Invertebrate Biology 137(2): 171–184. © 2018, The American Microscopical Society, Inc. DOI: 10.1111/ivb.12214
Male reproductive system of the arrow crab Stenorhynchus seticornis (Inachoididae) Mariana Antunes
,1,a Fernando J. Zara ,2 Laura S. L opez Greco Maria L. Negreiros-Fransozo1
,3 and
1
NEBECC Study Group on Crustacean Biology, Ecology and Culture, Institute of Biosciences, S~ao Paulo State University, Botucatu, S~ao Paulo 18618-970, Brazil 2 Invertebrate Morphology Laboratory (IML), Department of Applied Biology, Aquaculture Center (CAUNESP), and IEAMar, Univ. Estadual Paulista (UNESP), Jaboticabal, S~ao Paulo 14884-900, Brazil 3 Departamento de Biodiversidad y Biologıa Experimental, Facultad de Ciencias Exactas y Naturales, Instituto de Biodiversidad y Biologıa Experimental y Aplicada (IBBEA, CONICET-UBA), Universidad de Buenos Aires, Ciudad Universitaria, Buenos Aires C1428EGA, Argentina
Abstract. Our aim was to describe the reproductive system of males and the formation of sperm packages in the seminal receptacle (SR) of recently mated females of the arrow crab Stenorhynchus seticornis. The male reproductive system was analyzed, and was described using light microscopy and histological and histochemical methods. The first pair of gonopods was described by means of scanning electron microscopy. Additionally, the dehiscence of spermatophores was tested using samples obtained from the vas deferens of males and from the seminal receptacle of recently mated females. Testes were tubular type, and each vas deferens consisted of three regions: the anterior vas deferens (AVD), including a proximal portion that was filled with free spermatozoa and a distal portion contained developing spermatophores; the median vas deferens (MVD) that contained completely formed spermatophores; and the posterior vas deferens (PVD), which contained only granular secretions. The accessory gland, which was filled with secretions, was located in the transition region between the MVD and the PVD. The spermatophores from the MVD were of different sizes, and none of them showed dehiscence in seawater, whereas those spermatophores in contact with the seminal receptacle were immediately broken. The ultrastructure of the gonopods revealed the presence of denticles at the distal portion, which contribute to the mechanical rupture of the spermatophore wall during the transfer of sperm. The contents of the PVD and accessory gland of males are transferred together with the spermatophores, and are responsible for the secretions observed among the sperm packets in the SR of the female. We suggest that these secretions formed the layers found in the SR of recently mated females, and may play a role in sperm competition in arrow crabs. Additional key words: testis, vas deferens, accessory gland, dehiscence, Majoidea
Studies on morphology of the male reproductive system and the formation of spermatozoa and spermatophores in Decapoda are essential for the comprehension of the sexual maturation processes of species, as well as to enable assays of population monitoring in natural environments or species cultivation (Jerry 2001; Akarasanon et al. 2004; L opez Greco et al. 2007; Alfaro-Montoya 2010). The male reproductive system in brachyuran crabs, is composed of a pair of testes, which can be classified as a
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lobular or tubular (Sime o et al. 2009; Stewart et al. 2010; Zara et al. 2012; Nascimento & Zara 2013; Tiseo et al. 2014). Lobular testes are arranged in numerous seminiferous lobules (acini or cysts), with cells in a similar maturation stage, connected to a seminiferous tubule as a central axis. Tubular testes consist only of highly convoluted testicular tubules with cells in different maturation stages distributed in different strata, and the mature spermatozoa are released into the seminiferous tubule at the evacuation zone (Minagawa et al. 1994; Nagao & Munehara 2003; Sime o et al. 2009). The spermatozoa enter the vas deferens, which is divided into three
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regions known in brachyurans as anterior vas deferens (AVD), in which the spermatophores are produced; median vas deferens (MVD); and posterior vas deferens (PVD), in which the spermatophores and seminal fluid are usually stored (Krol et al. 1992; Tiseo et al. 2014). In many brachyuran species, the males develop accessory glands (AG), also known as caeca, diverticula, expansions, or outpockets found in MVD, PVD, or both (Johnson 1980; Beninger et al. 1988; Diesel 1989; Sime o et al. 2009; Zara et al. 2012; Tiseo et al. 2014). These glands can vary in their localization, morphology, and function along the vas deferens (Sime o et al. 2009). In species of Majoidea in which the glands are localized at the MVD, they are