acrosome reaction of cancer setosus molina, 1782 (decapoda: brachyura)

JOURNAL OF CRUSTACEAN BIOLOGY, 32(2), 181-189, 2012

ACROSOME REACTION OF CANCER SETOSUS MOLINA, 1782(DECAPODA: BRACHYURA)

Enrique Dupré 1,∗, Merari Goldstein 1, and Sergio Palma 2

1 Universidad Católica del Norte, Facultad de Ciencias del Mar, P.O. Box 117, Coquimbo, Chile2 Pontificia Universidad Católica de Valparaíso, Escuela de Ciencias del Mar, P.O. Box 1020, Valparaíso, Chile

A B S T R A C T

We describe the structural and ultrastructural sequence of events that occurs during the acrosome reaction process in Cancer setosusMolina, 1782. No change is observed in spermatophores extracted from the male vas deferens and incubated in different concentrations ofseawater; the spermatozoa are not released nor do they experience the acrosome reaction. Only spermatozoa from the masses in seminalreceptacles of females undergo acrosome reaction. The acrosome reaction begins with an elongation of the acrosomal vesicle after thespermatozoa are incubated for 15 to 20 min in seawater. During this elongation, the material in the perforatorial chamber is extruded andspread on the outer side walls of the acrosome forming a ring-shaped opening at the anterior region of the acrosome. The ring-shapedmaterial moves to the posterior region, thus increasing the diameter of the anterior opening of the acrosome. In the following stages, anacrosomal filament is extruded and extends beyond the anterior end of the acrosome. The distal end is enlarged and then divided into threethin terminal extensions.

KEY WORDS: acrosomal filament, Cancer setosus, seminal receptacle, spermatheca, spermatozoon, vasdeferens

DOI: 10.1163/193724011X615505

INTRODUCTION

During mating, male decapod crustaceans transfer spermato-zoa to the female via spermatophores. The spermatophoresare pedunculated for Anomura and non-pedunculated inBrachyura, Achelata, Astacidea, and Caridea. Brachyuranspecies present spherical spermatophores embedded in sem-inal fluid that are secreted by the proximal vas deferens.The spermatophores vary in size, contain from five to sev-eral hundred spermatozoa (Simeó et al., 2010), and aretransferred to the female together with the seminal fluidthrough the gonopods (modified pleopods). These modifiedappendages allow transfer of spermatophores directly to thefemale spermatheca, which is joined to the oviduct. Thespermatheca or seminal receptacle is a specialized structurethat receives the seminal fluid containing spermatophoresand permits the release of the spermatozoa from the sper-matophore; the spermatozoa can remain stored therein forlong periods without losing viability. Females of some crabspecies, e.g., Maja squinado (Herbst, 1788) and Menippemercenaria (Say, 1818), are known to experience betweenfour and 10 successive spawnings in the laboratory with-out having to copulate again (Cheung, 1968; Morgan et al.,1983), whereas others are able to store viable spermatozoafor periods of up to one year (Jivoff et al., 2007; Ander-son and Epifanio, 2010). An inseminated female can haveas many as eight batches of fertilized eggs without the inter-vention of any male. It is estimated that most females spawnthree successive times during an annual cycle (González-Gurriarán et al., 1998).

∗ Corresponding author; e-mail: [email protected]

Spermatozoa are released from the spermatophores in-side the spermatheca of the females. Uma and Subramo-niam (1979) indicate that the entry of low-molecular-weightsubstances through the vagina dissolves the spermatophorecover. Ryan (1967), Adiyodi and Anilkumar (1988), andDiesel (1989) report that the glandular epithelia of spermath-eca release enzymes that dissolve the spermatophores. How-ever, according to Beninger et al. (1988), the entry of seawa-ter through the vagina into the spermatheca may be enoughto cause the spermatophore to rupture and release sperm.

During ovulation, the spermatozoa stored in the spermath-eca are released into the oviduct and fertilize oöcytes in-side the oviduct. The spawned oöcytes then adhere to thepleopods as zygotes. To fertilize the oöcytes, the spermato-zoa must undergo an acrosome reaction that allows them tointeract with and penetrate the oöcyte. Reduced spermatozoafunction decreases fertilization efficiency, lowering recruit-ment for aquaculture as well as population replenishment innatural environments (Zhang et al., 2010). The acrosome re-action may be a good indicator of impaired function in thespermatozoa (Zhang et al., 2010).

The morphology of crustacean spermatozoa differs ac-cording to the corresponding taxonomic group. This mor-phological diversity determines specific reproductive andfertilization strategies (Pochon-Masson, 1983; Demestre etal., 1997). Brachyuran spermatozoa are generally character-ized by the presence of multiple arms radiating from thecell body and a complex spherical acrosome (Brown, 1966;Reger, 1970; Jamieson, 1989a, b, 1991; Medina, 1992; Med-

© The Crustacean Society, 2012. Published by Koninklijke Brill NV, Leiden DOI:10.1163/193724011X615505

182 JOURNAL OF CRUSTACEAN BIOLOGY, VOL. 32, NO. 2, 2012

ina and Rodríguez, 1992) that occupies most of the cell body.Although there have been many studies on the structure andultrastructure of brachyuran spermatozoa (Jamieson, 1991;Jamieson and Tudge, 2000; Tudge, 2009), only some ofthem have contributed to the understanding of the processof acrosome reaction and the way the spermatozoa penetratethe egg coat (Pochon-Masson, 1968; Goudeau, 1982; Med-ina, 1992; Kang et al., 2009; Zhang et al., 2010). Most ofthe studies on the spermatozoa structure of the genus Can-cer do not provide details that contribute to the understand-ing of the acrosome reaction process (Langreth, 1965, 1969;Brown, 1966; Pochon-Masson, 1968; Jamieson et al., 1997),although some do (Demestre and Fortuño, 1992; Moriyasuet al., 2000).

