Ultrastructure of Spermatozoa and Spermatophores of Old World

Ultrastructure of Spermatozoa and Spermatophores ofOld World Freshwater Crabs (Brachyura: Potamoidea:Gecarcinucidae, Potamidae, and Potamonautidae)

Sebastian Klaus,1* Christoph D. Schubart,2 and Dirk Brandis3

1Institut fur Zoologie, Universitat Heidelberg, INF 230, 69120 Heidelberg, Germany2Biologie I, Universitat Regensburg, Universitatsstr. 31, 93053 Regensburg, Germany3Zoologisches Museum, Universitat Kiel, Hegewischstr. 3, 24105 Kiel, Germany

ABSTRACT We investigated the ultrastructure of sper-matozoa and spermatophores of 19 palaeotropical fresh-water crab species [12 species of the Gecarcinucidae, 6of the Potamidae (Potamiscinae), and 1 species of thePotamonautidae (Deckeniinae: Hydrothelphusini)]. Theinvestigated Potamiscinae have densely packed coeno-spermic spermatophores with the exception of Thai-phusa sirikit and Johora singaporensis that exhibitcleistospermia. In contrast, in the Gecarcinucidae thespermatozoa are loosely embedded in a mucous matrix.The gecarcinucid and potamiscine sperm differ, further-more, in acrosomal structure and size. The acrosome inthe Gecarcinucidae is much smaller and spherical, whilethe larger acrosome in the Potamiscinae has the tend-ency to be depressed. In the Potamiscinae, an additionalmiddle acrosomal zone evolved between the acrosomeray zone and the outer acrosomal zone. Within theGecarcinucidae, a differentiation into two groups (Gecar-cinucinae and Parathelphusinae) is not supported by thepresent spermatological data. The sperm morphology ofHydrothelphusa aff. madagascariensis (Potamonautidae:Deckeniinae) differs from Potamonautes sidneyi (Pota-monautidae: Potamonautinae) in acrosomal size andshape, and in the absence of a periopercular rim. Acloser relationship of Deckeniinae and Gecarcinucidaecannot be confirmed by spermatology. J. Morphol.270:175–193, 2009. � 2008 Wiley-Liss, Inc.

KEY WORDS: spermatozoa; spermatophores; fresh-water crabs; Brachyura; Potamoidea

Brachyuran sperm cell morphology has beeninvestigated for more than 100 years (reviewed inFelgenhauer and Abele, 1991; Jamieson andTudge, 2000). Electron microscopy studies espe-cially improved our understanding of the morphol-ogy and function of brachyuran sperm cells. Theacrosomal reaction of the complex brachyuransperm cell during fertilization was resolved byelectron microscopic studies (Brown, 1966). Sper-matological investigations revealed both a con-served ground pattern of sperm cell morphologywithin the Brachyura, as well as variabilitybetween groups, mainly at the family level andabove (Felgenhauer and Abele, 1991; Jamieson,1994; Jamieson et al., 1995). Brachyuran sperma-

tozoal characters were used in several cladisticstudies (Jamieson, 1991, 1994; Jamieson et al.,1995) and largely support the system of classifica-tion of the Brachyura as suggested by Guinot(1978), that is, the grouping into Podotremata,Heterotremata s.lat., and Thoracotremata.

Brachyuran spermatozoa, as with decapodsperm cells in general, are aflagellate and immo-tile. The acrosome is spherical and consists of acentral perforatorial chamber (also called a ‘‘perfo-ratorium’’) that contains microtubule-like struc-tures and is surrounded by several acrosomalzones (see Fig. 1). We will term the electron-lucentzone surrounding the perforatorial chamber the‘‘inner acrosomal zone’’ (as per Jamieson, 1994).This zone is usually surrounded externally by the‘‘acrosome ray zone’’ in the investigated freshwatercrabs (but claimed to be absent in Potamon fluvia-tile, P. ibericum, and Potamonautes sidneyi by Gui-not et al., 1997). The ray zone is defined by its dis-tinct, coarse pattern and the potentially homolo-gous structure in podotreme crabs is called the‘‘fingerprint zone’’ (Guinot et al., 1998). Betweenthe acrosome ray zone and the prominent ‘‘outeracrosomal zone,’’ an additional zone can be distin-guished in some species. We term this the ‘‘middleacrosomal zone.’’ Apically, the acrosome is cappedwith an electron-dense operculum that contactsthe oocyte during a successful fertilization.Beneath the operculum the subopercular materialseparates operculum, inner acrosomal material,and perforatorial chamber, respectively. The acro-some is embedded in a cup-like nucleus, whilebetween acrosome and nucleus a thin layer of cyto-plasm remains, often accompanied by membrane-ous structures that are interpreted as vestigial

*Correspondence to: Sebastian Klaus, Institut fur Zoologie,Universitat Heidelberg, INF 230, D-69120 Heidelberg, Germany.E-mail: [email protected]

Published online 22 October 2008 inWiley InterScience (www.interscience.wiley.com)DOI: 10.1002/jmor.10678

JOURNAL OF MORPHOLOGY 270:175–193 (2009)

� 2008 WILEY-LISS, INC.

mitochondria (Jamieson, 1993). The nucleus hasseveral to many lateral arms that, in the Bra-chyura, mostly are without microtubules but con-tain chromatin.