called caeca, diverticula, or expansions (Diesel 1989; Sime o et al. 2009), but in species in which the glands are localized to the PVD they are called caeca or accessory glands (Diesel 1989; Sainte-Marie & Sainte-Marie 1999; Sime o et al. 2009). The AG also contributes to increase the amount of seminal fluid involved in the transfer of spermatophores, sperm packets, or sperm plugs (Diesel 1989; Sime o et al. 2009; Zara et al. 2012, 2014; Tiseo et al. 2014). The spermatophores are transferred from the ejaculatory ducts, which are connected to the penis, to the first pair of pleopods. The first and second male pleopods are modified and called gonopods (Beninger et al. 1991). The gonopods are used at mating in the transfer of the spermatophores to the seminal receptacle in the female (Bauer 1986). The peculiar shape of the gonopods and the presence of structures, such as setae and teeth, suggest that the gonopods have distinct functions during mating (Sal et al. 2011). The presence of denticles surrounding the distal opening of the ejaculatory channel of the first gonopod can contribute to the rupture of the spermatophores during sperm transfer, thus working as an additional mechanism for dehiscence (Rorandelli et al. 2008; Kienbaum et al. 2017). The dehiscence of spermatophores in the seminal receptacle (SR) may vary among species (Beninger et al. 1993). In Chionoecetes opilio (FABRICIUS 1788), the rupture of the spermatophore wall occurs by water absorption, when the spermatophore is exposed to seawater (Beninger et al. 1988; Moriyasu and Benhalima, 1998). In other brachyuran species, as observed in previous studies (Spalding 1942; Adiyodi & Anilkumar 1988; Zara et al. 2014), the rupture only occurs in the presence of secretions from the SR, during the dissolution of the sperm plug. Recently, Antunes et al. (2016) found sperm packets arranged in strata or layers in the SR of recently
mated females of Stenorhynchus seticornis (HERBST 1788). Here, we explore in greater detail the anatomy and histology of these male and female structures. The localization, insertion, and the morphological structure of setae, such as the presence of denticles surrounding the distal opening of the ejaculatory canal on the first gonopod of males of S. seticornis (Kienbaum et al. 2017), can allow for removal of the sperm content previously inserted by the last male to mate, therefore reducing the intensity of sperm competition in the SR (Beninger et al. 1991; Rorandelli et al. 2008). Although genetic analyses of broods from most Brachyura species demonstrate multiple paternity, the sperm stratification observed in the SR (attributed to dislodgement of the previous ejaculates to the dorsal region of the SR by the last male to mate) in females of S. seticornis (Antunes et al. 2016) can contribute to a single paternity of the brood, specifically in those species with ventral SR (Birkhead et al. 2009; Beninger et al. 1991; McKeown & Shaw 2008; Pardo et al. 2015). However, the mechanism of formation of stratified sperm packets is still unknown. The aim of this study was to analyze the detailed functional anatomy and histology of the male reproductive system of S. seticornis, focusing on the histochemistry of the vas deferens and accessory gland. We studied the ultrastructure of the first gonopod by means of scanning electron microscopy. In addition, we studied the mechanisms of dehiscence of spermatophores collected from the vas deferens (of males) and from the SR (of newly mated females) exposed to seawater.
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Methods Animals The crabs were manually collected by SCUBA diving in coastal areas of the Ubatuba municipality, including Rapada Island (23°250 00″S; 44°540 00″W) and Couves Island (23°250 00″S; 44°510 00″W), S~ ao Paulo State, Brazil, and individually placed in perforated plastic containers in a large aerated cooler for transport to the laboratory (Nebecc, UNESP) at Botucatu, State of S~ao Paulo, Brazil. The experiments were carried out using 10 randomly selected adult male–female pairs. Each individual from the pair was maintained isolated from the other by means of a plastic screen in the same tank (aquarium). Crabs were fed daily with pieces of penaeid shrimp and commercially packaged food for ornamental fishes. Ten aquaria were provided with a recirculating filtration system (12-h photoperiod,
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salinity of 35 ppt, and temperature of 261°C). The carapace width (CW) of males ranged 7.6–14.9 mm, and CW of females ranged 9.6–11.0 mm. Only mature individuals were used in this study; in small males, maturity was based on the presence of mature spermatozoa. In both sexes, maturity was also assessed by the degree of adherence of the abdomen to the thoracic sternite.