The acrosome reaction of invertebrates is initiated byionic, biochemical, or osmotic changes of the environmentor interactions between the spermatozoa and the oöcytecoat (Schackmann, 1989). The hairy crab, Cancer setosusMolina, 1782 is one of economic importance to the artisanalfishery along the southeastern Pacific coast. This speciesis endemic to the Pacific coast of South America from theEquator (0°S) to Taitao Peninsula, Chile (47°S). Cancersetosus is a benthic predator that inhabits the intertidalzone and the subtidal zone to 45 m deep. Despite itsimportance in regional artisanal fisheries, there are fewstudies on the reproduction of the hairy crab (Goldstein andDupré, 2010). The present study determines, for the firsttime in this species, the morphological changes experiencedby the sperm cells after being removed from the femalespermatheca and being placed in seawater. [Cancer setosusMolina, 1782 is probably a synonym of Romaleon polyodon(Poppig, 1836); see Ng et al. (2008) for details. Its use hereis tolerated only because of regional practice.]

MATERIALS AND METHODS

Animals

Mature male and female specimens of C. setosus werecaught in La Herradura Bay (29°58′S-71°22′W) by diversand kept in a 60-L tank with filtered seawater in an open-circuit aquarium. Before being dissected, each specimen wasanesthetized on ice for at least 30 min, until not respondingto external stimuli.

Sperm Collection

Spermatophores from the terminal regions of the vas defer-ens (males) and spermathecae (females) were removed andplaced in a Petri dish with seawater. The semen from the vasdeferens was placed in filtered seawater (FSW; 35 g/L) atdifferent dilutions (100-80-20%) to attempt osmotic ruptureof the spermatophores and subsequent release of the sper-matozoa. To obtain the spermatozoa from spermathecae, thespermatic mass was placed in FSW for 15 to 20 min andlater dyed with 1% methylene blue for light microscopy orprocessed for scanning electron microscopy.

Microscopy

For examination under light microscopy, the dyed sperma-tozoa were mounted on a slide and analyzed at a magnifi-cation range of 40× to 100×. The different stages of theacrosome reaction were registered with a digital camera, and

the morphometric parameters were measured with the soft-ware Image Pro-Plus 4.5. For scanning electron microscopy,spermatozoa from female spermathecae in different stagesof the acrosome reaction were fixed for 24 h in a solutionof 3% glutaraldehyde in FSW and washed three times for10 min each in FSW at 4°C, dehydrated through an ascend-ing ethanol series, point-critical dried with CO2 in a Samdri-780A, sputter-coated with gold in a JEOL JFC-1100, andexamined on a JEOL JSM-T300 scanning electron micro-scope.

RESULTS

Spermatophores from Spermathecae and Vas Deferens

Spherical spermatophores containing five to 30 spermato-zoa from vas deferens were observed (Fig. 1). After plac-ing the spermatophores in seawater for 20 min, however,the rupture or disintegration of the spermatophore wall wasnot obtained. In some of these spermatophores, the shapeof the spermatozoa was found to change within the sper-matophores, but the spermatophore wall remained intact.

Contrary to that which was previously observed in males,no spermatophores were observed sperm mass from the fe-male spermathecae, although a high number of free sperma-tozoa were embedded in a white gelatinous matrix. Whenthe spermatozoa were placed in seawater, only those fromthe spermathecae of the females underwent acrosome reac-tion.

Sperm Morphology

The spermatozoon is comprised of two different morpho-logical regions: the spheroidal acrosome in the anterior re-gion and the cup-shaped nucleus in the posterior region. Thespherical body of the spermatozoa (diameter: 3.5 ± 0.4 μm;n = 23) is due mainly to the spherical acrosome (diameter:3.4 ± 0.9 μm; n = 23). The nucleus surrounds the basalor posterior half of the acrosome (as evidenced by staining).The nucleus is a cup-shaped structure that extends from theequatorial region to the posterior region, which has a wrin-kled or striated surface. The nucleus is significantly thickeraround the equatorial region of the acrosome, forming an ex-

Fig. 1. Spermatophores from the male vas deferens in Cancer setosus withdifferent sizes.

DUPRÉ ET AL.: ACROSOME REACTION OF THE CRAB CANCER SETOSUS 183

Fig. 2. Scanning electron microscopy view of spermatozoa obtained from female spermathecae of Cancer setosus. A, Spermatic mass; B, Lateral view; C,Lateral and polar view; D, Anterior view; E, Posterior view. Acrosome (a); nucleus (n); operculum (o) observed as a depression on apical area of acrosome.

tension that surrounds it (Fig. 1A); from the lateral view, thisappears as two arms (Figs. 2A, 3A). The anterior region ofthe acrosome shows an apical cap or disc-shaped operculumthat is slightly curved with a central depression (Fig. 2A-D),covering the distal end of the acrosome tube. The perforato-rial chamber, which is observed inside the acrosome, aver-ages 2 μm in length and 0.69 μm in width and extends fromthe operculum to the posterior area of the nucleus (Fig. 3A-D). In spermatozoa extracted from the spermatheca, the op-erculum is concave or has a clearly pronounced pit (diame-ter: 0.76 μm) in the center (Figs. 1B-C, 2D).