Brachyuran spermatophores are considered tobe one of the most simple type in decapod crusta-ceans (Subramoniam, 1991). They are sphericaland consist of sperm masses that are enclosed by amucopolysaccharide matrix. In some brachyurans(e.g., Libinia emarginata, Pisidae and Carcinusmaenas, Portunidae), the spermatophores werereported to consist of two distinct layers with theouter layer consisting of chitinous material(Hinsch, 1991; Subramoniam, 1991). The forma-tion of the spermatophores takes place in the ante-rior vasa deferentia, and both apocrine and exo-crine secretion of the epithelia are described(Hinsch, 1991). After copulation, when the sperma-tophores are transferred to the female spermathe-cae, they are dissolved and the spermatozoareleased. Potentially, the seminal fluids may alsoplay a role in sperm plug formation (e.g., Ovalipesocellatus, Portunidae, Hinsch, 1986, 1991).

Several advantages of the mucopolysaccharide-enveloping of the spermatozoa were suggested,among them are mechanical protection, preventionof dehydration, an antimicrobial function andnutrition of the sperm (Hinsch, 1991; Subramo-niam, 1991). Based on the fact that the spermato-phores are dissolved after copulation in the genusGeryon (Geryonidae), Hinsch (1988) proposed thatthe spermatophores are mainly a packaging devicefor sperm transfer. A more complex function wasproposed by Beninger et al. (1993). They observedthat in Chionoecetes opilio (Majidae), the sperma-tophores perform a ‘‘differential dehiscence.’’ Inthis process, free spermatozoa from the initiallydehisced spermatophore are available for fertiliza-tion, while the still intact spermatophores storethe sperm in the female spermathecae. The sper-matophore pellicle was proposed to prevent exces-sive acrosome reactions and keeps the sperm forfuture fertilizations.

Although freshwater crabs represent one of themost diverse groups within the Brachyura, only afew studies on their spermatozoal and spermato-phore morphology have been conducted (Potamo-nautes sidneyi: Jamieson, 1993; Potamon fluviatile:Tudge and Justine, 1994; P. fluviatile and P. iberi-cum: Guinot et al., 1997; Potamiscus beieri: nodescription, but depicted in Brandis, 2000 as Pota-miscus sp.; Sinopotamon yangtsekiense: Du et al.,1999; Wang et al., 1999; Austrothelphusa trans-versa: no description, but depicted in Jamiesonand Tudge, 2000 as Holthuisana transversa).These previously investigated species belong to theAfrican family Potamonautidae, subfamily Potamo-nautinae (P. sidneyi) and to the Eurasian–NorthAfrican Potamidae, subfamily Potaminae (P. fluvia-tile, P. ibericum) and Potamiscinae (P. beieri, S.yangtsekiense), and to the Gecarcinucidae (sensuKlaus et al., 2006; A. transversa). Within the OldWorld freshwater crabs, spermatological data onthe African-Madagascan Deckeniinae (the Decke-niidae sensu Klaus et al., 2006) are still lacking.Also the spermatozoa and spermatophores of theneotropical Pseudothelphusoidea and the Tricho-dactylidae (Dilocarcinus septemdentatus: Matoset al., 1996) still remain largely unexplored.

Freshwater crabs are well adapted to theirlimnic environment, which also affects their modeof reproduction. They show direct developmentwith relatively few but large, lecithotrophic eggs.Earlier studies on freshwater crab spermatozoacould not detect correlations of sperm morphologywith their limnic habitat (Guinot et al., 1997).Nevertheless, it was proposed that the occurrenceof cleistospermia (spermatophores that containonly a single spermatozoon) could be an adaptationto reduce polyspermy, and therefore preventwastage of eggs (Guinot et al., 1997).

We understand the Old World freshwater crabsas one superfamily Potamoidea (as already kept asan option by Klaus et al., 2006) that includes thehere investigated species. This taxonomic approachis supported by the proposed potamoid monophyly(von Sternberg et al., 1999; Daniels et al., 2006;Klaus et al., 2006; Cumberlidge et al., 2008), therecognition of just two Asian families (the Gecarci-nucidae and the Potamidae; Klaus et al., 2006),and the still unresolved phylogenetic relationshipbetween the three potamoid families (Gecarcinu-cidae, Potamidae, and Potamonautidae; Danielset al., 2006; Cumberlidge et al., 2008).

