Morphology of Seminal Receptacle of the Harvested Golden Crab Chaceon Chilensis and Its Implication in the Fertilization Process

Morphology of seminal receptacle of the harvested golden crab Chaceon chilensis and its implication in the fertilization process

Luis Miguel Pardo,1,2,a Constanza Ceroni,1 Marcela Paz Riveros,1 Billy Ernst,3 and Josefa Pino4

1Instituto de Ciencias Marinas y Limnologicas, Laboratorio Costero Calfuco, Facultad de Ciencias, Universidad Austral de Chile, Valdivia, Chile 2Centro FONDAP de Investigacion en Dinamica de Ecosistemas Marinos de Altas Latitudes (IDEAL), Facultad de Ciencias, Universidad Austral de Chile, Valdivia, Chile 3Departamento de Oceanografıa, Universidad de Concepcion, Concepcion, Chile 4Programa de Magister en Pesquerıas, Departamento de Oceanografıa, Universidad de Concepcion, Concepcion, Chile

Abstract. The external and internal architecture of crab seminal receptacles are closely associated with mating behavior, sperm transfer, and storage patterns, and ultimately reproductive success. Therefore, describing their anatomy contributes to the development of a greater understanding of crab reproductive biology. In this study, we describe the anatomy and tissue characteristics of the seminal receptacle of the golden crab Chaceon chilensis,a harvested deep-sea species from the Juan Fernandez Archipelago (off central Chile). Females in the late mature stage were used as reference for histological description. Mor- phological features of the seminal receptacle included: (1) a dorsal receptacle–oviduct connection, (2) velum and septum separating the dorsal and ventral chambers, (3) extensive glandular epithelium in both chambers, (4) a ribbed chitin line, (5) a highly folded, bilobulated ventral chamber, and (6) a simple-type vagina. Some of these traits (2, 3, 4, 5) are novel for crab receptacles and could contradict the generalized idea about the pattern of sperm precedence associated with the location of the receptacle–oviduct connection. Exter- nal sperm plugs were not found in mature females, but variations in the color of opercula occluding the vulva were used to assess sexual maturity. The seminal receptacle of C. chilensis has an unusual internal morphology with vast sperm storage capacity and separate storage areas for sperm ejaculates, which could allow for cryptic female choice. In cases like these, male–male competition is usually intense and males deliver large quantities of sperm in each mating event, which makes them vulnerable to sperm depletion. Additional key words: reproduction, Brachyura, ﬁshery, sperm storage, mating