Acrosome Reaction Stages

After being incubated for 15 to 20 min in filtered seawater,the spermatozoa undergo a dramatic change of shape. Thischange is divided into five sequential steps, as detailed inFigs. 3, 4 and 5.

Stage 1: The spermatozoa shows the first evidence of theacrosome reaction, an anterior elongation of the acrosome,changing from spherical to pear-shaped. The equatorial zoneof the nucleus begins to move and concentrate (Fig. 3C), atthe back of the spermatozoa body (Figs. 3C-E, 4A-D).

Stage 2: The elongation of the acrosomal vesicle contin-ues until forming an anterior opening through which mate-rial is extruded from inside the acrosome and dispersedaround the outside of the acrosomal wall. This extrudedmaterial forms a ring around the opening. The nucleus con-

tinues the compaction in the back of the sperm body (Figs.3D, 4B).

Stage 3: The ring of extruded material moves throughthe outside walls of the acrosome to the posterior region,generating a larger-diameter opening in the anterior regionof the acrosome (Figs. 2E, 3D-F, 4C).

Stage 4: During this stage, the perforatorial chamber isviolently extruded, forming an acrosomal filament that isprojected beyond the anterior end of the acrosome in thecenter of the anterior opening. An arrowhead-shape structureoccurs at the distal end of the acrosomal filament (Figs.3F-G, 4D-H). The extruded material then continues movingover the outer wall of the acrosome and the nucleus isconcentrated in the posterior region of the acrosome (Figs.3G, 4H-I).

Stage 5: The most notable event of this stage is thetransformation of the distal end of the acrosomal filamentfrom an arrowhead-shaped structure to a trident-shapedstructure. The acrosome wall becomes thinner and only theoutermost part of the acrosome remains (Figs. 3H, 4J, 5F).

DISCUSSION

In species with internal fertilization such as C. setosus,the spermatophores are stored in the spermatheca of thefemale. However, the fertilization process requires thatspermatozoa be released from the acellular sheath of the


Fig. 3. Light microscopy view of different stages of acrosome reaction (AR) of Cancer setosus spermatozoa from female spermathecae stained withmethylene blue. A (unstained) and B, Side views of sperm without AR, showing spherical acrosomal vesicle (a), perforatorial chamber (at), and nucleusas an equatorial extension (n); C, Stage one: note anterior elongation (r) generated by extrusion of material from perforatorial chamber (at); D, Stage two:extruded material forms ring around anterior part of acrosome (r); E, Stage three: outer ring (r) moves toward back of acrosome and anterior openingbecomes wider than in previous stage; the perforatorial chamber (at) is still noticeable, and nucleus (n) starts to condense; F, Stage four (onset): materialinside perforatorial chamber extruded to form acrosomal filament (af), and acrosomal wall (aw) becomes thinner with only outermost part of acrosomeremaining as semi-transparent wall; G, Stage four: outermost wall of acrosome disappears and acrosomal filament (af) appears as arrowhead-like structureat distal end; H, Stage five: distal end of acrosomal filament transformed from arrowhead-shaped structure to trident-shaped structure in lateral view.

spermatophores (Hinsch, 1986; Diesel, 1989). This wasobserved in C. setosus, and the spermatozoa mass fromthe spermathecae of the females contained a large amountof spermatozoa embedded in a gelatinous matrix withoutthe presence of spermatophores or spermatophore walls.The disintegration of the spermatophores may be explainedby the action of secretions or “enzymes” released fromthe glandular epithelium of the spermathecae, as shownin different species (Ryan, 1967; Adiyodi and Anilkumar,1988; Alfaro et al., 2003, 2007). In other species such asChionoecetes opilio (Fabricius, 1788) (Beninger et al., 1988)and Inachus phalangium (Fabricius, 1775) (Diesel, 1989),

spermatozoa are released from the spermatophores by theaction of seawater entering the spermathecae through thevagina, and the glycoprotein secreted by the epitheliumof spermathecae helps control the bacterial flora (Beningeret al., 1993). However, the releasing of spermatozoa fromspermatophores by seawater is not supported by our results,as we never observed the dissolution of the spermatophoreswhen treated with seawater. On the other hand, Adiyodi andAdiyodi (1975) suggested that in addition to the secretionsproduced by the spermatheca, secretions introduced by themale during insemination would be useful for nutrition andpreservation of spermatozoa within the spermatheca.


Fig. 4. Scanning electron microscopy view of different stages of acrosome reaction (AR) of spermatozoa from spermathecae of Cancer setosus. A, Spermat the beginning of the AR, observe apical depression (o) and slightly condensed nucleus (n); B, Acrosomal elongation continues and extruded materialforms a ring (an) around anterior opening of acrosome; C, Ring of extruded material moves through outside wall of acrosome, generating a larger-diameteropening in anterior region, (r) ring-shaped material, (af) acrosomal filament in its initial stage; D, E, F, Material located inside perforatorial chamber extruded,forming acrosomal filament (af); G, Ring-shaped structure (r) forms large opening at anterior end of acrosome and arrowhead-shaped structure at distal endof acrosomal filament (af); H and I, Condensed nucleus (n) and acrosomal filament (af); J, Distal end of acrosomal filament forms trident-shaped structurein lateral view.


