Mar Biol (2007) 152:383–394

DOI 10.1007/s00227-007-0688-y

RESEARCH ARTICLE

Ovarian maturation of the multi-spawning spider crab Maja brachydactyla (Decapoda: Majidae) with special reference to yolk formation

Guiomar Rotllant · Eduardo González-Gurriarán · Luis Fernández · Khadra Benhalima · Enric Ribes

Received: 24 July 2006 / Accepted: 28 March 2007 / Published online: 28 April 2007© Springer-Verlag 2007

Abstract In the study of the reproductive biology of thespider crab Maja brachydactyla, the morphology of thefemale reproductive system and yolk formation have longbeen overlooked. Females spawn two or three times duringtheir annual reproductive cycle in northern Spain (Galicia).The ovaries consist of two lobes. The right and left lobesare connected by a small cross-lobe at the level of the heartand merge at the posterior edge. Before merging, the ova-ries descend to the ventral part of the body, joining thespermathecae in the vagina, which opens through a chitintube to the gonopore, located in the sternite, at the level ofthe third walking leg. No morphological changes havebeen observed between either the diVerent parts of the ova-ries or the diVerent annual spawning periods. At the start of

vitellogenesis, the oocyte of M. brachydactyla is character-ized by a large number of vesicles in the cytoplasm. Thesevesicles are surrounded by a unit membrane whose sizeincreases as the oocyte matures and contain Wne granularmaterial including a variable number of ovoid, electron-dense granules. The vesicles are of diverse origin, althoughmost of them develop directly from the mitochondria andthe Golgi complex (endogenous phase of vitellogenesis).In a subsequent phase, a series of substances (principallylipoproteins) are incorporated into the ooplasma by meansof micropinocytosis. These substances are also involved inyolk formation (exogenous phase of vitellogenesis). Duringvitellogenesis in M. brachydactyla, mitochondria play themost important role since they are not only the energeticcentre of the cellule, but they also act as containers of high-energy reserve substances: the yolk granules.

Introduction

The Majidae family, which contains approximately 900species, is widely distributed in marine waters (Brownellet al. 1977). Although the vast majority of majid crabs aresmall and have no direct economic importance, some spe-cies are subject to commercial exploitation (Brownell et al.1977). Commercially exploited majid varieties include thesnow crab, Chionoecetes opilio, and the common spidercrab, Maja brachydactyla (the NE Atlantic species previ-ously known as M. squinado, see Newman 1998 for taxo-nomic status). Therefore, these species have already beenquite widely studied.

Research has been done on the reproductive biology ofthe spider crab M. brachydactyla and M. squinado foundin the waters of the British Isles (Brosnan 1981; Edwards1979; Hartnoll 1965, 1969; Rodhouse 1984) and the Channel

Communicated by P.W. Sammarco.

G. Rotllant (&)IRTA, Carretera del Poblenou, s/n., Sant Carles de la Ràpita, 43540 Tarragona, Spaine-mail: guiomar.rotllant@irta.es

E. González-Gurriarán · L. FernándezDepartamento de Bioloxía Animal, Bioloxía Vexetal e Ecoloxía, Universidade da Coruña, Campus da Zapateira s/n, 15071 A Coruña, Spain

K. BenhalimaDepartment of Fisheries and Oceans, Invertebrate Fisheries Division, Science Branch, Maritimes Region, 343 Université Avenue, Moncton, NB, Canada E1C 5K4

E. RibesDepartment of Cell Biology, Faculty of Biology, University of Barcelona, Av. Diagonal, 645, 08028 Barcelona, Spain

123

384 Mar Biol (2007) 152:383–394

Islands (Meyer 1993), in French Brittany (Kergariou 1971,1975; Le Foll 1993), on the Asturian coast (N Spain)(García-Flórez and Fernández-Rueda 2000), on the Gali-cian coast (NW Spain) (González-Gurriarán et al. 1993,1995, 1998), on the Saharan coast (García Cabrera 1972),in Senegalese waters (Clotilde-Ba et al. 1997) and in theAdriatic Sea (Stevcic 1967, 1977). Among these works,only three preliminary studies have described the morphologyof their ovaries (García Cabrera 1972; Brosnan 1981;González-Gurrirán et al. 1993). In addition, González-Gurriarán et al. (1993) have determined four stages ofmaturity based on macroscopic criteria of their gonads.

Seasonal changes in the maturity of gonads in C. opiliowere described by Kon and Honma (1970a, b). The anat-omy of the female reproductive system was studied byElner and Beninger (1992), Lanteigne et al. (1996) and Sainte-Marie and Sainte-Marie (1998) and an ultrastructural andhistochemical study of the spermathecae was undertaken byBeninger et al. (1993). Furthermore, Diesel (1991) reviewedthe morphology of the spermathecae and their functionin several brachyurans and presented the Wrst descriptionof the spermathecae of M. brachydactyla.

The structure of the female reproductive tract and theoogenesis of brachyuran crabs have been reported for sev-eral crab species, such as Potamon dehaani (Ando andMakioka 1999), Pachygrapsus marmoratus (Pradeille-Rouquete 1975), Eriocheir sinensis (Hoestlandt 1948),Portunus sanguinolentus (Ryan 1967) and Ranina ranina(Minagawa et al. 1993). Brachyuran yolk formation hasalso been studied with electron microscopy (Hinsch andCone 1969; Eurenius 1973).

The present study focuses on the morphology and ultra-structure of the female reproductive system and oogenesisin M. brachydactyla. Yolk formation is the main feature ofvitellogenesis. In crustaceans, there is much controversy asto whether the synthesis of the yolk granules is endogenousor exogenous and the organ responsible for this synthesishas yet to be agreed upon.

Material and methods

Monthly samples were taken over a period of 18 monthsfrom commercial tangle net Wshing vessels operating oVthe Galician coast. Adult females (large abdomen) withcarapace lengths (CL) of between 130 and 160 mm wereselected and taken back to the laboratory alive, where theirwhole reproductive tracts (ovaries and spermathecae) wereimmediately removed and sampled.

For light microscopy, ovaries from diVerent parts (ante-rior, medium and posterior) were cut into small pieces andwhole spermathecae were Wxed in Bouin’s Xuid and dehy-drated in a series of graded concentrations of aqueous ethanol

solution. Samples were kept in 70% ethanol until theycould be processed. First, the samples were completelydehydrated. Next they were cleared in a xylene agent andembedded in paraYn. Blocks of tissue were sectioned seri-ally at 6 �m thickness using a Reichert-Jung rotary micro-tome and stained with hematoxylin–eosin or Masson’sTrichrome stain (Martoja and Martoja 1967). Light micros-copy examinations were performed under an OLYMPUSBX61® light microscope and photographed with a camerausing bright Weld optics.