pairs of crabs were randomly selected, and were anesthetized by chilling. From each male crab, we removed three samples of spermatophores immersed in seminal fluid from the vas deferens, and we removed three samples of spermatophores from the seminal receptacle of recently mated females. Spermatophores were transferred individually to excavated slides and diluted in 100 lL of seawater. In addition, three other samples of spermatophores from the vas deferens were diluted in 1 mL of matrix from the seminal receptacle of females, and three samples were diluted in 1 mL of secretion from the accessory gland (Fig. 1). Spermatophores were observed after 5, 7, 15, 30, 60, 70, 80, 90, 100, 110, and 120 min. The spermatophores were photographed under differential interference contrast (DIC) microscopy on a ZeissÒ Axio Imager Z2 microscope (Oberkochen, Germany).
Anatomy, histology, and histochemistry Six mature males were anesthetized by chilling; then, the dorsal carapace was removed, and the whole animal was directly fixed in 4% paraformaldehyde in 0.2 mol L1 sodium cacodylate buffer (pH=7.4) for 24 h. During the fixation time, the male reproductive system was photographed under stereomicroscope for anatomical description. Thereafter, the reproductive system samples were dissected and rinsed with the same buffer, dehydrated in an ascending ethanol series (70–95%), and embedded in LeicaÒ HistoResin (Wetzlar, Germany) (glycol methacrylate). After polymerization, the blocks were sectioned with a rotary microtome. The sections (5–7 lm) were mounted on slides and stained with hematoxylin and eosin (HE) (Junqueira & Junqueira 1983). Additionally, testes and vas deferens were stained for proteins with bromophenol blue (Pearse 1985) and xylidine Ponceau (Mello & Vidal 1980). Neutral and acid polysaccharides were stained with PAS and Alcian blue, respectively, as well as simultaneous staining with these dyes to determine whether neutral or acid polysaccharides were predominant in any tissue (Junqueira & Junqueira 1983).
Results Gross anatomy The testes in males of Stenorhynchus seticornis were a pair of highly convoluted tubules forming a compact block linked by a commissure that was difficult to preserve during the dissection (Fig. 2A). Due to the body architecture of this species, the testes examined were limited to the central part of the anterior cephalothoraxic region, near the stomach and hepatopancreas (Fig. 2B,C). Each vas deferens was divided into three regions: anterior
Scanning electron microscopy Samples of the vas deferens and first gonopod were transferred to a solution of Karnovsky fixative (2.5% glutaraldehyde and 2% paraformaldehyde) in 0.1 mol L1 sodium cacodylate buffer (pH 7.4) for at least 24 h, washed twice in the same buffer, dehydrated in ethanol series (50%, 75%, 95%, 100%), and critical point dried in EMS 850. The samples were mounted on aluminum stubs, and sputtercoated with gold using a Denton Vacuum Desk II. The micrographs were taken on a JEOL JSM-5410 scanning electron microscope (SEM) at 10–20 kV electron beam. Dehiscence experiments Spermatophores were induced to dehisce according to methods from Beninger et al. (1993). Three
Fig. 1. Schematic drawings of dehiscence experiments with samples of spermatophores taken from individuals of Stenorhynchus seticornis. Three samples of spermatophores immersed in seminal fluid were removed from the median vas deferens (MVD) of adult males, and three samples of spermatophores were removed from the seminal receptacle (SR) of recently mated females. Individual spermatophores were transferred to excavated slides and diluted in 100 lL of seawater. In addition, another three samples of spermatophores from the MVD were diluted in 1 mL of matrix from the seminal receptacle (SR) of females, and another three samples were diluted in 1 mL of secretion from the male accessory gland (AG).
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Fig. 2. Schematic drawing and gross morphology of the male reproductive system of Stenorhynchus seticornis. (A) Schematic drawing of the male reproductive system relative to the external outline of the cephalothorax (including the anterior rostrum and eyes). (B) Note the pair of testes (arrow), one located in each of the anterior cephalothorax margins, continuous with the pair of vas deferens, which extend longitudinally over the hepatopancreas and below the heart, toward the ventral posterior region of the body. (C) Morphology of the vas deferens, showing the different anatomical regions and accessory gland. (D) Details of accessory gland. AG, accessory gland; AVD, anterior vas deferens; MVD, median vas deferens; PVD, posterior vas deferens; T, testis.