The seminal receptacles of diverse brachyuran et al. 2003). In crabs, it has been hypothesized that crabs have been described in order to understand the seminal receptacle has a meso-ectodermal origin their role in sperm storage dynamics, fertilization, (McLay & Lopez-Greco 2011) and a generally simi- and multiple paternity (Hartnoll 1968; Sainte-Marie lar morphology among species (i.e., a sac that can et al. 2000; Becker et al. 2011; Gonzalez-Pisani et al. be elongate, globose, or kidney-shaped). However, 2012; Pardo et al. 2013). All of these aspects can fine anatomical configuration and details of internal influence the reproductive success of a species. The architecture of eubrachyuran seminal receptacles are receptacle structure that is unique to Eubrachyurans species specific. allows females to store ejaculates (sperm and semi- Golden crabs, which belong to the family Gery- nal liquid) to produce several broods in short time onidae, are deep-sea crabs mostly distributed on periods under favorable conditions (Fischer & continental slopes and are often intensively har- Thatje 2008) or produce broods without the need of vested around the world (Hastie 1995). In Chile, the a fresh sperm supply over long time periods (Hines golden crab Chaceon chilensis CHIRINO-GALVEZ & MANNING 1989 has been the focus of a small arti- aAuthor for correspondence. sanal fishery in the Juan Fernandez Archipelago E-mail: [email protected] since the early 2000s (Ernst et al. 2011). This fishery, 200 Pardo, Ceroni, Riveros, Ernst, & Pino operating at a depth of 300–1000 m, has shown a was determined according to criteria established pre- high prevalence of males in the catch (Guerrero & viously by Pino (2015). Arana 2009), which could indicate a partial spatial Six individuals were anesthetized by chilling, sac- segregation by sex, as in other geryonids (Melville- rificed, and their gonadal state determined according Smith 1987). to the method of Pardo et al. (2009). This catego- Little information has been reported on seminal rization was based principally on ovarian volume receptacle morphology of golden crabs. Seasonal and maturity of gametes as determined histologically variation in size and turgidity of the seminal recep- (oocytes in early, late, or post-vitellogenesis). The tacle has been described for Chaceon quinquedens lower section of the reproductive tract was carefully (SMITH 1879), Chaceon maritae (MANNING & extracted. A stereomicroscope was used to describe HOLTHUIS 1981), and Chaceon fenerii (MANNING & and photograph the gross external anatomy, includ- HOLTHUIS 1984) (Haefner 1977; Melville-Smith 1987); ing the connection between the seminal receptacle, however, these studies did not provide a detailed the oviduct, and vagina. The reproductive tract was description of the structure. Hinsch (1988), using then fixed in Bouin solution (picric acid, saturated light- and electron-microscopic techniques, provided aqueous solution; formalin, 40% aqueous solution; the first description of sperm and spermatophores acetic acid, glacial; 15:5:1 proportion) for at least inside receptacles of C. fenerii, but the study did 2 days. Samples were dehydrated through an not include a detailed description of the internal ascending ethanol series (50–100%), embedded in anatomy or histological characteristics of the recep- paraffin, and serial sections of 6 lm were cut for tacle. histological analysis. Sections were stained with This study provides a fine-scale description of the hematoxylin–eosin and Masson trichrome tech- morphology of the seminal receptacle and its associ- niques (Lynch et al. 1987). Finally, the histological ated structures from mature female specimens of sections were mounted using PermountÒ (Fisher C. chilensis, using different histological techniques Scientific GSA, Fair Lawn, NJ, U.S.A.) and to identify different components. Anatomical studies observed and photographed using a compound light of receptacles are an important first step toward microscope. addressing questions about sexual selection and providing information relevant to fisheries assessment Results (Sainte-Marie & Lovrich 1994; Hines et al. 2003; McLay & Lopez-Greco 2011; Pardo et al. 2016). The appearance of mature ovaries was similar to that of other brachyurans (Johnson 1980), laying Methods dorsal to the hepatopancreas and extending on either side along the anterior margin of the Mature female golden crabs were obtained in cephalothorax (Fig. 1A). Ovarian lobules connected Juan Fernandez Archipelago (33°350S; 78°400W) dorsally to the seminal receptacles. Each seminal from the commercial catch in November 2012 and receptacle had a cup-shaped appearance (Fig. 1B), sent live to Laboratorio Costero Calfuco at the with the ovary partially enveloping the receptacle on Universidad Austral de Chile in Valdivia. Maturity its dorsal side. The ventral side of the receptacle was

Fig. 1. Reproductive system of Chaceon chilensis. A. Dorsal view of the cephalothorax of a mature female with dorsal carapace removed to reveal the ovary in relation to the gills and heart. B. Isolated seminal receptacle showing its rela- tionship to ovary and vagina. C. Vulva (arrow) on ventral surface of female carapace. Scale: A=20 mm; B=5 mm; C=10 mm. G, gills; H, heart; Ov, ovary; RS, seminal receptacle; VA, vagina.

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the cephalothorax, close to the 5/6 sternal thoracic suture. In mature females, the vulvae were ovoid with a blackened margin (Fig. 1C). Dorsal and ventral chambers of seminal receptacles were clearly distinguishable in histological sections (Fig. 2A,B). All females analyzed showed ovaries in late vitellogenesis or post-vitellogenesis, with round yolk granules distributed homogeneously within oocytes (Fig. 3A); therefore, all females analyzed were classified as mature females. The zone, where ovaries connected with the receptacle (Fig. 3B), was composed of connective tissue that was tightly bound to the proximal oviduct (Fig. 3C), Fig. 2. Seminal receptacle of Chaceon chilensis. A. Sagit- which appeared not to open completely. Squamous tal histological section. B. Sketch showing internal regions cells derived from the epithelium of the dorsal of the seminal receptacle; each number indicates the area chamber (i.e., dense layer) occluded the receptacle of histological sections shown in subsequent figures. Scale entrance (Fig. 3D). bar=2 mm. DC, dorsal chamber; E, ejaculate; Ov, ovary; The dorsal chamber was externally covered by con- SE, septum; VA, vagina; VC, ventral chamber; VE, velum. nective tissue, followed by collagenous fibers and a dense layer of proliferative cells forming a stratified connected with a short, hardened vagina (Fig. 1B). squamous epithelium (Fig. 4A). Epithelial cells were When seminal receptacles were removed from the observed to slough off toward the lumen of the dorsal females, they were whitish in color with some chamber (Fig. 4B). The arrangement of these tissues translucent areas. left a channel for oocyte passage (Fig. 4C). The vagina was a simple short duct. Throughout At the transition zone between dorsal and ventral this whole structure, strong muscles were present to chambers, the epithelium of the dorsal chamber provide anchorage to the endophragm. The vulvae became glandular. This tissue is called modified dor- were located in the mid-zone of the ventral side of sal epithelium and it was composed of large, basally