For transmission electron microscopy, the tissues wereWxed for 3 h at 4°C in 2% paraformaldehyde–2.5% glutar-aldehyde mixture in 0.1 M cacodylate buVer (pH 7.2) (Kar-novsky 1965). They were post-Wxed for 1 h at 4°C in 1%osmium tetroxide in the same buVer and then dehydratedin acetone and embedded in Spurr’s low-viscosity plastic(Spurr 1969). Samples were kept in Spurr blocks until theycould be processed. Ultra-thin sections were cut on anultra-microtome, Leica Ultracut UCT®, stained with uranylacetate and lead citrate (Reynolds 1963) and then examinedwith a Jeol EM-1010® electron microscope at 80 kV.

For scanning electron microscopy, the tissues were Wxedfor 6 h, using 2% paraformaldehyde 2.5% glutaraldehyde,post-Wxed for 2 h in 2% osmium tetroxide and dehydratedin graded concentrations (of up to 100%) of ethanol for15 min. The samples were then dried at their critical pointwith CO2 using (CPD) and then sputter coated with gold–palladium (Goldstein et al. 2003). Coated samples werekept for microscopic observation. Observations were madeunder a Hitachi S-2300® scanning electron microscope at10–15 kV acceleration voltage.

Results

The female reproductive system is a bilaterally symmetricalbody, with the axis being the line running between the ros-trum and the most posterior edge of the animal. Each side iscomposed of an ovary, an oviduct, a spermatheca and avagina. The connective tissue holds the reproductive sys-tem Wrmly in place. Interiorly and dorsally, it extends frombehind the fourth antero-lateral carapace spines to the baseof the eyes and then medially to the stomach, comingtogether where it passes between the two mandible tendons.The two ovaries are joined by a commissure or isthmusbehind the stomach and in front of the heart. In the cardiacregion, at the level of the third walking leg, the ovaries sinkventrally under the pericardial sac and merge with the sper-matheca at the level of the vagina, passing through a veryshort oviduct. The ovaries then emerge dorsally at the levelof the Wfth walking leg, where they merge and run posteri-orly to the beginning of the abdomen (Fig. 1a, b). Whenripe ovaries (stage IV) were dissected and extended on a

123

Mar Biol (2007) 152:383–394 385

tray, the total length from the anterior edge to the posteriormerging point was approximately 35 cm with a width of6 mm (all anatomical measurements took the averageof three females having CLs of t150 mm) (Fig. 1c). Nomorphological changes were observed between either thediVerent parts of the ovaries or the diVerent annual spaw-nings. The germinal zones, which are located in the centreof the ovaries, produced oogonia. As they diVerentiate intooocytes, the oocytes and their encompassing follicular cellsbecome more peripherally located. The walls of the ovariesare formed by a thin layer of connective tissue (Fig. 2).

The spermathecae are approximately 30 mm long by10 mm wide and are divided into three functionally distinctregions, with no intervening anatomical separations. A Xat-tened pouch forms the distal and largest part of each sper-matheca. Its epithelium consists of a stratiWed layer of cells,

containing oval nuclei with homogenous chromatin. Itslumen is Wlled with spermatophores and spermatozoids thathave been stored after copulation (Fig. 9a). A vertical cen-tral column is observed in the middle of each spermatheca(Fig. 1c). The wall of the pouch is rough, distensible andmade up of spongy connective tissue. When present, sper-matophores or spermatozoids move freely in the lumen,which contains a small amount of seminal Xuid. Spermato-phores usually concentrate near the vagina, unless the sper-matheca is full. The vagina is a muscular chamber wherepassing oocytes are fertilized by the spermatozoids storedin the distal part of the spermatheca (Fig. 9b). The ventralend of the vagina is formed by a chitin tube. This tube iscomposed of a layer of chitin-lined columnar epithelialcells resting on a base membrane and is bounded on theoutside by a connective tissue that joins the vagina to thegonopore located in the sternite, at the level of the thirdwalking leg (Fig. 1c).

Four stages of gonad maturity were originally deter-mined based on macroscopic criteria and histologicalexaminations by González-Gurriarrán et al. (1993). Thisdescription has since been completed by observations usinglight microscopy, TEM and SEM; the results are presentedbelow. Primary vitellogenesis corresponds to stages I throughIII and secondary vitellogenesis to stage IV.

Stage I

Underdeveloped gonads are white or light cream in colour.The ovaries contained mainly oogonia and early previtello-genic oocytes. The smallest oocytes remain at the centre ofthe ovaries, whereas the largest ones appear to be moreperipherally located. Larger oocytes present a maximum

Fig. 1 General view and internal anatomy of the female spider crab Maja brachydactyla. a Location of the female reproductive tract. b Longitudi-nal view of the female reproductive system. c Diagram of the female ovaries showing the position of the spermathecae and the oviduct

Fig. 2 Scanning electron micrograph showing the general morphol-ogy of the ovary in stage III of the spider crab Maja brachydactyla.Fc follicular cells, Gz germinal zone, Ov vitellogenic oocyte, W wall

123

386 Mar Biol (2007) 152:383–394

mean diameter of 264 �m, with perfectly visible roundnuclei and nucleoli that are large in comparison to the sizeof their respective oocytes (Fig. 3a).

Stage II

Developing gonads are cream to pale orange in colour.They contained oogonia, previtellogenetic and primaryvitellogenic oocytes. The oocytes are ellipsoid at this stageand are clearly surrounded by follicular cells that arealready present in stage I, but are not easily visible. Oocytespresent a maximum mean diameter of 453 �m. Previtello-genic and primary vitellogenetic oocytes contain clearlydeWned nuclei (Fig. 3b).

Stage III

Gonads are widely spread throughout the cephalothoraciccavity and are orange coloured. Ovaries contain oogonia,previtellogenetic and primary vitellogenetic oocytes, aswell as follicular cells. The nuclei are spherical and havespherical nucleoli. Yolk granules are present on the periph-ery of the oocytes (Fig. 3c). Some secondary vitellogenicoocytes are found in the later phases of stage III (Fig. 4d).Larger oocytes have a maximum mean diameter of 673 �m.

In the cytoplasm of the primary vitellogenic oocytes, theGolgi vesicles and bright vesicles merge into type-1 yolkgranules, while vitellogenic secondary oocytes incorporateexternal material by pinocytosis, forming type-2 yolk gran-ules. A central germinal zone remains clear but is sur-rounded by secondary vitellogenic oocytes (Fig. 3d).