Fig. 5. Diagrams of acrosome reaction of Cancer setosus. A, Spermwithout acrosome reaction; B, Stage one: anterior elongation of acrosome;C, Stage two: everted material of subacrosomal region forms ring aroundopening of anterior acrosome; D, Stage three: perforatorium extrudedthrough anterior acrosome opening; E, Stage four: perforatorium extendedinto acrosomal filament; F, Stage five: distal end of acrosomal filamentexpands into trident-like structure in lateral view.

With respect to the spermatophores from the vas defer-ens, our results suggest that the presence of a secretion (per-haps an enzyme) in the spermatheca is necessary to breakdown the wall of the spermatophore and release the sperm,as demonstrated by the mass of spermatozoa without sper-matophores found in the spermathecae.

Spermatozoa Morphology

Most Brachyura spermatozoa consist of a spherical acro-somal complex with a perforatorial chamber inside, a sub-acrosomal region, and three or more arms radiating from thecup-shaped nucleus. The acrosome is composed of severalconcentric layers showing varying electron density undertransmission electron microscopy. An external, low-densitylayer forms the main portion of the acrosome and cov-ers another higher-density layer that forms the perforato-rial chamber, which contains many microtubules-like packedin a membrane (Hinsch, 1988). This description coincideswith the morphology of the spermatozoa of Cancer bore-alis Stimpson, 1859 (Langreth, 1965, 1969; Benhalima etal., 2002), Cancer pagurus Linnaeus, 1758 (Jamieson et al.,1997), and, in general, also with the morphology of C. se-tosus. However there are many other species as Latreillop-sis gracilipes Guinot and Richer de Forges, 1981 (Jamieson,1994; Jamieson and Tudge, 2000); Geryon fenneri Manningand Holthuis, 1984; Geryon quinquedens Smith, 1879 (Hin-sch, 1988); Uca tangeri (Eydoux, 1835) (Medina, 1992);Eriocheri sinensis Milne-Edwards, 1853 (Kang et al., 2009)and, Scylla serrata (Forskål, 1775) (Zhang et al., 2010) thatcoincide with the species of Cancer.

The spermatozoa of some species show three or moredistinct nuclear arms radiating from the body and sometimescontaining microtubules. However, this characteristic is notconsistent with the spermatozoa of C. setosus, which have,rather than arms, a single continuous projection around theequatorial region of the acrosome that contains the nuclearmaterial. The lack of arms has also been observed for

Dendrobranchiata such as Aristeus antennatus (Risso, 1816)(Demestre et al., 1997), Peisos petrunkevitchi Burkenroad,1945 (Scelzo and Medina, 2004), and Petalomera lateralis(Gray, 1831) (Jamieson, 1990). Medina (1992) suggests thatthe arms are important for the adherence of spermatozoa tothe egg coat during fertilization. Therefore, the spermatozoaof C. setosus might use a different strategy for adhering tothe oöcyte.

Acrosome Reaction

Oöcyte fertilization requires that the spermatozoa undergoan acrosome reaction allowing the spermatozoa to interactwith and penetrate the oöcyte. Because reduced spermato-zoa function decreases fertilization efficiency, it also low-ers recruitment for aquaculture as well as population replen-ishment in natural environments. Acrosome reaction is po-tentially a good indicator of impaired spermatozoa function(Zhang et al., 2010).

The acrosome reaction consists of an initial eversion ofthe acrosomal material and a secondary eversion of the mate-rial found within the perforatorial chamber (Jamieson et al.,1997; Kang et al., 2009; Simeó et al., 2010; Zhang et al.,2010). These two phases of the acrosome reaction were alsoobserved in C. setosus, with the first phase corresponding tothe eversion of the inner acrosomal material to form a ring ofextruded material in the anterior zone of the acrosome. Thisleft a front opening that increases in diameter as the acro-somal material is extruded. The second phase comprises theexplosive extrusion of the acrosomal filament. The filamentextrusion could be generated by tubules located inside thetube, as suggested by Hinsch (1969) for Libinia. Polymeriza-tions of microtubules-like generate a forward projection ofthe perforatorial chamber material (Talbot and Chanmanon,1980; Hinsch, 1988; El-Sherief, 1991), although Tudge etal. (1994) showed that tubulin molecules were present in C.pagurus. Other actin-associated molecules such as the pro-teins α-actinin, tropomyosin, and spectrin were also identi-fied within the spermatozoon of C. pagurus using specificpolyclonal antibodies (Tudge et al., 1994).

The acrosome reaction of C. setosus ends with theformation of a trident-shaped structure (in lateral view) atthe distal end of the acrosomal filament (present study).This structure was not mentioned in any other Brachyuranspecies.

Another notable aspect of the spermatozoa of C. setosusextracted from female spermathecae was the occurrence ofthe acrosome reaction in seawater without the presence ofoöcytes or in a solution of oöcytes, as previously noted forother species such as U. tangeri (Medina, 1992), E. sinensis(Kang et al., 2009), and S. serrata (Zhang et al., 2010).However it is necessary to test if the presence of oöcyte oroöcyte water trigger the acrosome reaction in C. setosus.