Fig. 3. Histological details of area (1) in Fig. 2B. A. Mature oocytes of Chaceon chilensis. B. Transition zone between ovary and the dorsal chamber of seminal receptacle; areas within rectangles are magnified in C and D and stained with Masson’s trichrome. C. Oviduct with cylindrical epithelium around fibroblasts and collagen. D. Desquamate cell aggregation. Scale: A=100 lm; B=500 lm; C, D=50 lm. CF, collagen fibers; CT, connective tissue; DC, desquamate cells; DL, dense layer; Oo, oocytes; OVD, oviduct.

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Fig. 4. Dorsal chamber of seminal receptacle; A–C show histological details from area (2) in Fig. 2B, and D shows histological details from area (3) in Fig. 2B. A. Mesoderm-derived tissue from dorsal chamber; area within rectangle is magnified in B. Stained with Masson’s trichrome. B. Stratified epithelium (dense layer); arrow indicates pyknotic nucleus from desquamated cells. C. Channel for passage of oocytes; stained with hematoxylin–eosin. D. Modified dorsal epithelium. Scale: A=100 lm; B=100 lm; C=400 lm; D=50 lm. AS, apocrine secretion; CL, collagen layer; CT, connective tissue; DL, dense layer; LDC, lumen of dorsal chamber; MDT, modified dorsal epithelium.

Fig. 5. Ventral chamber of seminal receptacle; histological details from Fig. 2B. A. Detail from area (4) in Fig. 2B. Transition zone between dorsal and ventral chamber showing epithelial folds; stained with hematoxylin–eosin. B–D. Detail from area (5) in Fig. 2B. B. Glandular invagination of ventral epithelium; area within rectangle is magniﬁed in C; stained with Masson’s trichrome. C. Holocrine glandular epithelium. D. Invagination of glandular ventral epithelium with secretions in direct contact with ejaculate. E. Detail from area (6) in Fig. 2B. Ridged cuticle overlies non- secretory simple columnar epithelium; arrow indicates cuticle ribs; stained with Masson’s trichrome. Scale: A=300 lm; B=200 lm; C=50 lm; D=200 lm; E=50 lm. C, cuticle; CE, columnar epithelium; E, ejaculate; F, folds; GHE, glandular holocrine epithelium; HS, holocrine secretion; LDC, lumen of dorsal chamber; LVC, lumen of ventral chamber; Sp, sperms; VL, velum. multinucleated cells that release their secretions into The ventral chamber of the seminal receptacle the receptacle0s lumen (Fig. 4D). In this transition was clearly bilobulated (U-shaped), showing a blind zone, it was possible to observe the characteristic lobule and another lobule that connected to the dor- folds formed by columnar epithelium internally sal chamber (Fig. 2A); both contained ejaculates lined by a thick cuticle of chitin (Fig. 5A). These composed of free sperm and seminal liquid. Most of folds projected internally toward the lumen of the ectoderm-derived region was internally lined the receptacle, forming a distinguishable velum with a thick cuticle, except for two invaginations (Fig. 5A). that exhibited a holocrine glandular epithelium

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and the procuticle, was composed of a wider and less dense band (Fig. 6C). The vagina ran directly from the receptacle to the vulva and was ensheathed along its entire length by a layer of connective tissue con- taining muscle ﬁbers. A simple inner columnar epithelium was present and the lumen of the vagina was short and tubular with a dense cuticle, especially close to the vulva (Fig. 6D).