Stage IV

Completely mature gonads Wll the cephalothoracic cavity:they are bright orange in colour. Ovaries are in a pre-ovula-tory phase and are mainly Wlled by secondary vitellogeneticoocytes. However, a germinal zone containing oogoniesand previtellogenic oocytes can still be observed in the cen-tre (Fig. 3e). Follicular cells are extremely Xat and diYcultto observe. Large and well-developed secondary vitello-genic oocytes present a maximum mean diameter of845 �m and the whole cytoplasm is clearly occupied bydiVerentiated, type-2 yolk granules. Mature oocytes havean irregular shape, and their vitelline envelopes are welldeveloped (Fig. 3f).

During the initial phases of primary vitellogenesis,oocytes usually have one or more nucleoli 5.5–6 �m indiameter in the peripheral area and near the nuclear enve-lope (Fig. 4a). The nucleoli of the oocytes during primary

Fig. 3 Light micrographs showing cross-section through the ovary during the reproductive cycle: the spider crab Maja brachydactyla. a Stage I. b Stage II. c Beginning of stage III. d Advanced stage III. e Central zone of the ovary in stage IV. f Peripheral zone of the ovary in stage IV. MagniWca-tions: a, b, d, e, f = £50; c = £75. Fc follicle cells, Gz germinal zone, N nucleus, Og oogonies, Opv previtellogenic oocyte, Ov1 primary vitellogenic oocyte, Ov2 secondary vitellogenic oocyte, Vb bright vesicle, Y1 type 1 yolk granule, Y2 type 2 yolk granule, W wall

123

Mar Biol (2007) 152:383–394 387

vitellogenesis become oval due to the segregation of theirnucleolar components, which starts during the previousphase of previtellogenesis. As a consequence of nucleolarsegregation, a series of electron-dense materials spreadfrom the nucleoli to form nucleolar satellites (Fig. 4c). Dur-ing this phase, multiple nucleolar vacuoles are sometimesobserved in both Wbrillar and granular components (Fig. 4a).The size of these vacuoles increased with time, but theirnumber diminished, so when the oocytes began secondaryvitellogenesis only one large vacuole appeared and theircontents were transferred to the cytoplasm, forming spheri-cal electron-dense masses or nuage (Fig. 4c).

During primary vitellogenesis, some electron-denseextranuclear masses move away from the nuclear envelopeand settle in the central area of the cytoplasm. There, theyare associated with the mitochondrial aggregates giving riseto intermitochondrial cement (Fig. 4d). As the process of

vitellogenesis develops, the electron-dense extranuclearmasses diminish in size before Wnally disappearing towardsthe end of the exogenous phase of vitellogenesis. The num-ber of mitochondria in the perinuclear location increasesduring vitellogenesis, parallel to nuclear emissions. Simi-larly, mitochondrial groups are usually found in the areaaround the nuclear envelope (Fig. 4d). During primaryvitellogenesis, it is possible to observe how a certain num-ber of mitochondria begin internal regression, with altera-tions in their cristae. These convert into bright vesicles,which become Wlled with a substance with a granularappearance during secondary vitellogenesis. The substance,which was electron dense, gives rise to a number of yolkgranules (Fig. 5a–c).

At the beginning of primary vitellogenesis, the oocytesare characterized by a large number of dark vesicles in theircytoplasms (Fig. 4a, e). The vesicles are of multiple

Fig. 4 Transmission electron micrographs showing the initial phase:primary vitellogenesis of the spider crab Maja brachydactyla.a General view of an oocyte showing a nucleus with several nucleolusand a cytoplasm rich in bright vesicles and type-1 yolk granules.b Detail of bright vesicles and Golgi complex. c Section through thenucleus. d Note the mitochondria surrounding the electron dense

material or “intermitochondrial cement”. e Detail of a type-1 yolkgranule, the cytoplasm contains mitochondria and Golgi complex.Note that the arrows indicate fusion of Golgi-derived vesicles withyolk granules. ci intermitochondrial cement, G Golgi complex, Mmitochondria, N nucleus, Ng nuage, Nu nucleolus, Ne nuclear enve-lope, St nucleolar satellites, Vb bright vesicle, Y1 type 1 yolk granule

Fig. 5 Transmission electron micrographs showing the formationsequence for type-1 yolk granule in the spider crab Maja brachydac-tyla. a Note the mitochondria located near the bright and granulate

vesicle. b Arrows show granules inside the vesicles. c Note the fusionwith the granule vesicle and yolk vesicle. M mitochondria, Vb brightvesicle, Vg granulate vesicle

123

388 Mar Biol (2007) 152:383–394

origins, although most of them developed directly from themitochondria and the Golgi complex (Fig. 4b, e). Thesevesicles are surrounded by a unit membrane whose sizeincreases as the oocyte matures (Fig. 6a). In short, the for-mation of type-1 yolk granules has a purely endogenousorigin. The bright vesicles originate in the perinuclear areaand then migrate to the periphery of the cytoplasm andsequestrated material from the Golgi complex (Fig. 4b, e).The material incorporated into the bright vesicles mergeand form bigger granules (Fig. 4e), producing completelyelectron-dense masses of type-1 yolk granules (Fig. 6c).

During primary vitellogenesis, nucleolar segregation isaccompanied by a marked increase in the nuage. When theoocytes enter vitellogenesis, they are clearly diVerentiatedby the increase in nucleolar segregation observed in thecytoplasm, together with the presence of a greater numberof electron-dense masses with a granular-Wbrillar aspectnear the nuclear envelope: these granules measure approxi-mately 12 nm in diameter (Fig. 6a). Type-1 yolk granulesare formed near the perinuclear membrane and bright vesi-cles are found in the peripheral part of the cytoplasm(Fig. 6a–c). Microvilli appear in the oocyte membrane.Then, the second phase of vitellogenesis is prepared for thesequestration of external material (Fig. 6b).

In the following phase—secondary vitellogenesis—thewall of the ovary is reinforced by somatic cells so that it isable to house a large number of mature oocytes (Fig. 7a).The oocytes are then surrounded by follicular cells that feedthem in order to produce yolk granules (Fig. 7b). The for-mation of type-1 yolk granules continues during secondary

vitellogenesis. Hence, developed mitochondria are alsopresent (Fig. 7c, d). Furthermore, a series of other sub-stances (principally lipoproteins) are incorporated into theooplasma by means of pinocytotic activity (Fig. 6b). Thesesubstances are involved in the formation of type-2 yolkgranules. Hence, the formation of type-2 yolk granules dur-ing secondary vitellogenesis corresponds to an exogenousphase. In summary, during secondary vitellogenesis, type-1and type-2 yolk granules are found together in the cyto-plasm of the oocytes (Fig. 7e).