It has been proposed that the mechanism that causesthe spermatozoa to change shape is due to ions present inthe seawater that activate the polymerization of cytoskele-tal proteins. This mechanism has been suggested for Rhyn-chocinetes typus Milne-Edwards, 1837 (Dupré and Schaaf,1996; Dupré and Barros, 2011), the crab E. sinensis (Kanget al., 2009), and the lobster Homarus americanus Milne Ed-wards, 1837 (Talbot and Chanmanon, 1980; Gesteira andHalcrow, 1988). For the last two species and the shrimp


Sicyonia ingentis (Burkenroad, 1938) the main cause of theacrosome reaction is Ca++. The first stage depends on Ca++and is inhibited by high pH (Clark et al., 1981a, b), and thesecond phase is dependent on a decrease in intracellular pH(Griffin et al., 1987). In species of the shrimp genera Litope-naeus, Byrdie, and Trachypenaeus, as well as Litopenaeusoccidentalis (Streets, 1871) (Alfaro et al., 2003, 2007), thespermatozoa only changed shape if a solution of seawaterwith oöcytes was added to the sperm.

Several authors have suggested that the propulsion ofmicrotubules inside the perforatorial chamber is a mecha-nism that favors the movement of nuclear and subacroso-mal material toward the front, where eversion occurs dur-ing the fertilization of acrosomal material (Talbot and Chan-manon, 1980; Hinsch, 1986, 1988). The presence of micro-tubules inside the perforatorial chamber has been describedfor many crabs spermatozoa including Callinectes sapidus(Rathbun, 1896) (Brown, 1966), Pinnixia sp. (Reger, 1970),Cancer (Langreth, 1965), Ovalipes ocellatus (Herbst, 1799)(Hinsch, 1986), Pilodius areolatus (Milne Edwards, 1834)(Tudge, 2009), and C. pagurus (Jamieson et al., 1997). Sim-ilar mechanisms may be involved in the acrosome reactionfor acrosomal filament formation in P. setosus.

Acrosomal Reaction Stages

This study identified five stages in the C. setosus acrosomereaction. Although these stages may overlap with the twomain stages (elongation of the acrosomal vesicle and extru-sion of the acrosomal filament) reported for U. tangeri (Med-ina, 1992; Medina and Rodriguez, 1992), Portunus trituber-culatus (Miers, 1876) (Zhu et al., 2004), E. sinensis (Kanget al., 2009), H. americanus (Talbot and Chanmanon, 1980),and S. serrata (Zhang et al., 2010), dividing the two mainevents of the acrosome reaction into other stages affordsgreater understanding of the thinning of the acrosomal walland the nuclear condensation that occurs in the second stageof the P. setosus acrosome reaction. These have also been de-scribed for the shrimp A. antennatus (Demestre et al., 1997),the lobster H. americanus (Talbot and Chanmanon, 1980),and the crab E. sinensis (Kang et al., 2009).

In the final stage of the acrosome reaction, the distalend of the acrosomal filament is transformed into a trident-like structure (in lateral aspect) and the acrosomal vesiclewalls become thinner, leaving only the outer portion. Thesecharacteristics show that the acrosome reaction in C. setosusis not comparable to that described for C. pagurus (Jamiesonet al., 1997), although they might not belong to the samegenus (see Ng et al., 2008: 53). Nor has this feature of theC. setosus acrosome reaction has been found in U. tangeri(Medina, 1992), E. sinensis (Kang et al., 2009), S. serrata(Zhang et al., 2010), C. sapidus (Brow, 1966), Carcinusmaenas (Linnaeus, 1758) or C. pagurus (Jamieson et al.,1997).

Another important aspect of the acrosome reaction ofC. setosus is the absence of a plasma membrane coveringthe anterior opening at the beginning of the reaction,suggesting that the fusion of membranes could occur duringthe sperm-egg interaction or while gathering the extrusionof the acrosomal filament. Similar results were obtained inH. americanus (Talbot et al., 1991), in which membranefusion occurs before the extrusion of the acrosomal filament

because, according to those authors, observations with atransmission electron microscope did not show a membranesurrounding the filament being formed.

For a better understanding of the mechanism by whichthe perforatorium is ejected and the driving force for the ex-trusion of the acrosomal material during the acrosome re-action, transmission electron microscopy studies identifyingthe ions that generate the acrosome reaction are also needed.

ACKNOWLEDGEMENT

This study was financed by FONDEF Grant D05 I-10246.

REFERENCES

Adiyodi, K., and R. Adiyodi. 1975. Morphology and cytology of theaccessory sex glands in invertebrates. International Review of Cytology43: 353-398.

, and G. Anilkumar. 1988. Arthropoda-Crustacea, pp. 261-318.In, K. G. Adiyodi and R. G. Adiyodi (eds.), Reproductive Biology ofInvertebrates. Vol. 3. Wiley and Sons, New York.

Alfaro, J., K. Ulate, and M. Vargas. 2007. Sperm maturation in theopen thelycum shrimp Litopenaeus (Crustacea: Decapada: Penaeoidea).Aquaculture 270: 436-442.

, N. Muñoz, M. Vargas, and J. Komen. 2003. Induction of spermactivation in open and closed thelycum penaeoid shrimps. Aquaculture216: 371-381.

Anderson, J., and C. Epifanio. 2010. Mating and sperm storage of the Asianshore crab Hemigrapsus sanguineus. Journal of Shellfish Research 29:497-501.

Benhalima, K., D. Duggan, and D. Robichaud. 2002. Reproductive biologyof male jonah crab, Cancer borealis Stimpson, 1859 (Decapoda, Can-cridae) on the Scotian shelf, northwestern Atlantic. Crustaceana 75: 891-913.

