangalore, India, pp decapod Inachius phalangium (Majidae): a male ghost spider 522±544 crab that seals o€ rivals' sperm. J Zool, Lond 220: 213±224 Sasikala SL, Subramoniam T (1987) On the occurrence of acid Diesel R (1991) Sperm competition and mating behavior in Bra- mucopolysaccharides in the spermatophores of two marine chyura, with special reference to spider crabs (Majidae). In: prawns, Penaeus indicus (Milne-Edwards) and Metapenaeus Bauer RT, Martin JW (eds) Crustacean sexual biology. monoceros (Fabricius) (Crustacea: Macrura). J exp mar Biol Columbia University Press, New York, pp 145±163 Ecol 113: 145±153 Elner RW, Beninger PG (1995) Multiple reproductive strategies in Sastry AN (1983) Ecological aspects of reproduction. In: Vernberg snow crab, Chionoecetes opilio: physiological pathways and FJ, Vernberg WB (eds) The biology of Crustacea. Vol. 8. En- behavioral plasticity. J exp mar Biol Ecol 193: 93±112 vironmental adaptations. Academic Press, New York, pp 179± Felgenhauer BE (1992) External anatomy and integumentary 270 structures. In: Harrison FW, Humes AG (eds) Microscopic Se vigny J-M, Sainte-Marie B (1996) Electrophoretic data support anatomy of invertebrates. Vol. 10. Decapod Crustacea. Wiley- the last-male precedence hypothesis in the snow crab, Chio- Liss, New York, pp 7±43 noecetes opilio (Brachyura: Majidae). J Shell®sh Res 15: 437± Fingerman M (1992) Glands and secretion. In: Harrison FW, 440 Humes AG (eds) Microscopic anatomy of invertebrates. Vol. Spalding JF (1942) The nature and formation of the spermato- 10. Decapod Crustacea. Wiley-Liss, New York, pp 345±394 phore and sperm plug in Carcinus maenas. Q Jl microsc Sci Garth JS (1958) Brachyura of the Paci®c coast of America. 83:399±422 Oxyrhyncha. Allan Hancock Pacif Exped 21: 1±854 Stephensen K (1946) The Branchyura of the Iranian Gulf. Ap- George MJ (1963) The anatomy of the crab Neptunus sang- pendix: the male pleopoda of the Brachyura. In: Danish sci- uinolentus Herbst. Part IV. Reproductive system and embryo- enti®c investigations in Iran, Part IV. Ejnar Munksgaard, logical studies. J Madras Univ (Sect B) 33: 289±304 Copenhagen, pp 57±237 Haefner PA (1985) The biology and exploitation of crabs. In: Stevenson JR (1985) Dynamics of the integument. In: Bliss DE, Provenzano AJ (ed) The biology of Crustacea. Vol. 10. Eco- Mantel LH (eds) The biology of Crustacea. Vol. 9. Integument, nomic aspects, ®sheries, and culture. Academic Press, New pigments, and hormonal processes. Academic Press, New York, York, pp 111±166 pp 1±42 Halcrow K (1993) Pore canal systems and associated cuticular Subramoniam T (1991) Chemical composition of spermatophores microstructures in amphipod crustaceans. In: Horst MN, in decapod crustaceans. In: Bauer T, Martin JW (eds) Crusta- Freeman JA (eds ) The crustacean integument: morphology cean sexual biology. Columbia University Press, New York, and biochemistry. CRC Press, Boca Raton, Florida, pp 39± pp 308±321 77 Subramoniam T (1993) Spermatophores and sperm tranfer in Hartnoll RG (1969) Mating in the Brachyura. Crustaceana 16: marine crustaceans. Adv mar Biol 29: 129±214 161±181 Talbot P, Demers D (1993) Tegumental glands of Crustacea. In: Hartnoll RG (1975) Copulatory structure and function in the Horst MN, Freeeman JA (eds) The crustacean integument: Dromiacea, and their bearing on the evolution of the Bra- morphology and biochemistry. CRC Press, Boca Raton, chyura. Pubbl Stazi zool Napoli 39(Suppl): 657±676 Florida, pp 152±191 Johnson PT (1980) Histology of the blue crab, Callinectes sapidus: Waage JK (1979) Dual function of the damsel¯y penis: sperm a model for the Decapoda. Praeger Publishers, New York, removal and transfer. Science 203: 916±918 pp 327±367 Wenner A, Kuris A (eds) (1991) Crustacean egg production. Lanteigne C, Beninger PG, Gionet C (1996) Ontogeny of the fe- A A Balkema Inc, Rotterdam male primary sexual characters in the majid crabs Chionoecetes Williams A (1965) Marine decapod cructaceans of the Carolinas. opilio and Hyas coarctatus. J Crustacean Biol 16: 501±514 Fish Bull US 65: 1±271