In the Wnal phases of secondary vitellogenesis, theoocyte is surrounded by a vitelline envelope (Fig. 8a, b),also known as a chorion, which protects the egg whileallowing the ovary to pass through the oviduct. During thisphase, follicular cells are completely Xattened and supportthe nutrition of the oocyte (Fig. 8a, b). In the later stage oftype-2 yolk granule development, numerous lipid dropletsand masses of glycogen particles appear in the ooplasm andare present in the mitochondria (Fig. 8c, d).

Immature oocytes are always found in ovaries in stagesII, III and IV gonads, but only in very small proportions.The formation of yolk granules during vitellogenesis is acontinuous process, starting with an endogenous phase andending with an exogenous phase.

Ovaries carry the mature oocytes through a short ovi-duct into the spermatheca. The spermatheca has a thickwall, in which we observe a multi-stratiWed epithelium thatsecretes a substance that helps to maintain the spermato-phores. Spermatophores are distributed between the walland the central column of the spermatheca. This column

Fig. 6 Transmission electron micrographs showing the Wnal phase:primary vitellogenesis of the spider crab Maja brachydactyla.a General view of the oocyte showing type-1 yolk granule; note thebright vesicles on the periphery around the large nucleus. Arrow indi-cates the presence of the nucleus envelope. b Detail of the oocyte

membrane in which micropinocytosis was present. Arrows indicate thepresence of pinocytotic vesicles. c High magniWcation of type-1 yolkgranule and bright vesicles. Fc follicular cells, mv microvilli, N nucle-us, Ne nuclear envelope, Ng nuage, Vb bright vesicle, Y1 type-1 yolkgranule, W wall

123

Mar Biol (2007) 152:383–394 389

and the connective tissue keep the spermatheca vertical(Fig. 9a). At the level of the vagina, the spermatheca issurrounded by a muscular layer that would have extrudedthe oocytes during oviposition. Internally, a cuticular layerenvelops the spermatophore mass (Fig. 9b), while ultra-structural observations show that the mass present in thevagina contains a large quantity of spermatozoids (Fig. 9b,inset). Although oocytes should have entered the vagina inorder to be fertilized, we did not observe any, presumablybecause of the short period of time during which ovulationoccurred.

Discussion

In M. brachydactyla, as in other Brachyuran crabs studied,the ovaries are conWned to the broad, convex cephalotho-rax. They are H shaped, with a central bridge of ovariantissue and a pair of short oviducts connected to the sper-mathecae that join the genital pores on the sternum (c.f.Adiyodi and Subramoniam 1983). In spider crabs, includ-ing M. brachydactyla, the spermatheca—a specialized areaof the genital duct that receives the spermatophores wherespermatozoa are stored—presents the ventral-type form

Fig. 7 Transmission electron micrographs showing formation of the type-2 yolk granule during secondary vitellogenesis of the spider crab Maja brachydactyla. a Electron dense wall of the oocyte showing two somatic cells. b Detail of the secondary vitellogenic oocyte with diVerent types of yolk granules surrounding the follicle cell. c Note the cytoplasm containing numerous mitochondria of diVerent sizes. d Formation of granulate vesicles. e High magniWcation showing type-2 yolk granules. Fc follicular cells, M mitochondria, N nucleus, Vg granulate vesicle, Y2 type-2 yolk granule, W wall

Fig. 8 Transmission electron micrographs showing an advanced phase of oocytes: secondary vitellogenesis in the spider crab Maja brachydactyla. a The space between the oocytes and follicle cells is Wlled with the vitellin envelope or chorion. b The space between the oocytes and follicle cells is Wlled with the Xocculent material that forms the vitellin envelope. c High magniWcation showing a large lipid droplet near the type-2 yolk granule. d Note the presence of glycogen particles near type-2 yolk granule. Fc follicle cells, Gl glycogen, L lipid droplet, M mitochondria, N nucleus, Ve vitelline envelope, Y2 type 2 yolk granule, W wall

123

390 Mar Biol (2007) 152:383–394

previously described by Hartnoll (1968). To our knowl-edge, R. ranina is the only species in which there is nointernal connection between the spermatheca and theoviduct (Minagawa et al. 1993). The fertilization of theoocytes in this species must therefore be external. InM. brachydactyla, as in E. sinensis (Hoestlandt 1948), theovaries do not end at the junction between the spermathecaand the oviduct, as found in other crab species studied.Instead, the ovaries are joined to the spermatheca on theventral side, close to the gonopore, and are then posteriorlyextended to reach the dorsal part of the cephalothorax. InE. sinensis (Hoestlandt 1948), the two lobes run separatelyuntil they reach the end of the cephalothorax. In M. brachy-dactyla, however, the two lobes merge and sink into theabdomen together. This could be a strategy to elongate theovaries in order to accumulate more oocytes and therebyincrease the fecundity of this species.

The spermathecae play a key role in the reproductivebehaviour of majids (Diesel 1991; González-Gurriaránet al. 1998; Sainte-Marie and Sainte-Marie 1998), as theymake it possible for sperm to be stored from one or morecopulations by several males. This allows the subsequentfertilization of consecutive broods without copulation inbetween. In C. opilio and Hyas coarctatus, the dorsalregion of the spermatheca is blind in immature females anddevelops progressively as the crab matures (Lanteigne et al.1996). In M. brachydactyla, González-Gurriarán et al.(1998) showed seasonal patterns of spermatheca Wlling. Atthe beginning of the reproductive cycle (September–Janu-ary), most primiparous females lacked sperm in their sper-mathecae during stages I and II. Their spermathecae werefullest from February to May, coinciding with gonad stagesII and IV, while their contents decreased progressively asthe cycle continued (from June to September). Ovigerousfemales retained sperm in their spemathecae, although lev-els of fullness were slightly lower than in non-ovigerousfemales. Both new and older sperm masses (which were

distinguished by colour and size) were observed in the sper-mathecae. In cases where spermathecae contained multiplemasses, they were arranged in a parallel direction along themajor axis. They were retained in the highly elastic dorsalpart of the receptacle that connects to the vagina. In thesespecies, the vagina is typically a deXated tube with lateralmuscles. In M. brachydatyla, it is through the vagina thatthe oviduct joins the spermathecae to the ovaries. Hartnoll(1968) classiWed this as a ventral-type spermatheca andobserved this type in all the Majid species studied. Thevagina connects to the ventral part of the spermatheca,which is a cuticle layer.