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(AVD), median (MVD), and posterior (PVD). The AVD, immediately posterior to the testis, was a thin, sinuous, and whitish tubule (Fig. 2A,C). The MVD was enlarged, less convoluted, and also whitish and milky in color. The transition from MVD to PVD was marked by the set of accessory glands (AG), classified as simple branched tubular glands (Fig. 2C,D). The PVD surface showed many undulations, and was more translucent white than the MVD (Fig. 2D).
the spermatophores wall was thin and inconspicuous (Fig. 4E,F). Only in secretion samples taken from the MVD was it possible to visualize the extremely thin spermatophore wall (Fig. 4G,H). Under light microscopy, the eosinophilic luminal secretion was reactive to proteins and neutral and acid polysaccharides, while the wall of the spermatophores stained intensely to all compounds (Fig. 4I–L). The single columnar epithelium of the MVD was lying on a thicker layer of musculature than the AVDd (Fig. 4I). The transition from MVD to PVD was characterized by the opening of the accessory glands. In longitudinal sections, the spermatophores were visible stored in the MVD, while the PVD was exclusively filled with secretion without any spermatophores (Fig. 5A). The accessory gland was a simple branched tubular gland with a blind end filled with a compact, mosaic-like secretion (Fig. 5B,C). The accessory gland showed a sinuous, simple squamous epithelium, cells of which had flat nuclei, and a thick layer of musculature (Fig. 5B–E). The muscle fibers were obliquely distributed in different directions (Fig. 5C,D). The glandular opening in the PVD was characterized by a simple cubic epithelium, and the cells showed an eosinophilic cytoplasm similar to the main gland tubule (Fig. 5F). Three distinct types of luminal secretions were found in the AG: S1 secretions were homogeneous, finely granular, acidophilic and eosinophilic glycoproteins that contained neutral polysaccharides; S2 secretions were composed of a basophilic matrix of acidic and neutral polysaccharides; S3 secretions formed globular structures that were exclusively reactive to proteins (Fig. 5E,G–I). The PVD showed a simple, sinuous, squamous epithelium lying on a layer of musculature similar to the accessory gland, but thicker than in the MVD (Fig. 5H). The luminal secretion seemed more fluid and less compact than that observed in the accessory gland. The three PVD secretion types were similar to those described for the AG (Fig. 5J–M).
Histology, histochemistry, and ultrastructure The testes resembled the tubular type, with a tubule slightly wider in certain regions (Fig. 3A). Germ cells in the same spermatogenesis stage were found together in specific tubule areas (Fig. 3B). The spermatogonia were localized in the periphery of the seminiferous tubule (Fig. 3C). The primary spermatocytes showed large nuclei depicting different stages of the meiotic prophases (Fig. 3D). The secondary spermatocytes showed a small nucleus with homogeneously stained chromatin (Fig. 3E). Spermatids developed a nucleus with a rounded shape and a larger PAS-positive acrosome vesicle. During spermiogenesis, the accessory cells (Sertoli cells) also showed large nuclei and cytoplasm (Fig. 3F). Mature spermatozoa were released into the lumen of the seminiferous tubule. The acrosome vesicle was large and occupied most of the cell, while the nucleus almost surrounded the acrosome vesicle, forming a very thin C-shaped structure with radial arms (Fig. 3G). The AVD could be divided into two distinct portions, the proximal (AVDp) and the distal (AVDd), based on differences in the luminal content. The AVDp showed a simple squamous or cubic epithelium lying on a thin layer of musculature (Fig. 4A). The spermatozoa were grouped in a compact mass of cells without a wall. Among the spermatozoa, there was an eosinophilic secretion that reacted negatively to proteins and was intensely reactive to neutral polysaccharides (Fig. 4A, B). At the AVDd, the simple epithelium was formed of tall columnar cells (Fig. 4C). The eosinophilic secretion stained intensely to PAS and aided the separation of the spermatozoa in large masses, forming round or elliptical spermatophores with a very thin glycoprotein wall reaching to the MVD (Fig. 4C). The MVD, completely filled with spermatophores, appeared immersed in a homogeneous secretion. Fractured MVD under SEM showed the spermatozoa organized in spermatophores; however, the wall of
Gonopod morphology The ultrastructure of the first gonopod showed many denticles, particularly abundant at the apex, that were distributed in opposite directions; these outgrowths occur inside the aperture of the ejaculatory canal (Fig. 6E–G). A prominent process was present, externally, at the subterminal aperture of the ejaculatory canal (Fig. 6F). The remaining basal two-thirds of the gonopod channel were smoothly surfaced (Fig. 6I).