Discussion The morphological configuration of the seminal receptacle in females of Chaceon chilensis corre- sponds to a dorsal-type receptacle (Diesel 1991), associated with a simple-type vagina (Hartnoll 1968). For mature females, the morphology of the receptacle shows characteristic features such as the development of abundant glandular epithelium in both chambers, velum and septum separating the dorsal and ventral chambers, a chitin line with ribs, and a highly folded bilobulated ventral chamber. Most of these traits have implications for sperm storage capacity or how and where sperm are used Fig. 6. Histological details of seminal receptacle shown in during the fertilization process. Fig. 2B. A, B. Detail from area (7) in Fig. 2B. A. Sperm The site where the oviduct connects to the recep- aggregations in the ventral chamber. B. Large circular tacle strongly influences the potential for sperm muscular fibers close to the receptacle–vagina connection; competition in polyandrous species (Diesel 1991). – stained with hematoxylin eosin. C, D. Detail from area For example, if the oviduct opens to the ventral (8) in Figure 2B. C. Vaginal entrance, non-secretory sim- chamber close to the vaginal connection, the recep- ple columnar epithelium overlaid by procuticle and epicuticle; stained with hematoxylin–eosin. Area within tacle configuration promotes last-male sperm prece- rectangle is magnified in D. D. Vaginal section, showing dence. The opposite is true where the receptacle- the thick cuticle, columnar epithelium, muscular fibers oviduct connection is dorsal, the first male transfer- and connective tissue; stained with Masson’s trichrome. ring its ejaculate has better chances of fertilizing Scale: A=20 lm; B=300 lm; C=100 lm; D=50 lm. E, oocytes (McLay & Lopez-Greco 2011). However, in ejaculate; Epc, epicuticle; LVC, lumen of ventral cham- both cases, ejaculate stratification can fail when a ber; C, cuticle; CE, columnar epithelium; CL, collagen large volume of ejaculate is transferred, causing layer; CT, connective tissue; M, muscular fibers; Prc, pro- sperm from different males to mix (Jivoff 2003; Jen- cuticle; Sp, sperms; LV, lumen of vagina. sen & Bentzen 2012). When females mate with several males during the mating season or store sperm from a previous mat- (Fig. 5B–D). Underlying the cuticle, there was a ing, the presence of two closely connected lobules in non-secretory simple columnar epithelium with large the ventral chamber of C. chilensis may mean that basal nuclei followed by connective tissue (Fig. 5E). sperm precedence is not solely dependent on the Free sperm and sperm aggregations were detected position of the oviduct opening. This novel internal in the ventral chamber of the females studied. Sper- receptacle morphology for a brachyuran crab could matophore walls were not distinguishable, so sperm potentially allow for post-copulative female choice. aggregations appeared not to be spermatophores Musculature along the receptacle’s wall and (Fig. 6A). attached to the internal skeleton may play an impor- At the base of the receptacle–vagina connection, tant role in this choice, moving non-motile sperm two large circular muscular fibers could be observed and oocytes to the fertilization site (Hartnoll 1968; (Fig. 6B). The cuticle increased in thickness and com- Hayer et al. 2014). Part of the receptacle’s inner wall plexity toward the vagina, where two layers could be appears to be composed of collagen fibers, providing defined (blue stained by Masson’s trichrome); the epi- more elasticity to the receptacle (Zara et al. 2014). cuticle, a fine and dense line inside the vaginal lumen Large muscular fibers are present around the ventral