Unlike in other crabs such as P. trituberculatus, inM. brachydactyla no histological diVerences are observedbetween oocytes from diVerent parts of the ovary (Hama-saki et al. 2004). The germinal zone is central in M. brachy-dactyla, as reported in other Majoidea crabs such as Libiniaemarginata (Hinsch and Cone 1969) and C. opilio andH. coarctatus (Lanteigne et al. 1996), and as reviewed intrue crabs by Meusy and Payen (1988) and Pochon-Masson(1994). In the freshwater crab P. dehaani (Ando andMakioka 1999), the ovarian lumen is diVerentiated into twoportions with diVerent functions—the oogenetic pouchesfor oocyte growth and the central ovarian lumen as a pas-sage for the ovulated eggs. In cross sections of the anteriorarea of each ovarian sac, the germinal zones are located atthe necks of well-developed oogenetic pouches radiallyarranged around a very narrow central ovarian lumen.However, these zones were reported to be randomly scat-tered throughout the ovarian epithelium in the posteriorregions of the ovarian sacs. Ando and Makioka (1999)described the formation of oocytes in the freshwater crab asfollows: oogonia from the germinal zone was surroundedby ovarian epithelium forming separated oogenetic pouches;early previtellogenic oocyte left the germinal zone andmoved to the periphery with small somatic interstitial cellsand mature eggs were ovulated from the oogenetic pouches

Fig. 9 Light micrographs showing sections through the spermathecaeof the spider crab Maja brachydactyla. a Transversal section of theupper part showing a general view of the spermathecae. Note thepresence of stratiWed epithelium, free spermatozoid in the lumen andthe central area occupied by a gelatinous material forming the centralcolumn. b Section through a vagina stained with hematoxylin and

eosin to show the muscle lining of the cuticle layer. The lumen is Wlledwith spermatophores. Inset Transmission electron micrographsshowing the structure of the spermatozoids embedded in the spermato-phores. MagniWcations: a = £190; b = £50. Cc central column, Cucuticle, Ep epithelium, Mu muscle, Sph spermatophore, Sz spermato-zoid

123

Mar Biol (2007) 152:383–394 391

into the ovarian lumen. Oogenetic pouches were onlydescribed in the ovaries of P. dehaani. In other true crabspecies studied, such as M. brachydactyla, oogonia origi-nated in the central germinal zone and then migrated to theperiphery, while oocytes matured and were encompassedby their follicular cells (cf. Jensen et al. 1996; Fernández-Vergaz et al. 2000; López-Abellán et al. 2002; Smith et al.2004). Perhaps, the small number of larger eggs producedduring each spawning in P. dehaani could be related to itssimpler ovarian structure with respect to the other crab spe-cies studied.

M. brachydactyla has a complex biological cycle, withvarying habitat requirements and changes in behaviouraccording to the speciWc point in its life history (González-Gurriarán et al. 2002). Moreover, the spider crab undergoesa terminal molt (Sampedro et al. 1999) that divides its lifehistory into stages, with diVerent growth and reproductiveperiods. Gonad maturation starts after the pubertal molt,where females reach sexual maturity (in late summer orearly autumn). In December, specimens with gonads in latestages of development begin to appear and females spawnfor the Wrst time in late winter and early spring. From Apriluntil September a large percentage of ovigerous femalesreach more advanced stages of maturity in preparation forthe next spawning cycle (González-Gurriarrán et al. 1993).These authors determined four stages of maturity in theovaries of M. brachydactyla, while in this study we havecarried out a detailed examination at the morphological andultrastructural levels, placing special emphasis on yolk for-mation. Maturity in M. brachydactyla is a continuous pro-cess. Stage I represents an immature phase and furtherstages in the vitellogenic process lead to the ovary beingmainly composed of fully mature oocytes in stage IV. Aspent phase was reported in another spider crab species,C. opilio (Kon and Honma 1970a; Chiba and Honma 1972;Sainte-Marie and Sainte-Marie 1998), while oosorption inoocytes retained in the ovary of recently spawned goldencrabs (Chaceon fenneri) was observed by Hinsch (1992).However, no spent phase was observed in M. brachydactylafrom the Galician coast. M. brachydactyla from this areawas estimated to have three consecutive broods during theyearly cycle (González-Gurriarrán et al. 1993). In the caseof animals caught oV the Galician coast and studied in cap-tivity, up to Wve fertile broods were observed (Iglesias et al.2002) and the time between hatching and the followingspawning was 3.4 days on average (González-Gurriarránet al. 1998). This consecutive spawning and the short periodbetween larval hatching and the next spawning might indi-cate that when spent phases exist, they tend to be very shortand are not representative of the whole cycle. Furthermore,the number of eggs is usually very high, suggesting that theovary is completely empty at the beginning of each spawn-ing and that atretic oocytes are diYcult to Wnd.

The morphological changes in the nucleolus observedin oocytes of M. brachydactyla show the same patterns asdescribed by Hubert (1972) and Azevedo and Coimbra(1980). During oogonia and the diplotenic oocyte phases,and before synthesis activity has taken place, the nucleo-lus is compact; therefore, its Wbrillar and granular com-ponents are diYcult to distinguish. In previtellogenicoocytes, nuclear segregation phenomena were observedand compact nucleolar masses appeared to be similar to thenucleolar satellites observed in Labidocera aestiva (Blades-Eckelbager and Youngbluth 1984). These masses headdirectly towards the nuclear envelope. During primaryvitellogenesis, the Wrst yolk granules are produced in thecytoplasm and low electron-density spaces appear in thenucleolus, which correspond to nucleolar vacuoles. Asvitellogenesis progresses, these nucleolar vacuoles increasein volume, while at the same time their numbers decrease.When the oocyte reaches secondary vitellogenesis, itsnucleus therefore only presents a single large central vacu-ole. From these observations, it is possible to deduce thatthroughout the vitellogenesis process, there is a continuousexodus of nucleolar material towards the cytoplasm in theperinuclear area, leading to the formation of electron-denseand granulo-Wbrillar masses in the cytoplasm. On accountof their structure and location, these masses are considerednucleolar emissions and are referred to as nucleolus-like-bodies or nuage depending on the terminology utilized.During the oogenesis of M. brachydactyla, there is a grad-ual increase in nuclear emissions, which are most accentu-ated in the previtellogenesis and primary vitellogenesisphases. These emissions become smaller and even disap-pear as secondary vitellogenesis progresses and the oocytecytoplasm is Wlled with vitellogenic granules. Similarresults to the ones described in M. brachydactyla have alsobeen reported in other crustacean species (Orchestia gam-marellus: Zerbib 1980; Centropages typicus: Arnaud et al.1982; L. aestiva: Blades-Eckelbarger and Youngbluth1984). These nucleolar emissions are accompanied by cel-lular hyperplasia: the nucleus and nucleolus increase in vol-ume and in the cytoplasm, the mitochondria change theirmorphology and increase in number, inducing the forma-tion of yolk granules. A similar increase in the number ofmitochondria during vitellogenesis has also been observedin other crustacean species (C. typicus: Arnaud et al. 1982;L. aestiva: Blades-Eckelbarger and Youngbluth 1984).