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Fig. 3. Histology of the male reproductive system of Stenorhynchus seticornis, stained with hematoxylin and eosin. (A) Tubular testis with regionalized maturation zones, with cells at the same stage of development, and lumen of the seminiferous tubule (arrows). (B) Maturation zone shows primary spermatocytes (SPC 1) randomly distributed along the testis and spermatids (ST). (C) Spermatogonia (SPGO, indicated by arrows) in the seminiferous tubule. (D) SPC1 in different stages of meiotic prophase. (E) Secondary spermatocytes (SPC2). (F) Spermatids (black arrow), with PASpositive acrosomal vesicle, and Sertoli cells (black arrowhead) during spermiogenesis process. (G) Spermatozoa in the lumen of the seminiferous tubule, with complete acrosomal vesicle (white arrow) and radial arms (white arrowheads).
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Fig. 4. Histology of the male reproductive system of Stenorhynchus seticornis. (A,B) Proximal anterior vas deferens (AVDp), with agglomerate of free spermatozoa immersed in negative eosinophil secretion for proteins. The walls of the AVDp are a simple cubic epithelium with a thin muscle layer (indicated by the black arrowhead in panel A). (C) Formation of spermatophores in the distal anterior vas deferens (AVDd), with eosinophilic secretion reacting intensely to PAS stain (black arrowhead). (D) Median vas deferens (MVD) with spermatophores and columnar epithelium, with nucleus occupying the central region (black arrowhead). (E,F) SEM of a fractured MVD, showing the spermatozoa. (G,H) SEM of spermatophores are covered with a thin membrane. (I) The MVD has a simple columnar epithelium, with a muscle layer thinner than in the AVD (black arrow), the secretions of the MVD is eosinophilic (black arrowheads). (J–L) Secretions (black arrowheads) of the MVD are intensely reactive for proteins, neutral polysaccharides, and acids.
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Spermatophore dehiscence The spermatophores obtained from the MVD and maintained in seawater or in secretion from the AG retained an intact wall and did not show dehiscence after 120 min (Fig. 6A,B). On the other hand, the contents obtained from the SR of female crabs showed only free spermatozoa which formed round masses and lacked the spermatophore wall (Fig. 6C, D). This material, when observed directly under DIC optics, showed the presence of free spermatozoa with up to five radial arms, one of them being the posterior median process (Fig. 6D).
Discussion The testes in males of Stenorhynchus seticornis are limited to the central cephalothoracic region due to the peculiar body shape in this species (i.e., individuals are elongated along the anteroposterior axis). The male reproductive system in other species of Brachyura typically occupies the cephalothoracic region, near the midgut gland, extending along both sides of the carapace (Krol et al. 1992; Sime o et al. 2009). The commissure observed by Mouchet (1931), which connects the left and right testes, is present in S. seticornis but is extremely slender and was easily broken during the dissection of the studied specimens. Each testis has the same pattern previously described for other Majoidea (Kon & Honma 1970; Sapelkin & Fedoseev 1981; Sime o et al. 2009) and is classified as tubular, comprising only one small, highly convoluted tube (Minagawa et al. 1994; Nagao & Munehara 2003), similar to a few other species of Majoidea, Grapsoidea, and Xanthoidea (Binford 1913; Tiseo et al. 2014). By contrast, the majority of brachyuran species show testes of the lobular type (Sime o et al. 2009; Zara et al. 2012; Nascimento & Zara 2013). The tubular testes of S. seticornis were composed of several areas that resembled the organization found in lobular testes, but inside the same tubule. Each area is surrounded by accessory cells, and releases spermatozoa into the interior of the seminiferous duct (Gupta & Chatterjee 1976; Hinsch 1988; Minagawa et al. 1994; Suganthi & Anilkumar 1999; Moriyasu et al. 2002; Nagao & Munehara 2003; Garcia & Silva 2006; Cuartas & Petriella 2007; Jivoff et al. 2007; Castilho et al. 2008; Stewart et al. 2010; Zara et al. 2012). Although the testes in males of S. seticornis are of the tubular type, the germ cells are haphazardly distributed, with no delimitation of specific zones for each cellular activity, such as proposed