In this study, we describe spermatozoal andspermatophore morphology of potamoid freshwatercrabs with the focus on the Asian Gecarcinucidae(12 species), but also including representatives ofthe Potamidae (the Asian subfamily Potamiscinae,six species) and one species of the MadagascanHydrothelphusini (Potamonautidae: Deckeniinae:Hydrothelphusa aff. madagascariensis). Spermato-logical data for the Australian Gecarcinucidae, the

Fig. 1. Diagrammatic drawing of a freshwater crab sperma-tozoon with nomenclature of its morphology.

176 S. KLAUS ET AL.

Journal of Morphology

Potamonautidae, subfamily Potamonautinae andthe Potamidae, subfamily Potaminae are availablethrough the studies of Jamieson (1993), Jamiesonand Tudge (2000), and Guinot et al. (1997).

MATERIALS AND METHODS

The investigated freshwater crab species were purchased ataquarists or collected on field trips to Malaysia, Singapore, andIndonesia during 2005 (Table 1). For transmission electron mi-croscopy, vasa deferentia and spermathecae (Malayopotamon cf.brevimarginatum) were fixed in 4% glutaraldehyde, either phos-phate- or cacodylate-buffered (both pH 7.4). After several wash-ing steps with cacodylate buffer, the tissue was postfixed with1% osmium tetroxide for 2 h. Cacodylate and maleate buffer(pH 5.2) washing steps were followed by en-bloc staining with1% uranyl acetate overnight. After dehydration through agraded series of ethanol, the tissue was infiltrated and embed-ded with Spurr’s resin. Thin-sections (75 nm) were cut with adiamond knife, collected on copper grids (200 mesh, coated witha Formvar1 support film if required). The sections were post-stained with aqueous lead citrate for 1 min. Electron micro-graphs were taken on a Zeiss EM10 transmission electronmicroscope. Acrosomal measurements were taken with a slidingcaliper from the negative.For light microscopy, a spermatheca of Oziothelphusa ceylo-

nensis was fixed with a mixture of formalin, acetic acid, mercu-ric chloride, and trichloroacetic acid (‘‘SuSa’’ according to Hei-denhain, 1917), dehydrated through a series of ethanol and af-ter treatment with methyl benzoate embedded in Paraplast1.Sections of 8 lm thickness were cut on a sliding microtome andstained trichromatically according to Goldner (1938).

RESULTS

Especially for the vasa deferentia that werefixed under tropical field conditions, the ultrastruc-tural analysis did not yield well-resolved images,notably at higher resolution (beyond 10,0003).

The outer spermatozoal cell membranes and thechromatin were often already in a state of decay.Nevertheless, the structure of the acrosome wasalways sufficiently preserved for morphologicalcomparison. Measurements of acrosomal lengthand width and opercular width and height aregiven in Figures 2 and 3, respectively.

Potamidae: Potamiscinae

Diagrammatic drawings of a longitudinal sagit-tal section of potamiscine spermatozoa are given inFigure 4. The acrosomes of the Potamiscinae areslightly depressed in shape, the acrosomal lengthto width (AL : AW) ranging from 0.7 to 0.8. Thesmallest acrosome is found in Malayopotamon cf.brevimarginatum with a mean acrosomal width(AW) of 3.4 6 0.3 lm and a mean acrosomal length(AL) of 2.4 6 0.2 lm (n 5 7) (Fig. 2, no. 2). At theupper limit of acrosomal size ranges is Thaiphusasirikit with AW 5 5.2 6 0.3 lm and AL 5 4.0 60.3 lm (n 5 5) (Fig. 2, no. 6). The perforatorialchamber is of moderate diameter, ranging approxi-mately from 1/4 to 1/5 of the total AW (M. cf. brevi-marginatum: 1/3; T. sirikit: 1/11).

The acrosomes of the investigated Potamiscinaehave a complex zonation, with the acrosome rayzone always externally adjacent to a middle andan outer acrosomal zone. The operculum and acro-some ray zone are linked via a ‘‘tongue andgroove’’ connection (Fig. 5F). A low circular ridgeat the border between outer and inner acrosomalzone interdigitates with a circular groove on thebasal side of the operculum. This kind of connec-