In M. brachydactyla oocytes, mitochondrial aggrega-tions are often observed near the nucleus. These are typi-cally formed by four or more mitochondria that surround anelectron-dense material similar to the nuage masses whichAndre (1962) called intermitochondrial cement. Ultrastruc-tural studies of mitochondrial aggregations have mainlybeen carried out on amphibians (Clerot 1968; Eddy 1975;Kalt 1973) and teleost Wshes (Clerot 1976). These authors

123

392 Mar Biol (2007) 152:383–394

suggest a nuclear origin for this intermitochondrial cementand consider mitochondrial aggregations to be the result ofthe multiplication of mitochondria.

The formation of yolk granules in M. brachydactylashows the same patterns described in other decapod species(Homarus americanus: Kessel 1968; L. emarginata: Hinschand Cone 1969; Cancer pagurus: Eurenius 1973; E. sinen-sis: Dhainaut and De Leersnyder 1976; Astacus leptodacty-lus: Zerbib 1979). Vitellogenesis is divided into twosuccessive phases that clearly diVerentiate the oocytegrowth period. Primary vitellogenesis is characterized byendogen protein yolk accumulation, while in secondaryvitellogenesis extracellular protein substances are incorpo-rated through pinocytosis.

In M. brachydactyla, primary vitellogenesis—the endog-enous phase—is a continuous process, corresponding toyolk synthesis involving the participation of cytoplasmicstructures such as the Golgi complex and mitochondria.The participation of the endoplasmic reticulum cannot beruled out either. The Golgi complex, which exhibits a lowlevel of development during the previtellogenic phase, con-siderably increases in size during vitellogenesis. In this pro-cess, Golgian vesicles merge—with each other and withother endoplasmic vesicles—to form the type-1 yolk gran-ules. Kessel (1968) and Anderson (1964) consider that theGolgi complex plays an important role in stocking compo-nents and participating in the synthesis of yolk granulemembranes. Golgian vesicles that contain hydrolyticenzymes may contribute to the process of autophagia,which may be related to the formation of bright vesiclesin oocyte cytoplasm during the primary vitellogenesis ofM. brachydactyla. This has been observed in C. typicus(Arnaud et al. 1982) and in L. aestiva (Blades-Eckelbargerand Youngbluth 1984). These bright vesicles could originatefrom autophagosomes, since transitional forms betweenmitochondria and bright vesicles are observed. The partici-pation of the Golgi complex in the formation of yolk

granules in crustaceans has been studied by several otherauthors including Hinsch and Cone (1969), Eurenius(1973), Dhainaut and De Leersnyder (1976), Beams andKessel (1980) and Arnaud et al. (1982).

In M. brachydactyla oocytes, the formation of pinocyto-sis vesicles in oolema in the vitellogenesis exogen phase iscomplemented by an increase in the number of microvilliand the development of the vitelline envelope. Further-more, morphological changes are observed in the follicularcells that surround the oocytes. These enlarge and theircytoplasms acquire the appearance of a typical secretor cell,since the endoplasmic reticulum is well developed. Thepinocytosis vesicles, which contain amorphous materiallocated in the cortical zone of their ooplasm, merge primar-ily with the endogen yolk granules, giving rise to a hetero-synthetic yolk and type-2 yolk granules. The exogenousorigins of several components of these yolk granules andtheir incorporation through the process of pinocytosis havebeen described at the morphological and histochemical lev-els in several crustacean species. The Wrst of these studiesfocused on the spider crab L. emarginata and was con-ducted by Beams and Kessel (1963) and Hinsch and Cone(1969).

The main objective of this study was to determine thepossible mechanisms involved in yolk granule formationduring vitellogenesis in M. brachydactyla. These aresummarized in Fig. 10. Vitellogenesis starts with anendogenous phase in which vesicles have multiple origins,although most of them develop directly from the mitochondriaand Golgi complex. This process ends with an exogenousphase in which a series of substances (principally lipo-proteins) are incorporated into the ooplasma by micropi-nocytosis. The participation of mitochondria in yolkformation has been considered in several copepods spe-cies. In this paper, we have conWrmed that mitochondriaare the origin of the bright vesicles throughout the auto-phagia process and that type-1 and type-2 yolk granules

Fig. 10 Schematic diagram illustrating the hypothetical sequence of events during the process of yolk formation: the spider crab Maja brachydactyla. Fc follicle cells, G Golgi complex, Ly lysosomes, M mitochondria, mv microvilli, N nucleus, Ne nuclear envelope, ng nuage, Nu nucleolus, Ol oolemma, St nuclear satellites, Vb bright vesicle, Ve vitelline envelope, VG Golgi vesicle, Vg granulate vesicle, Vp pinocytic vesicle, Y1 type-1 yolk granule, Y2 type-2 yolk granule, W wall

123

Mar Biol (2007) 152:383–394 393

are formed by these vesicles during primary and second-ary vitellogenesis.

Acknowledgements Support for this work was provided by severalprojects from the Consellería de Pesca, Marisqueo e Acuicultura of theXunta de Galicia (PE608A 94/2-0 among others), the SpanishDGICYT project (PB94-0501), the Spanish CICYT Projects (MAR97-0446 and REN2001-0729) and the Centre de Referència en Aqüicul-tura of the Catalan Government.