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by Nagao & Munehara (2003) and found in other brachyurans with the same type of testes (Binford 1913; Erkan et al. 2009; Sime o et al. 2009, 2010). In S. seticornis, specific zones of cellular activity are observed in the transverse section. However, in other species of brachyurans, such as in Maja brachydactyla BALSS 1922, the seminiferous tubule is divided along the long axis into three zones, called germination, transformation, and evacuation zones. Each zone has distinct contents and performs a different role throughout spermatogenesis (Sime o et al. 2009). Spermatogenesis in S. seticornis followed the general pattern described for other species of Brachyura (Castilho et al. 2008; Santos et al. 2009; Zara et al. 2012; Tiseo et al. 2014). Like M. brachydactyla (Sime o et al. 2009), S. seticornis did not show diminishing of the nucleus volume, as observed in Portunidae (Stewart et al. 2010; Nascimento & Zara 2013) and in species of Ucididae such as Ucides cordatus (LINNAEUS 1763), studied by Castilho et al. (2008). When mature, the spermatozoa of S. seticornis show five lateral arms, similar to the sperm morphology of Inachus phalangium (FABRICIUS 1775) (Rorandelli et al. 2008) and other Brachyura (Felgenhauer & Abele 1990). According to Jamieson et al. (1998), the morphology of spermatozoa in brachyuran crabs seems to be a functional adaptation to the encapsulation inside the spermatophore or to the contact between the sperm and the oocyte. In addition to the lateral arms, a median-posterior extension may have nuclear material, with or without microtubules (Tudge et al. 2014). Such extensions observed in S. seticornis were also observed in Libinia emarginata LEACH, 1815 and Pitho lherminieri (DESBONNE IN DESBONNE & SCHRAMM 1867), according to Hinsch (1973) and Tudge et al. (2014). The AVD region is classified as proximal (AVDp) and distal (AVDd) in S. seticornis, and in some other crab species such as Goniopsis cruentata (LATREILLE 1803), M. brachydactyla, Callinectes danae SMITH 1869, and Callinectes ornatus ORDWAY 1863 (Garcia & Silva 2006; Sime o et al. 2009; Zara et al. 2012; Nascimento & Zara 2013). Formation of spermatophores begins in the AVDp and ends at the AVDd, which also functions in spermatophore storage (Sime o et al. 2009). These two regions also differ in the thickness of the epithelium and in types of secretions. According to Nascimento & Zara (2013), the changes in the epithelium are related to the volume of the secretions. The eosinophilic secretions present among the sperm packages in the AVD of S. seticornis were also detected in
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Fig. 5. Histology of the male reproductive system of Stenorhynchus seticornis. (A) Transition region between the median vas deferens (MVD) and posterior vas deferens (PVD), marked by the opening of the accessory gland (AG). (B) AG, filled only with secreted material. (C) The AG is covered by many muscle fibers (black arrow), and the lumen is completely filled with secreted material (black arrowhead). (D) Details of the muscle fibers of the AG. (E) The AG has a muscle layer that is thinner than in the AVD (black arrow) and is filled with three types of secretions: S1 secretions are homogeneous, finely granular, acidophilic and eosinophilic glycoproteins with neutral polysaccharides that are weakly reactive to proteins; S2 secretions are basophilic and acidic and neutral polysaccharides; S3 secretions are proteins that are less eosinophilic. (F–I) The AG becomes narrower and lined with cubic epithelium and is wrapped in thinner muscles (black arrow) in the region closest to the PVD opening. (J) The epithelium of the PVD is simple, sinuous, and set in a thick muscular layer (black arrow). (K–M) PVD filled with three distinct secretions S1–S3.