TABLE 1. Specimens used for preparation of the vas deferens

Species Provenance

GecarcinucidaeGeithusa pulchra (Ng, 1989) Malaysia, Pulau RedangHeterothelphusa fatum (Ng, 1997) Singapore, AquaristOziothelphusa ceylonensis (Fernando, 1960)a Sri Lanka, AquaristOziothelphusa sp. South India, AquaristParathelphusa convexa (De Man, 1879) Indonesia, Java, GarutParathelphusa aff. maindroni (Rathbun, 1902) Indonesia, S-Sumatra, near LampungPhricotelphusa gracilipes (Ng and Ng, 1987) Malaysia, Pulau LangkawiSartoriana spinigera (Wood-Mason, 1871) Nepal, Mechi provinceSayamia bangkokensis (Naiyanetr, 1982) Thailand, AquaristSiamthelphusa improvisa (Lanchester, 1901) Malaysia, Pulau LangkawiSomanniathelphusa sp. Thailand, AquaristTerrathelphusa kuhli (De Man, 1883) Indonesia, Java, Cibodas

Potamidae: PotamiscinaeGeothelphusa albogilva (Shy, Ng and Yu, 1994) Taiwan, AquaristJohora singaporensis (Ng, 1986) SingaporeLarnaudia beusekomae (Bott, 1970) Thailand, AquaristMalayopotamon cf. brevimarginatum (De Man, 1892)b Indonesia, S-Sumatra, Danau RanauPudaengon thatphanom (Ng and Naiyanetr, 1995) Thailand, AquaristThaiphusa sirikit (Naiyanetr, 1992) Thailand, Aquarist

Potamonautidae: Deckeniinae: HydrothelphusiniHydrothelphusa aff. madagascariensis (A. Milne-Edwards, 1872) Madagascar, Aquarist

aOziothelphusa ceylonensis: both vas deferens (ultrastructure) and spermatheca (histology).bMalayopotamon cf. brevimarginatum: only spermatheca for ultrastructure.

ULTRASTRUCTURE OF FRESHWATER CRAB SPERMATOZOA 177


tion is found in all investigated potamiscine sper-matozoa, although shallowly developed in Johorasingaporensis and Thaiphusa sirikit.

Coenospermic spermatophores are alwaysdensely packed with several sperm cells and irreg-ularly shaped, while cleistospermic spermato-phores (only one sperm cell per spermatophore)are spherical (see Guinot et al., 1997). Both typesof spermatophores occur within the Potamiscinae.The spermatophores are enclosed by a distinct pel-licle that consists of several layers (see Fig. 6).Coenospermic spermatophores have a pellicle withthree layers, first an electron-dense layer (Fig. 6A,no. 1), followed by electron-lucent material (Fig.6A, no. 2) and a third, denser layer (Fig. 6A, no.3). In Larnaudia beusekomae, diffuse electron-dense appendages are visible on the surface oflayer one (Fig. 6B, arrows). The cleistosperm sper-matophores of Johora singaporensis and Thai-phusa sirikit show a more complex pellicle. A thinelectron-dense layer (Fig. 6B, no. 1) is followed bya thicker layer (Fig. 6B, no. 2), resembling the firstelectron-dense layer in L. beusekomae. As in L.beusekomae, this layer is followed by more elec-tron-lucent material (Fig. 6B, no. 3). Basal to thislayer a second thin, electron-dense layer is situ-

ated (Fig. 6B, no. 4), followed by a broader layer ofundefined material (Fig. 6B, no. 5). Where thespermatophore pellicle of T. sirikit is disrupted,the layers two, three, and five extrude, while thelayers one and four seem to stay intact (Fig. 6B,star). This argues for the extruding layer beingviscous while the thin electron-dense layers areprobably more rigid.

Larnaudia beusekomae (Figs. 7A–C,5F, 4, and 6A)

The acrosome of Larnaudia beusekomae isslightly depressed with an acrosome length towidth ratio of 0.8 6 0.1 (n 5 7). The operculum isimperforate and more convex than in the otherpotamiscines (Fig. 3, no. 1). A periopercular rim isabsent. The perforatorial chamber is surroundedby a thin inner acrosomal zone and a cylindricalacrosome ray zone that connects apically to theoperculum. The ray zone is externally adjacent toa very thin electron-dense middle acrosomal zone.The outer acrosomal zone is prominent and homo-geneous. The nuclear arms are situated laterally.L. beusekomae exhibits coenospermia.

Fig. 2. Acrosome length plotted against acrosome width. 1, Larnaudia beusekomae; 2, Malayopotamon cf. brevimarginatum; 3,Geothelphusa albogilva; 4, Pudaengon thatphanom; 5, Johora singaporensis; 6, Thaiphusa sirikit; 7, Potamon fluviatile; 8, Potamonibericum; 9, Hydrothelphusa aff. madagascariensis; 10, Potamonautes sidneyi; 11, Phricotelphusa gracilipes; 12, Sartoriana spinigera;13, Oziothelphusa ceylonensis; 14, Oziothelphusa sp.; 15, Terrathelphusa kuhli; 16, Parathelphusa convexa; 17, Parathelphusa aff.maindroni, 18, Geithusa pulchra; 19, Heterothelphusa fatum, 20, Siamthelphusa improvisa; 21, Somanniathelphusa sp.; 22, Sayamiabangkokensis. Circles, Potamidae; squares, Gecarcinucidae; hexagons, Potamonautidae. Measurements of P. sidneyi, P. fluviatile, andP. ibericum are taken from the literature, standard deviation not indicated.