Beninger, P. G., C. Lanteigne, and R. W. Elner. 1993. Reproductiveprocesses revealed by spermatophore dehiscence experiments and byhistology, ultrastructure, and histochemistry of the female reproductivesystem in the snow crab Chionoecetes opilio (O. Fabricius). Journal ofCrustacean Biology 13: 1-16.

, R. W. Elner, T. P. Foyle, and H. Odense. 1988. Functional anatomyof the male reproductive system and the female spermatheca in thesnow crab Chionoecetes opilio (O. Fabricius) (Decapoda: Majidae) anda hypothesis for fertilization. Journal of Crustacean Biology 8: 322-332.

Brown, G. 1966. Ultrastructural studies of sperm morphology and sperm-egg interaction in the decapod Callinectes sapidus. Journal of Ultrastruc-tural Research 14: 425-440.

Burkenroad, M. D. 1945. A new sergestid shrimp (Peisos petrunkevitchii,n. gen., n. sp.), with remarks on its relationships. Transactions of theConnecticut Academy of Arts and Sciences 36: 553-591.

Cheung, T. S. 1968. Transmoult retention of sperm in the adult female stonecrab Menippe mercenaria (Say). Crustaceana 15: 117-120.

Clark, W. H. Jr., M. G. Kleve, and A. I. Yudin. 1981a. An acrosome reactionin natantian sperm. Journal of Experimental Zoology 218: 279-291.

, A. I. Yudin, and M. G. Kleve. 1981b. Primary binding in thegametes of the marine shrimp Sicyonia ingentis. Journal of Cell Biology91: 174a.

Demestre, M., and J. Fortuño. 1992. Reproduction of the deepwater shrimpAristeus antennatus (Decapoda: Dendrobranchiata). Marine EcologyProgress Series 84: 41-51.

, N. Cortadellas, and M. Durfort. 1997. Ultrastructure of the spermof the deep-sea Decapoda Aristeus antennatus. Journal of Morphology234: 79-87.

Diesel, R. 1989. Structure and function of the reproductive system ofthe symbiotic spider crab Inachus phalangium (Decapoda, Majidae):observation on the sperm transfer, sperm storage and spawning. Journalof Crustacean Biology 9: 266-277.

Dupré, E., and C. Barros. 2011. In vitro fertilization of the rock shrimp,Rhynchocinetes typus (Decapoda, Caridea): a review. Biological Re-search 44: 125-133.

, and G. Schaaf. 1996. Influence of ions on the unfolding ofthe spermatozoa of the rock shrimp, Rhynchocinetes typus. Journal ofExperimental Zoology 274: 358-364.


El-Sherief, S. 1991. Fine structure of the sperm and spermatophores ofPortunus pelagicus (L.) (Decapoda, Brachyura). Crustaceana 61: 271-279.

Eydeaux, F. 1835. Nouvelle espèce de Gélasime. Magasin de Zoologie 5(7):4 pp.

Fabricius, J. C. 1775. Systema Entomologiae, sistens Insectorum Classes,Ordines, Genera, Species, adjectis Sysnonymis, Locis, Descriptionibus,Observationibus. Officina Libraria Kortii, Flensburgi et Lipsiae, 832 pp.

Fabricius, O. 1788. Beskrivelse over den store Gronlandske krabbe. NyeSamling af det Kongelige Danske Videnskabers Selskabs Skrivter,Kongelige Danske Videnskabernes Selskab 3: 181-190.

Forskål, P. 1775. Discriptiones Animalium Avium, Amphibiorum, Piscium,Insectorum, Vermium; quae in Itinere orientali observavit. Petrus Forskål.Post mortem auctoris editit Carsten Niebuhr. Adjuncta est materialMedica Kahirina: xxxiv, 164 pp. Hafniae.

Gesteira, T., and K. Halcrow. 1988. Influence of some external factors onthe acrosome reaction in the spermatozoa of Homarus americanus MilneEdwards, 1837. Journal of Crustacean Biology 8: 317-321.

Goldstein, M., and E. Dupré. 2010. Sistema reproductivo de hembras ymachos en Cancer setosus (Molina, 1782) (Decapoda: Brachyura). LatinAmerican Journal of Aquatic Research 38: 274-280.

González-Gurriarán, E., L. Fernández, J. Freire, and R. Muiño. 1998.Mating and role of seminal receptacles in the reproductive biologyof the spider crab Maja squinado (Decapoda, Majidae). Journal ofExperimental Marine Biology and Ecology 220: 269-285

Goudeau, M. 1982. Fertilization in a crab (Carcinus maenas). 1. Earlyevents in the ovary and cytological aspects of the acrosome reaction andgamete contact. Tissue and Cell 14: 97-101.

Gray, J. E. 1831. Description of a new genus, and some undescribed speciesof Crustacea, pp. 39-40. In, J. E. Gray (ed.), The Zoological Miscellany.Part I. London.

Griffin, F., W. Clark, J. Crowe, and L. Crowe. 1987. Intracellular pHdecreased during the in vitro induction of the acrosome reaction in thesperm of Sicyonia ingentis. Biological Bulletin 173: 311-323.

Guinot, D., and B. Richer de Forges. 1981. Homolidae, rares ou nou-veaux, de l’Indo-Pacifique (Crustacea, Decapoda, Brachyura). Bulletindu Muséum national d’Histoire naturelle, Paris 3(4): 523-581.