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Fig. 6. Spermatophores, spermatozoa, and first gonopod from male of Stenorhynchus seticornis. Spermatophores taken from the MVD remained intact after 5 min (A) and 120 min (B) in seawater. (See Fig. 1 for experimental setup.) (C) Spermatozoa agglomerates from the seminal receptacle after 5 min in seawater. (D) Spermatozoa agglomerates shown in DIC (arrows indicate radial arms). (E) Diagrams of the first pleopod (gonopod) of male crab. (F) Note the presence of a prominent process (white arrow) at the subterminal ejaculatory canal opening. (G) Detail of the opening at the apex of the first gonopod, covered by denticles. (H) Detail of denticles that line the opening of the first gonopod. (I) Inner overview of the ejaculatory canal, showing completely flat wall. d, denticles; Ec, ejaculatory canal; eco, ejaculatory canal opening; s, simple setae.
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individuals of C. danae and C. ornatus, and are involved in the formation of spermatophores (Sime o et al. 2009; Zara et al. 2012; Nascimento & Zara 2013). Once they are formed, the spermatophores of S. seticornis are stored in the MVD, which absorbs products secreted by the AVD during the process of packaging spermatozoa into spermatophores (Hinsch & Walker 1974). The thickening of the muscle layer in the MVD region may have a relationship to the movement of spermatophores to posterior regions of the vas deferens by means of muscle contractions (Ryan 1967). The PVD may have several functions among different species (Krol et al. 1992; Sime o et al. 2009; Zara et al. 2012; Nascimento & Zara 2013), including storage of the spermatophores and seminal fluid, secretion formation, and phagocytosis of spermatozoa and spermatophores. Benhalima & Moriyasu (2000) proposed that all of these functions may be important in Chionoecetes opilio, including phagocytosis and re-absorption of excess spermatophores and spermatozoa in a region of high cellular activity in the vas deferens. In S. seticornis, the PVD is filled only with secretions, while storage of spermatophores is limited to the MVD. A similar distribution of spermatophores and secretions was also found in Libinia spinosa (MILNE EDWARDS 1834) (Sal Moyano et al. 2010). The location of the accessory glands in males of S. seticornis, at the transition between the MVD and PVD, is similar to that in other crabs previously studied, such as Pachygrapsus transversus (GIBBES, 1850) and P. gracilis (SAUSSURE, 1857) (Tiseo et al. 2014). The structure of the accessory glands in S. seticornis is similar to that observed in I. phalangium by Diesel (1989), who described these glands as numerous ceca at the PVD. In other crab species, such as C. opilio studied by Beninger et al. (1988) and Benhalima & Moriyasu (2000), there are also ceca at the AVD, MVD, and PVD. On the other hand, the accessory glands in males of M. brachydactyla are localized at the MVD, and composed of 7–8 highly branched and enlarged diverticula, which are connected to the dorsal region of the PVD (Sime o et al. 2009). In other crab species, the same structure was described as expansions (e.g., in G. cruentata, studied by Garcia & Silva [2006]), or evaginations (e.g., in U. cordatus, studied by Castilho et al. [2008]) of the vas deferens. Tiseo et al. (2014) classified the accessory glands from Pachygrapsus transversus and P. gracilis as exocrine tubular structures, which are not complex, but instead form branched structures that are connected to the epithelium of the vas deferens and release
their products inside the lumen. Considering the literature on morphology, localization, and function of the accessory glands of crab species, and despite the distinct terminology adopted by various authors, these glands are very similar among the studied species. Thus, we think that these names (ceca, AG, etc.) refer to the same homologous structure that is involved in the production of important substances for spermatophore and seminal fluid formation. The secreted material that surrounds sperm packages in the SR of females of S. seticornis (Antunes et al. 2016) has the same histochemical features as the secretions of the AG and PVD of males investigated in the present study. Such similarities observed in S. seticornis support the hypothesis that the secretions produced by the accessory glands in males delimit the sperm packets in layers, and are transferred together with the spermatophores at mating. Diesel (1989, 1991) and Sainte-Marie et al. (2000) described this process for I. phalangium and C. opilio, respectively. This hypothesis can be supported by the observation that, compared to other regions of the vas deferens, the accessory glands and the PVD have a thicker layer of muscles, which should be capable of compressing the epithelium and facilitating the release of secretions that are transferred along with the sperm package at mating. By contrast, in those species that do not have accessory glands in the vas deferens, for instance, L. spinosa described by Sal