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Malayopotamon cf. brevimarginatum(Figs. 7D,E and 4)

Of the investigated potamid spermatozoa, thoseof Malayopotamon cf. brevimarginatum from southSumatra have the smallest acrosome. It isdepressed (AL/AW 5 0.7 6 0.06, n 5 7), the operc-ulum is imperforate, and a periopercular rim isabsent. The acrosome ray zone is broad and cylin-drical. A prominent, more electron-dense outeracrosomal zone and a middle acrosomal zone canalso be distinguished. The middle acrosomal zonedoes not attach to the operculum and surroundsthe acrosome ray zone like a ring. In the outeracrosomal zone, patches of more electron-densematerial can frequently be observed and are prob-ably not an artifact. There are several lateral nu-clear arms. As the investigated spermatozoa origi-nate from a female spermatheca, spermatophores,if present, would have been dissolved.

Geothelphusa albogilva (Figs. 7F–H and 4)

The acrosome of Geothelphusa albogilva is likein Malayopotamon cf. brevimarginatum, remark-ably depressed (AL/AW 5 0.7 6 0.06, n 5 7), and

the operculum only slightly bulging centrally (Fig.3, no. 3). Beneath the outer rim of the operculuma less electron-dense zone is situated, probably avestigial periopercular rim. The middle acrosomalzone is thin and cylindrical and attaches directlyto the operculum as any subopercular materialseems to be absent. The acrosome ray zone isprominent, surrounding cylindrically the wide per-foratorial chamber, and apically attaching to theoperculum. As in M. cf. brevimarginatum, theouter acrosomal zone can be distinguished into aninner lighter area and an outer denser one. Theinner area is outwardly convex and reaches api-cally the vestigal periopercular rim. At the base ofthe acrosome, the perforatorial chamber bulgeslaterally toward the outer acrosomal zone. Thenuclear arms are situated laterally. Geothelphusaalbogilva exhibits densely packed coenospermia.

Pudaengon thatphanom (Figs. 7I–K and 4)

The acrosome of Pudaengon thatphanom is alsodepressed (AL/AW 5 0.7 6 0.05, n 5 5). The acro-some ray zone is broad but contacts, in contrast toGeothelphusa albogilva, the overlying operculum

Fig. 3. Operculum height plotted against operculum width. 1, Larnaudia beusekomae; 2, Malayopotamon cf. brevimarginatum;3, Geothelphusa albogilva; 4, Pudaengon thatphanom; 5, Johora singaporensis; 6, Thaiphusa sirikit; 7, Potamon fluviatile; 8, Pota-mon ibericum; 9, Hydrothelphusa aff. madagascariensis; 10, Potamonautes sidneyi; 11, Phricotelphusa gracilipes; 12, Sartorianaspinigera; 13, Oziothelphusa ceylonensis; 14, Oziothelphusa sp.; 15, Terrathelphusa kuhli; 16, Parathelphusa convexa; 17, Parathel-phusa aff. maindroni, 18, Geithusa pulchra; 19, Heterothelphusa fatum, 20, Siamthelphusa improvisa; 21, Somanniathelphusa sp.;22, Sayamia bangkokensis. Circles, Potamidae; squares, Gecarcinucidae; hexagons, Potamonautidae. Measurements of P. sidneyi, P.fluviatile, and P. ibericum are taken from the literature, standard deviation not indicated.



only marginally. The acrosome ray zone loses elec-tron density, outwardly, toward the middle acroso-mal zone, thus forming an intermediate zoneabsent in the other investigated potamiscine sper-matozoa. The middle acrosomal zone is very thinand cylindrical. Within the outer acrosomal zone,an inner, less electron-dense area can be identifiedthat is outwardly convex and attaches to the mid-dle acrosomal zone. Pudaengon thatphanom alsoexhibits densely packed coenospermia.