Invertebrate Biology vol. 136, no. 2, June 2017 204 Pardo, Ceroni, Riveros, Ernst, & Pino chamber and the vaginal opening, and their contrac- Pisani et al. 2012). The ventral chamber has been tion could help the displacement of ejaculates inside called the insemination chamber, while the dorsal the receptacle. chamber is referred to as a sperm storage chamber For this kind of receptacle, males probably trans- (Diesel 1989). However, sites for sperm storage and fer large ejaculate loads, filling the ventral chamber fertilization seem to be very variable among species; and increasing their chances of paternity. This idea for example, histological evidence from Libinia spi- is supported by the unusually extended copulatory nosa and Leurocyclus tuberculosus indicates that the periods reported for golden crabs (Mori & Relini dorsal chamber has both functions (Sal Moyano 1982; Elner et al. 1987) and because the vasa defer- et al. 2010; Gonzalez-Pisani et al. 2012). Moreover, entia of mature males of C. chilensis have been intra-ovarian gamete mixing and fertilization have found with depleted sperm reserves (Pino 2015), sug- been proposed for Chionoecetes opilio (Sainte-Marie gesting they probably do not economize sperm as et al. 2000), and external fertilization has been pro- do other brachyurans (Sainte-Marie et al. 2008). In posed for Metacarcinus magister (Jensen & Bentzen this scenario, males of C. chilensis could be vulnera- 2012). ble to sperm depletion under a classic sex-biased For C. chilensis, sperm storage and fertilization fishery management (Pardo et al. 2015). probably both occur in the ventral chamber, because Although seminal receptacles change in appear- free sperm were exclusively found there and mature ance depending on the stage of ovary development oocytes necessarily cross the ventral chamber in and sperm load (Sainte-Marie & Sainte-Marie 1998; route to the vagina. During this passage, gamete Pardo et al. 2013; Zara et al. 2014), in this study, mixing and fertilization likely take place. However, focused solely on one developmental stage, several the analysis of recently mated females is absolutely particularities can be observed in the receptacle of needed to confirm this hypothesis, because sperm C. chilensis. First, the oviduct connection with the may fill both chambers of the receptacle if males dorsal chamber was seemingly occluded by cells in transfer large ejaculates or if old ejaculates are dis- desquamation (holocrine secretion) from the dense placed by fresh ejaculate (Sal Moyano et al. 2010). layer, which form an aggregation of cell debris in One of the most remarkable and novel traits of the lumen of the dorsal chamber. This tissue seems the seminal receptacle in females of C. chilensis is a not to be specialized as “valve-like tissue” (Lee & thick septum in the center of the receptacle, which Yamazaki 1990) or “holocrine transfer tissue” gives a U shape to the ventral chamber. This septum (Becker et al. 2011), but could have the same func- was composed of multilayered squamous cells and tion, preventing sperm from accessing the oviduct. connective tissue, but the ventral part was internally Similar processes have been observed in the portu- lined by a cuticle layer. The ventral part of the sep- nid Aranaeus cribrarius (Zara et al. 2014). Second, tum shows some invaginations, which have glandu- the entire internal surface of the ventral chamber of lar epithelium that release their content directly to C. chilensis has an unusually high density of folded the ventral chamber where sperm are stored. This epithelium. Third, the ventral epithelium showed internal composition of the seminal receptacle is invagination, with glandular cells releasing secre- novel for brachyurans, but given the lack of fine- tions to the lumen of the ventral chamber. Secre- scale knowledge of the receptacle morphology in tions in seminal receptacles are composed of most species, especially Gerionydae, these features polysaccharides and glycoproteins, which have been may not be unique. associated with sperm and spermatophore conserva- For the individuals analyzed (i.e., mature tion (Johnson 1980; Becker et al. 2011; Zara et al. females), neither an external sperm plug (protruding 2014) and could have a similar function in C. chilen- from the vulvae) nor an internal sperm plug were sis. Fourth, the ventral cuticle has superficial inter- observed. However, an internal sperm plug can only nal ribs, undescribed in other crabs. These last three be observed in females recently mated, which was traits increase the volume of the ventral chamber not the case for those analyzed in this study. An giving it more capacity to receive large ejaculates internal sperm plug would disappear during ovarian from one or more males. development at the same time that the seminal Most receptacles described in crabs are undivided, receptacle changes its morphology, as reported in as in cancrid (Orensanz et al. 1995; Jensen et al. other species with dorsal-type receptacles (Bawab & 1996; Pardo et al. 2013) and pinnoterid receptacles El-Sherief 1988; Hinsch 1988; Wolcott et al. 2005; (Becker et al. 2011), or have a velum dividing the Zara et al. 2014). dorsal and ventral chambers, typically described in Sperm plugs have been used as a reliable indicator spider crabs (Majoidea) (Diesel 1989; Gonzalez- of prior mating (Oh & Hankin 2004) and to identify

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