References

Adiyodi RG, Subramoniam T (1983) Arthropoda—Crustacea. In:Adiyodi KG, Adiyodi RG (eds) Reproductive biology of inverte-brates, vol I: oogenesis, oviposition, and oosorption. Wiley, NewYork, pp 443–495

Anderson E (1964) Oocyte diVerentiation and vitellogenesis in theroach Periplaneta americana. J Cell Biol 20:131–155

Ando H, Makioka T (1999) Structure of the ovary and mode of oogene-sis in a freshwater crab Potamon dehaani. J Morphol 239:107–114

Andre J (1962) Contribution à la connaissance du chondriome. Ètudede ses modiWcacions ultraestructurales pendant la spermato-genèse. J Ultrastruct Res Suppl 3:1–185

Arnaud J, Brunet M, Mazza J (1982) Ètude de l’ovogenèse chez Centro-pages typicus. (Copepoda, Calanoida). Reprod Nutr Dév 22(3):537–555

Azevedo C, Coimbra A (1980) Evolution of nucleoli in the course ofoogenesis in a viviparous teleost (Xiphophorus helleri). Biol Cell38:43–48

Beams HW, Kessel RG (1963) Electron microscope studies on devel-oping crayWsh oocytes with special references to the origin ofyolk. J Cell Biol 18:621–649

Beams HW, Kessel RG (1980) Ultrastructure and vitellogenesis in the oo-cyte of the crustacean, Oniscus asellus. J Submicrosc Cytol 12:17–27

Beninger PG, Lanteigne C, Elner RW (1993) Reproductive processesrevealed by spermatophore dehiscence experiments and by histol-ogy, ultrastructure, and histochemistry of the female reproductivesystem in the snow crab Chionoecetes opilio (O. Fabricius).J Crustacean Biol 13(1):1–16

Blades-Eckelbarger PI, Youngbluth MJ (1984) The ultrastructure ofoogenesis and yolk formation in Labidocera aestiva (Copepoda:Calanoida). J Morphol 179:33–46

Brosnan DM (1981) Studies on the biology, ecology and Wshery of thespider crab Maia squinado Herbst (1768) of the west coast or Ire-land. PhD thesis, University College, Galway, pp 133

Brownell WN, Provenzano Jr AJ, Matinez M (1977) Culture of theWest Indian spider crab (Mithrax spinosissimus) at Los Roques,Venezuela. Proc World Maricult Soc 8:157–168

Chiba A, Honma Y (1972) Studies on gonad maturity in some marineinvertebrates. 7. Seasonal changes in the ovary of the lined shorecrab. Bull Jpn Soc Sci Fish 38:323–327

Clerot JC (1968) Mise en evidence par cytochimie ultrastructurale del’émision de protéines par le noyau d’auxocytes de Batraciens.J Microscopie 7:973–992

Clerot JC (1976) Les groupements mitochondriaux des cellules germi-nales des poissons Téléostéens Cyprinidés. I. Ètude ultrastructu-rale. J Ultrastruct Res 54:461–475

Clotilde-Ba FL, Diatta Y, Capapé C (1997) Observations sur huitespèces comestibles de crustacés décapodes des eaux sénégalais-es (Afrique de l’ouest). Bol Mus Mun Funchal 49(282):171–187

Dhainaut A, De Leersnyder M (1976) Etude cytochimique et ultra-structurale de l’evolution ovocitaire du crabe Eriocheir sinensis.I. Ovogenèse naturelle. Arch Biol 87:261–282

Diesel R (1991) Sperm competition and the evolution of mating behav-ior in Brachyura, with special reference to spider crabs (Deca-poda: Majidae). In: Martin RGB, Martin JW (eds) Crustaceansexual biology. University Press, New York, pp 145–163

Eddy EM (1975) Germ plasm and the diVerentiation of the germ cellline. In: Bourne GH, Danielli JF (eds) International review ofcytology, vol 43. Academic, New York, pp 229–280

Edwards E (1979) Preliminary results of an investigation on a newWshery for spider crabs (Maia squinado) along the south coast ofEngland. ICES ShellWsh Committee CM 1979/K14 9pp

Elner RW, Beninger PG (1992) The reproductive biology of snowcrab, Chionoecetes opilio: a synthesis of recent contributions. AmZool 32:524–533

Eurenius L (1973) An electron microscope study on the developingoocytes of the crab Cancer pagurus L. with special reference toyolk formation (Crustacea). Z Morph Tiere 75:243–254

Fernández-Vergaz V, López Abellán JL, Balguerías E (2000) Morpho-metric, functional and sexual maturity of the deep-sea red crabChaceon aVinis inhabiting Canary Island waters: chronology ofmaturation. Mar Ecol Prog Ser 204:169–178

García Cabrera C (1972) Estudio biológico pesquero del Centollo enaguas del litoral sahariano. Bol Inst Esp Oceanogr 156:1–36

García-Flórez L, Fernández-Rueda P (2000) Reproductive biology ofspider crab females (Maja brachydactyla) oV the coast of Asturias(north-west Spain). J Mar Biol Assoc UK 80:1071–1076

Goldstein J, Newbury D, Joy D, Lyman Ch, Echlin P, Litshin E, Saw-yer L, Michael J (2003) Scanning electron microscopy and x-raymicroanalysis, 3rd edn. Kluwer Academic/Plenum Publishers,New York, pp 647–673

González-Gurriarán E, Fernández L, Freire J, Muiño R (1993) Repro-duction of the spider crab Maja squinado (Brachyura: Majidae) inthe southern Galician coast (NW Spain). ICES:15pp

González-Gurriarán E, Freire J, Parapar J, Sampedro MP, Urcera M(1995) Growth at moult and moulting seasonality of the spidercrab, Maja squinado (Herbst) (Decapoda: Majidae) in experimen-tal conditions: implications for juvenile life history. J Exp MarBiol Ecol 189:183–203

González-Gurriarán E, Fernández L, Freire J, Muiño R (1998) Matingand role of seminal receptacles in the reproductive biology of thespider crab Maja squinado (Decapoda: Majidae). J Exp Mar BiolEcol 220:269–285

González-Gurriarán E, Freire J, Bernárdez C (2002) Migratory pat-terns of female spider crabs Maja squinado detected using elec-tronic tags and telemetry. J Crustacean Biol 22(1):91–97

Hamasaki K, Imai H, Akiyama N, Fukunaga K (2004) Ovarian devel-opment and induced oviposition of the overwintering swimmingcrab Portunus trituberculatus (Brachyura: Portunidae) reared inthe laboratory. Fish Sci 70:988–995

Hartnoll RG (1965) The biology of spider crabs: a comparison of Brit-ish and Jamaican species. Crustaceana 9:1–16

Hartnoll RG (1968) Morphology of the genital ducts in female crabs.J Linn Soc (Zool) 47:279–300