Johora singaporensis (Figs. 5A–C and 4)

The spermatozoa of this potamiscine crab are of aslightly depressed shape (AL/AW 5 0.8 6 0.04, n 511). The nuclear arms are situated equatorially in a

very regular way. The acrosome of Johora singaporen-sis also shows a complex zonation. The inner acroso-mal zone is more prominent than in the previouslydescribed species. The acrosome ray zone and themiddle acrosomal zone are cylindrical, similarly thick,and much thinner than the prominent outer acroso-mal zone. Also, in contrast to the previously describedpotamiscines, the outer acrosomal zone is less electrondense than the inner one. The operculum is depressedand is the only one of the investigated species with anapical perforation (Fig. 5C). Opercular width andheight are smallest among the Potamiscinae (Fig. 3,no. 5). A periopercular rim is missing. Johora singa-porensis exhibits only cleistospermia, with every sin-gle spermatozoon being encapsulated by a thin doublemembrane.

Fig. 4. Diagrammatic drawings of potamid and potamonautid (Hydrothelphusa aff. madagas-cariensis) spermatozoa (longitudinal sagittal view). Scale bar 5 1 lm.

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Fig. 5. Potamid (A–F) and potamonautid (G–I) spermatozoa and spermatophores. TEM. (A–C) Johora singaporensis. (D–F)Thaiphusa sirikit. (G–I) Hydrothelphusa aff. madagascariensis. First column longitudinal sagittal section, second column cross sec-tion. (C) Perforated operculum of Johora singaporensis (arrow). (F) Larnaudia beusekomae. (I) Corrugated surface of Hydrothel-phusa aff. madagascariensis spermatozoon with electron-lucent corona (arrow). ar, acrosome ray zone; ia, inner acrosomal zone;ma, middle acrosomal zone; nu, nucleus; na, nuclear arm; oa, outer acrosomal zone; op, operculum; sw, spermatophore wall; tg,‘‘tongue and groove’’ connection of operculum and outer acrosomal zone. Scale bar 5 1 lm or as indicated.

Fig. 6. The potamiscine spermatophore pellicle. TEM. (A) Larnaudia beusekomae (coenospermia). 1, outer electron-dense layer;2, electron-lucent material; 3, inner electron-dense layer; arrows, extraspermatophoral appendages. (B) Thaiphusa sirikit (cleisto-spermia). 1, outer thin electron-dense layer; 2, thick electron-dense layer; 3, electron-lucent material; 4, inner thin electron-denselayer; 5, undefined electron-dense material; star, extrusion of layers 2, 3, and 5; nu, nucleus of sperm cell.



Thaiphusa sirikit (Figs. 5D–F, 4, and 6B)

The spermatozoa of Thaiphusa sirikit closelyresemble those of Johora singaporensis, but exceedall other investigated spermatozoa in size (Fig. 2,no. 6). Its acrosome is slightly depressed (AL/AW

5 0.8 6 0.03, n 5 5), the operculum is asdepressed as the operculum of J. singaporensis butis imperforate. The larger opercular height in T.sirikit compared to J. singaporensis is the result ofa thicker operculum, and not because of shape dif-

Fig. 7. Potamid spermatozoa and spermatophores. TEM. (A–C) Larnaudia beusekomae. (D, E) Malayopotamon cf. brevimargi-natum. (F–H) Geothelphusa albogilva. (I–K) Pudaengon thatphanom. First column longitudinal sagittal section, second columncross section, and last column spermatophores. ar, acrosome ray zone; ia, inner acrosomal zone; ma, middle acrosomal zone; nu, nu-cleus; na, nuclear arm; oa, outer acrosomal zone; tg, ‘‘tongue and groove’’ connection of operculum and outer acrosomal zone. Scalebar 5 1 lm or as indicated.

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ferences (Fig. 3, no. 6). A periopercular rim isabsent. The acrosome ray zone is slightly convexto the outside and is attached to the perforatorialchamber anteriorly and posteriorly. In contrast toJ. singaporensis, the ray zone is more electrondense than the middle acrosomal zone. The latteris very thin. Rudiments of the thickened ring sur-round the posterior opening of the perforatorialchamber. A pair of parallel situated centriolescould also be detected. The nuclear arms are situ-ated equatorially. As in J. singaporensis, onlycleistospermia could be identified in T. sirikit.

Potamonautidae: Deckeniinae:HydrothelphusiniHydrothelphusa aff. madagascariensis (Figs.5G–I and 4)

The spermatozoa of this Madagascan species arelarge, as indicated by acrosomal measurements(Figs. 2 and 3, no. 9). The acrosome proportionsare slightly elongate (AL/AW 5 1.1 6 0.09, n 5 9),and the operculum is strongly apically projected(Fig. 3, no. 9). No evidence for the existence of nu-clear arms could be detected, although they havebeen found in all other brachyurans with theexception of some podotreme crabs (Jamieson,1994). The outer acrosomal zone is prominent and,in contrast to the other here investigated sperma-tozoa, is inwardly convex. The more electron-dense, tubule-like, structured acrosome ray zonebetween the outer acrosomal zone and the perfora-torium broadens apically and contacts the outerrim of the operculum. Inner and middle acrosomalzones are absent, and the acrosome ray zone con-tinuously attaches to the perforatorial chamber,the subopercular zone and the outer acrosomalzone. A periopercular rim could not be identified.Any evidence for spermatophores, either of cleisto-or coenospermic type, is lacking. Nevertheless,around the corrugated outer margin of the sper-matozoa a thin and light zone is situated thatcould be interpreted as vestigal spermatophorematrix (Fig. 5I).