Herbst, J. F. W. 1782-1804. Versuch einer Naturgeschichte der Krabben undKrebse, nebst einer systematischen Beschereibung ihrer verschiedenenArten. Vol. 3(4). Berlin and Stralsund. 1-274, 1-226, 1-216 pp.

Hinsch, G. 1969. Microtubules in the sperm of the spider crab, Libiniaemarginata L. Journal of Ultrastructural Research 29: 525-534.

. 1973. Sperm structure of Oxyrhyncha. Canadian Journal ofZoology 51: 421-426.

. 1986. Comparison of sperm morphologies, transfer and spermmass storage between two species of crab, Ovalipes ocellatus andLibinia emarginata. International Journal of Invertebrate Reproductionand Development 10: 79-87.

. 1988. Ultrastructure of the sperm and spermatophores of thegolden crab Geryon fenneri and a closely related species, the red crabG. quinquedens, from the eastern Gulf of Mexico. Journal of CrustaceanBiology 8: 340-345.

Jamieson, B. 1989a. Ultrastructural comparison of the spermatozoa ofRanina ranina (Oxystomata) and of other crabs exemplified by Portunuspelagicus (Brachygnatha) (Crustacea, Brachyura). Zoomorphology 109:103-111.

. 1989b. The ultrasctructure of the spermatozoa of four species ofxanthid crabs (Crustacea, Brachyura, Xanthidae). Journal of Submicro-scopic Cytology and Pathology 21: 579-584.

. 1990. The ultrastructure of the spermatozoa of Petalomera lateralis(Gray) (Crustacea, Brachyura, Dromidacea) and its phylogenetic signif-icance. International Journal of Invertebrate Reproduction and Develop-ment 17: 39-45.

. 1991. Ultrastructure and phylogeny of crustacean spermatozoa.Memories of Queensland Museum 31: 109-142.

. 1994. Phylogeny of the Brachyura with particular reference tothe Podotremata: evidence from a review of spermatozoal ultrastructure(Crustacea, Decapoda). Philosophical Transactions of the Royal Societyof London B 345: 373-393.

, and C. Tudge. 2000. Crustacea-Decapoda, pp. 1-95. In, B. G. M.Jamieson (ed.), Reproductive Biology of Invertebrates. Progress in MaleGamete Ultrastructure and Phylogeny. Vol. 9, Part C. John Wiley andSons, Chichester.

, D. Guinot, C. Tudge, and B. Richer de Forges. 1997. Ultrastructureof the spermatozoa of Corystes cassivelaunus (Corystidae), Platepistomananum (Cancridae) and Cancer pagurus (Cancridae) supports recogni-tion of the Corystoidea (Crustacea, Brachyura, Heterotremata). Helgo-länder Meeresuntersuchungen 51: 83-93.

Jivoff, P., A. H. Hines, and L. S. Quackenbush. 2007. Reproductive biologyand embryonic development, pp. 255-298. In, V. S. Kennedy and L. E.Cronin (eds.), The Blue Crab Callinectes sapidus. Maryland Sea GrantCollege, College Park, Maryland.

Kang, X., G. Li, S. Mu, M. Guo, and S. Ge. 2009. Acrosome reaction ofChinese mitten-handed crab Eriocheir sinensis (Crustacea: Decapoda)spermatozoa: promoted by long-term cryopreservation. Aquaculture 295:195-199.

Langreth, S. 1965. Ultrastructural observations on the sperm of the crabCancer borealis. Journal of Cell Biology 27: 56a-57a.

. 1969. Spermiogenesis in Cancer crabs. Journal of Cell Biology 13:575-603.

Linnaeus, C. 1758. Systema Naturae per Regna Tria Naturae, SecundumClasses, Ordines, Genera, Species, cum Characteribus, Differentiis, Syn-onymis, Locis (edit. 10). Vol. 1. Laurentii Salvii, Holmiae [Stockholm].823 pp.

Manning, R. B., and L. Holthuis. 1984. Geryon fenneri, a new deep-watercrab from Florida (Crustacea: Decapoda: Geryonidae). Proceedings ofthe Biological Society of Washington 97: 666-673.

Medina, A. 1992. Structural modifications of sperm from the fidler crabUca tangeri (Decapoda) during early stages of fertilization. Journal ofCell Biology 12: 610-614.

, and A. Rodríguez. 1992. Spermiogenesis and sperm structure inthe crab Uca tangeri (Crustacea, Brachyura), with special reference tothe acrosome differentiation. Zoomorphology 111: 161-165.

Miers, E. J. 1876. Descriptions of some new species of Crustacea, chieflyfrom New Zealand. Annals and Magazine of Natural History 17(4): 218-229.

Milne Edwards, H. 1834-1840. Histoire naturelle des Crustacés, com-prenant l’anatomie, la physiologie et la classification de ces animaux.Librairie Encyclopédique de Roret. Vols. 1-3. Librairie Encyclopédiquede Roret, Paris. 532 pp.

. 1853. De la famille des ocypodides (Ocypodidae) suite. Annalesdes Sciences Naturelles, Série 3 (Zoologie) 20: 163-228.

Morgan, S., J. W. Goy, and J. D. Costlow Jr. 1983. Multiple ovipositionsfrom single matings in the mud crab Rhithropanopeus harrisii. Journalof Crustacean Biology 3: 542-547.