Hartnoll RG (1969) “Mating in the brachyura.” Crustaceana 16:161–181Hinsch GW (1992) Ovary of the golden crab, Chaceon fenneri: post-

spawning and oosorption. J Morphol 211:1–6Hinsch GW, Cone V (1969) Ultrastructural observations of vitellogen-

esis in the spider crab, Libinia emarginata L. J Cell Biol 40:336–342

Hoestlandt H (1948) Recherches sur la biologie de l’Eriocheir sinensis H.Milne-Edwards (crustacé brachyoure). Ann Inst Ocean 24:1–116

Hubert J (1972) Ultrastructure du nucleoli au cours du stade diplotènedans les ovocytes du Lézard Lacerta vivipara. Jacquin Arch AnatMicrosc 61(4):325–338

Iglesias J, Sánchez FJ, Moxica C, Fuentes L, Otero JJ, Pérez JL (2002)Datos preliminares sobre el cultivo de larvas y juveniles de cento-lla Maja squinado Herbst, 1788 en el Centro OceanográWco de

123

394 Mar Biol (2007) 152:383–394

Vigo del Instituto Español de Oceanografía. Bol Inst Esp Ocea-nogr 18(1–4):25–30

Jensen PC, Orensanz JM, Amstrong DA (1996) Structure of the femalereproductive tract in the dungeness crab (Cancer magister) andimplications for the mating system. Biol Bull 190:336–349

Kalt MR (1973) Ultrastructural observations on the germ line of Xeno-pus laevis. Zeits Zellforsch 138:41–62

Karnovsky MJ (1965) A formaldehyde–glutaldehyde Wxative of highosmolarity for use in electron microscopy. J Cell Biol 27:137–138

Kergariou G (1971) L’araignee de mer, Maia squinado L., sur le lit-toral de Bretagne. Bull Inst Pêches Marit 205:11–19

Kergariou G (1975) Contribution à l’étude de la reproduction de l’ ara-ignée de mer, (Maja squinado H.). ICES, ShellWsh and BenthosCommittee CM 1975/K:34 8pp

Kessel RG (1968) Fine structure of annulate lamellae. J Cell Biol36:658–664

Kessel RG (1968) Mechanism of protein yolk synthesis and depositionin Crustacean oocytes. Z Zellfrorsch Mikrosk Anat 89:17–38

Kon T, Honma Y (1970a) Studies on the maturity of the gonad in somemarine invertebrates-III. Seasonal changes in the ovary of the tan-ner crab. Bull Jpn Soc Sci Fish 36(10):1021–1027

Kon T, Honma Y (1970b) Studies on the maturity of the gonad in somemarine invertebrates -IV. Seasonal changes in the testes of thetanner crab. Bull Jpn Soc Sci Fish 36(10):1028–1033

Lanteigne C, Beninger PG, Gionet C (1996) Ontogeny of female pri-mary sexual characters in the majid crabs Chionoecetes opilio andHyas coarctatus. J Crustacean Biol 16(3):501–514

Le Foll D (1993) Biologie et exploitation de l’araignée de mer Majasquinado Herbst en Manche Ouest. PhD thesis, Université deBretagne Occidentale, IFREMER, 524pp

López-Abellán LJ, Balguerías E, Fernández-Vergaz V (2002) Life his-tory characteristics of the deep-sea crab Chaceon aYns popula-tion oV Tenerife (Canary Islands). Fish Res 58:231–239

Martoja R, Martoja M (1967) Initiations aux techniques de l’histologieanimale. Masson et Cie, Paris, pp 350

Meusy JJ, Payen GG (1988) Female reproduction in Malacostracancrustacean. Zool Sci 5:217–265

Meyer CG (1993) The biology and Wshery of the spider crab (Majasquinado) around Jersey (Channel Islands).PhD thesis, Universityof Plymouth, Plymouth, 116pp

Minagawa M, Chiu JR, Kudo M, Ito E, Takashima F (1993) Femalereproductive biology and oocyte development of the red frogcrab, Ranina ranina, oV Hachijojima, Izu Islands, Japan. Mar Biol115:613–623

Newman V (1998) A review of the Maja squinado (Crustacea: Deca-poda: Brachyura) species-complex with a key to the easternAtlantic and Mediterranean species of the genus. J Natl Hist32:1667–1684

Pochon-Masson J (1994) Les Gamétogenèses. In: Forest J (ed) Traitéde Zoologie Pierre-P Grassé, vol VII: Crustacés, Fascicule I:Morphologie, Physiologie, Reproduction, Systématique. Masson,Paris-Milan-Barcelona, pp 727–783

Pradeille-Rouquette M (1975) Étude de la fonction de reproductionchez les femelles du crabe Pachygrapsus marmoratus (F.) et dediVérents facteurs qui lui sont liés (I). Cah Biol Mar 17:387–403

Reynolds ES (1963) The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J Cell Biol 17:208–212

Rodhouse DM (1984) Experimental Wshing for the spider crab, Maiasquinado: sea and laboratory trials. J Mar Biol Assoc UK 64:251–259

Ryan EP (1967) Structure and function of the reproductive system ofthe crab Portunus sanguinolentus (Herbst) (Brachyura: Portuni-dae) II. The female system. Proceedings of the Symposium onCrustacea. Mar Biol Assoc India Ernakulam. Part II: 522–544

Sampedro MP, González-Gurriarán E, Freire J, Muiño R (1999) Mor-phometry and sexual maturity in the spider crab Maja squinado(Decapoda: Majidae) in Galicia, Spain. J Crustacean Biol19(3):578–592

Sainte-Marie G, Sainte-Marie B (1998) Morphology of the sperma-theca, oviduct, intermediate chamber, and vagina of the adultsnow crab (Chionoecetes opilio). Can J Fish Aquat Sci 76:1589–1604

Smith KD, Potter IC, Hesp SA (2004) Comparisons between the repro-ductive biology of females of two species of deep sea crabs thatlive in diVerent water depths. J ShellWsh Res 23(3):887–896

Spurr AR (1969) A low viscosity epoxy resin embedding medium forelectron microscopy. J Ultrastruct Res 81:341–350

Stevcic Z (1967) A short outline of the biology of the spinous spidercrab. Bull Sci Conseil Acad RSF Yugoslavie Sect A 12:313–314

Stevcic Z (1977) Contribution a la connaissance de la preoduction del’araigné de mer (Maja squinado). Rapp Comm int Mer Medit24:177–178

Zerbib C (1979) Etude ultrastructurale de l’ovocyte en vitellogenèsechez les Ecrevisses Astacus astacus et Astacus leptodactilus. IntJ Invert Reprod 1:289–295

Zerbib C (1980) Ultrastructural observation of oogenesis in theCrustacea Amphipoda Orchestia gammarelus (Pallas). TissueCell 12:47–62

123