Gecarcinucidae

Diagrammatic drawings of longitudinal saggittalsections of many gecarcinucid spermatozoa aregiven in Figure 8. The acrosomal shape of theGecarcinucidae is spherical or slightly depressed(AL/AW 5 0.8–1.0). Oziothelphusa sp. from Indiahas the smallest acrosome of the investigatedgecarcinucids (AW 5 1.98 6 0.2 lm, AL 5 1.72 60.2 lm, n 5 9; Fig. 2, no. 14) and Parathelphusaaff. maindroni the largest (AW 5 2.93 6 0.2 lm,AL 5 2.49 6 0.2 lm, n 5 12; Fig. 2, no. 17). Theperforatorial chamber is of moderate relative size(the diameter approximately one-third of the total

AW). Spermatophores, if present, are irregular orspherical (cleistospermic) and always consist of amucous matrix in which the spermatozoa are em-bedded. A complex spermatophore pellicle, as inthe potamiscines, is absent. Only in Phricotel-phusa gracilipes, Terrathelphusa kuhli, and Siam-thelphusa improvisa (Fig. 9G) could a pellicleconsisting of two thin, electron-dense layers beidentified. Although in the other gecarcinucids acomparable structure was not identified, its ab-sence can be attributed to the poor preservation.

Phricotelphusa gracilipes (Figs. 8 and 10A–C)

The spermatozoa are of relatively small size andthe acrosomes are spherical in shape (AL/AW 5

1.0 6 0.03, n 5 12). The perforatorial chamber isbroad in diameter, about one-third of the AW. Theacrosomal ray zone is outwardly convex, its struc-ture being very distinct and more granular-likethan the typical ‘‘fingerprint’’ style. A middle acro-somal zone is absent. The operculum is imperfo-rate and bulges out, and a thin periopercular rimcan be identified. The nuclear arms are very smalland distributed over the whole surface of thenucleus, in some cases only apically aroundthe operculum. Several spermatozoa are looselyembedded in a coenospermic homogeneous matrix.

Sartoriana spinigera (Figs. 8 and 10D–F)

The spherical spermatozoa of Sartoriana spini-gera (AL/AW 5 1.0 6 0.07, n 5 9) are of largersize than in Phricotelphusa gracilipes. The acro-some ray zone is relatively thin and cylindrical. Amiddle acrosomal zone is absent, the outer acroso-mal zone is homogeneous and less electron densethan the ray zone. The periopercular rim is veryprominent and situated more under the edge ofthe operculum than surrounding it. Nuclear armsare found laterally and basally. Only cleistosper-mic single spermatozoa that are enveloped by athin membrane were found.

Oziothelphusa ceylonensis (Figs. 8 and 10G–I)and Oziothelphusa sp. (Figs. 8 and 10J–L)

Of the genus Oziothelphusa two species wereinvestigated, O. ceylonensis from Sri Lanka andone undetermined species from India. The sperma-tozoa of both species are very similar in theirstructure, but not in their size. The acrosome ofOziothelphusa sp. is the smallest of the here inves-tigated spermatozoa (mean AW 5 1.98 6 0.2 lm,AL 5 1.72 6 0.2 lm, n 5 9; Fig. 2, no. 14) and sig-nificantly smaller than O. ceylonensis (mean AW 52.82 6 0.2 lm, AL 5 2.22 6 0.2 lm, n 5 5; Fig. 2,no. 13). The acrosome of O. ceylonensis is slightlydepressed (AL/AW 5 0.8 6 0.04, n 5 5), while it is



nearly spherical in Oziothelphusa sp. (AL/AW 50.9 6 0.09, n 5 9). The acrosome ray zone is of thefingerprint-type in both species, outwardly convexand does not reach the subopercular zone. A mid-dle acrosomal zone is absent. The outer acrosomalzone is more electron dense than the ray zone. Theoperculum is gently and centrally bulging. There

is no periopercular rim. The nuclear arms arearranged at the basal side of the spermatozoon. InOziothelphusa sp. they have a small diameter,occur in high number, and appear in longitudinalsagittal section as if arranged in two rows. In bothspecies, the coenospermic spermatozoa are packedin a homogeneous matrix.