Moyano et al. (2010), the spermatozoa are free in the SR of females, despite being contained in a sperm gel, with no formation of layers. These possible functions of the accessory glands and the PVD suggest a potential role in the formation of the sperm packages, which were observed in the SR of newly mated females. The existence of accessory glands and the absence of spermatophores in the PVD are consistent with a possible strategy by males to reduce sperm competition. The spermatophores of different sizes in the MVD did not show dehiscence by hydration in seawater, nor when they were diluted in secretions from the SR. Nevertheless, spermatophores obtained from the SR of newly mated females showed sperm packages without a surrounding wall, which characterizes the agglomerates of spermatophores. Thus, it is probable that there might be an unknown mechanism acting in the rupture of the spermatophores during their transfer from the PVD to the SR. The existence of a large number of denticles (Kienbaum et al. 2017) at the tip of the first gonopods in males could cause the rupture of the spermatophores during their transfer to the SR, as previously proposed
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by Rorandelli et al. (2008). This mechanism is consistent with the extremely fine wall surrounding each of the spermatophores of S. seticornis and I. phalangium, as described here and by Rorandelli et al. (2008), respectively. Beninger et al. (1988) found two types of spermatophores which differed in the composition of the wall surrounding the spermatophore, and suggested that some of these structures might break at the time of mating, while others might persist and be stored in the SR. In another study, Beninger et al. (1991) observed that, due to the narrow opening of the first gonopod (~40 lm in C. opilio), the spermatophores should undergo a significant pressure and mechanical force during sperm transfer, with the larger spermatophores (50–200 lm in diameter) subjected to higher stress. In our experiments, immersion in seawater was not sufficient to disrupt the walls of spermatophores; both mechanical and chemical action seem necessary for the release of spermatozoa, as shown by Beninger et al. (1993) in C. opilio. According to Diesel (1989), the glandular epithelium of the SR can produce enzymes responsible for the rupture of spermatophore membranes. Additionally, the morphology of the gonopods of S. seticornis, with spines or denticles at their tips distributed in opposite directions, may contribute to the rupture of the spermatophore during sperm transfer (Kienbaum et al. 2017), as observed by Rorandelli et al. (2008) in I. phalangium. Considering all of these aspects of spermatophore transfer and release of spermatozoa in brachyurans, the variation in thickness and composition of the spermatophore wall are consistent with the patterns of dehiscence we observed in S. seticornis. The origin, function, and chemical composition of the spermatophores in Decapoda are not yet completely understood. In Portunoidea, for instance, the external layer of the spermatophore contains chitin and acidic polysaccharides, while the seminal fluid contains carbohydrates, proteins, and lipids (Subramoniam 1991). It is not clear whether these compounds come from secretions of the vas deferens, or from the spermatophores themselves. Our histochemical analysis of spermatophores from individuals of S. seticornis corroborate a previous report, which found that spermatophore walls from individuals of the blue crab, C. danae, are composed of glycoproteins (Zara et al. 2012). In many species of Majoidea, the release of oocytes can occur just after mating, and the immediate dehiscence of spermatophores may be an important part of male reproductive success because
fertilization can take place immediately if the oocytes are mature enough to be extruded (Beninger et al. 1988). In this way, the males of S. seticornis may increase paternity, and these processes in S. seticornis may serve as a model in the study of other Majoidea.
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Acknowledgments. We thank IML technicians for laboratory assistance, NEBECC researchers A. L. Castilho, V. J. Cobo, T. M. Davanso, and I. R. R. Moraes for their assistance in the arrow crab samplings. We also thank the Electron Microscopy Laboratory of FCAV, UNESP Jaboticabal and Marcia F. Mataqueiro for the technical support. This research was supported by Coordination for the Improvement of Higher Education Personnel CAPES (CAPES-MINCYT #217/2012) to LSLG and MLNF; (CAPES-CIMARII #1989/2014-23038.004309/ 2014-51) to FJZ; Brazilian National Council for Scientific and Technological Development (CNPq–Universal #481435/2011-5) to MLNF; (CNPq-Universal #486337/ 2013-8) to FJZ; and S~ao Paulo Research Foundation FAPESP (Biota #2010/50188-8; #2016/25344-2) to FJZ. This study was carried out in compliance with current Brazilian state and federal regulations on wild animal sampling (MMA-SISBIO license #13383).
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