Moriyasu, M., K. Benhalima, D. Duggan, P. Lawton, and D. Robichaud.2000. Reproductive biology of the male jonah crab, Cancer borealisStimpson, 1859 (Decapoda, Cancridae) on the Scotian Shelf, northwest-ern Atlantic. Crustaceana 75: 891-913.

Ng, P. K. L., D. Guinot, and P. J. F. Davie. 2008. Systema Brachyurorum:Part I. An annotated checklist of extant brachyuran crabs on the world.Raffles Bulletin of Zoology 17: 1-286.

Pochon-Masson, J. 1968. L’ultrastructure des spermatozoides vésiculaireschez les crustacés décapodes avant et au cours de leur dévaginationexpérimentale. I. Brachyoures et Anomoures. Annales des SciencesNaturelles Zoology 10: 1-100.

. 1983. Arthropoda-Crustacea, pp. 407-449. In, K. G. Adiyodi andR. G. Adiyodi (eds.), Reproductive Biology of Invertebrates: Spermato-genesis and Sperm Function. Vol. 2. John Wiley and Sons, Chichester.

Rathbun, M. J. 1896. The genus Callinectes. Proceedings of the UnitedStates National Museum 18: 349-375.

Reger, J. 1970. Studies on the fine structure of spermatids and spermatozoaof the crab, Pinnixia sp. Journal of Morphology 132: 89-100.

Risso, A. 1816. Histoire naturelle des Crustacés des environs de Nice.Librairie Grecque-Latine-Allemande, Paris. 175 pp. Plates 1-3.

Ryan, E. 1967. Structure and function of the reproductive system of thecrab Portunus sanguinolentus (Herbst) (Brachyura: Portunidae). II. Fe-male system. Proceedings of the Symposium on Crustacea, Ernakulam.Marine Biological Association of India 2: 522-524.

Say, T. 1818. An account of the Crustacea of the United States. Journal ofthe Academy of Natural Sciences, Philadelphia 1[2(1)]: 235-253.

Scelzo, M., and A. Medina. 2004. A Dendrobranchiate, Peisos petrunke-vitchi (Decapoda, Sergestidae), with reptant-like sperm: a spermiocladis-tic assessment. Acta Zoologica 85: 81-89.

Schackmann, W. 1989. Ionic regulation of the sea urchin sperm acrosomereaction and stimulation by egg derived peptides, pp. 3-28. In, H. Schat-


ten and G. Schatten (eds.), The Cell Biology of Fertilization. AcademicPress, New York.

Simeó, C., K. Kurtz, G. Rotlant, M. Chiva, and E. Ribes. 2010. Sperm ul-trastucture of the spider crab Maja brachydatyla (Decapoda: Brachyura).Journal of Morphology 271: 407-417.

Smith, S. I. 1879. The stalk-eyed crustaceans of the Atlantic coast of NorthAmerica north of Cape Cod. Transactions of the Connecticut Academyof Arts and Sciences 5: 27-136.

Stimpson, W. 1859. Notes on North American Crustacea, No. 1. Annals ofthe Lyceum of Natural History of New York 7: 49-93.

Streets, T. H. 1871. Descriptions of five new species of Crustacea fromMexico. Proceedings of the Academy of Natural Sciences of Philadelphia1871: 225-227, Plate II.

Talbot, P., and P. Chanmanon. 1980. Morphological features of the acro-some reaction of lobster (Homarus) sperm and the role of the reactionin generating forward sperm movements. Journal of Ultrastructural Re-search 70: 287-297.

, and G. Summers. 1978. The structure of sperm from Palinurus,the spiny lobster, with special regard to the acrosome. Journal ofUltrastructural Research 64: 341-351.

, B. Poolsanguan, H. Al-Hajj, and W. Poolsanguan. 1991. Gameteinteraction during in vitro fertilization of lobster (Homarus americanus)oocyte. Journal of Structural Biology 106: 125-134.

Tudge, C. 2009. Spermatozoal morphology and its bearing on decapodphylogeny, pp. 101-119. In, J. W. Martin, K. A. Crandall, and D. L.Felder (eds.), Decapod Crustacean Phylogenetics. Crustacean Issue.Vol. 18. CRC Press, Boca Raton.

, P. Grellier, and J. L. Justine. 1994. Actin in the acrosome of thespermatozoa of the crab, Cancer pagurus L. (Decapoda, Crustacea).Molecular Reproduction and Development 38: 178-186.

Uma, K., and T. Subramoniam. 1979. Histochemical characteristics of sper-matophore layers of Scylla serrata (Forskâl) (Decapoda: Portunidae). In-ternational Journal of Invertebrate Reproduction 1: 31-40.

Zhang, Z., H. Cheng, Y. Wang, S. Wang, F. Xie, and S. Li. 2010. Acrosomereaction of sperm in the mud crab Scylla serrata as a sensitive toxicitytest for metal exposures. Archives of Environmental Contamination andToxicology 58: 96-104.

Zhu, D. F., C.-L. Wang, H.-W. Yu, and S. Zhou. 2004. Morphologicaland structure changes of sperm during the acrosome reaction in theswimming crab Portunus trituberculatus. Acta Zoologica Sinica 50: 800-807.

RECEIVED: 2 July 2011.ACCEPTED: 14 September 2011.

acrosome reaction of cancer setosus molina, 1782 (decapoda: brachyura)

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