Fig. 8. Diagrammatic drawings of gecarcinucid spermatozoa (longitudinal sagittal view).Scale bar 5 1 lm.

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Terrathelphusa kuhli (Figs. 8 and 11A–C)

The spermatozoa of Terrathelphusa kuhli arenearly spherical (AL/AW 5 0.9 6 0.07, n 5 13).The zoning of the acrosome is similar to Oziothel-phusa ceylonensis. the acrosome ray zone is out-wardly convex, and the outer acrosomal zone ho-mogeneous. In contrast to the spermatozoa of thegenus Oziothelphusa, the perforatorial chamberhas a larger diameter. The operculum is stronglyconvex but not particularly apically bulging (Fig.3, no. 15). A periopercular rim is absent. The nu-clear arms are situated laterally often having alarge diameter. The many coenospermic spermato-zoa are situated in a homogeneous matrix. Withinthe spermatophores, clusters of spherical electron-

lucent, globular structures with an electron-densecore are visible.

Parathelphusa convexa (Fig. 11D–F) andParathelphusa aff. maindroni (Fig. 11G–I)

Spermatozoa of two closely related species of thegenus Parathelphusa were investigated, P. convexafrom Java and P. aff. maindroni from south Suma-tra. The acrosomal structure in both species is verysimilar. The acrosome ray zone is outwardly convex,apically reaching the subopercular material in P.convexa but not in P. aff. maindroni. A middle acro-somal zone is absent. The operculum bulges cen-trally (more distinct in P. convexa). The operculum

Fig. 9. Gecarcinucid spermatozoa and spermatophores. TEM. (A–C) Geithusa pulchra. (D–F) Heterothelphusa fatum. (G–I)Siamthelphusa improvisa. First column longitudinal sagittal section, second column cross section, and last column spermatophores.ar, acrosome ray zone; ‘‘ma,’’ middle acrosomal zone-like layer; nu, nucleus; na, nuclear arm; oa, outer acrosomal zone; pr, perioper-cular rim; sw, spermatophore wall. Scale bar 5 1 lm or as indicated.



Fig. 10. Gecarcinucid spermatozoa and spermatophores. TEM. (A–C) Phricotelphusa gracilipes. (D–F) Sartoriana spinigera.(G–I) Oziothelphusa sp. India. (J–L) Oziothelphusa ceylonensis. First column longitudinal sagittal section, second column cross sec-tion, and last column spermatophores. ar, acrosome ray zone; ia, inner acrosomal zone; nu, nucleus; na, nuclear arm; oa, outeracrosomal zone; pr, periopercular rim. Scale bar 5 1 lm or as indicated.

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in P. aff. maindroni shows a comparatively thinelectron-dense zone that is connected to the outerspermatozoal membrane by columnar structures,not detected in the other investigated freshwatercrabs. A periopercular rim is present. Nuclear armsare many in both species and are situated laterally.The spermatozoa are of similar size, with the acro-some of P. convexa being spherical (AL/AW 5 1.0 60.05, n 5 7), while the acrosome of P. aff. maindroniis slightly depressed (AL/AW 5 0.8 6 0.04, n 5 12).The spermatozoa of both species are coenospermi-cally packed in a homogeneous matrix.

The following five species are closely relatedwithin the Gecarcinucidae. We will term this

assemblage the ‘‘Somanniathelphusa-group’’ (seeKlaus et al., 2009), and their close phylogeneticrelationship is reflected by a very similar spermmorphology.

Geithusa pulchra (Figs. 8 and 9A–C)

The acrosome of the spermatozoa of Geithusapulchra is spherical (AL/AW 5 1.0 6 0.07, n 5 9).In contrast to all other investigated species of the‘‘Somanniathelphusa-group,’’ the operculum dis-tinctly bulges out (Fig. 3, no. 18). A periopercularrim is only weakly developed, and there is a prom-inent subopercular zone. The acrosome ray zone is,

Fig. 11. Gecarcinucid spermatozoa and spermatophores. TEM. (A–C) Terrathelphusa kuhli. (D–F) Parathelphusa convexa. (G–I)Parathelphusa aff. maindroni. First column longitudinal sagittal section, second column cross section, and last column spermato-phores. ar, acrosome ray zone; ia, inner acrosomal zone; nu, nucleus; na, nuclear arm; oa, outer acrosomal zone; pr, periopercularrim. Scale bar 5 1 lm or as indicated.



also in contrast to the other spermatozoa of thisgroup, not cylindrical but outwardly convex. Amiddle acrosomal zone is absent. There are manynuclear arms that are located on the apical, lat-eral, and basal side of the spermatozoon. Geithusapulchra exhibits coenospermia with the spermato-phores showing a strong zonation with a less elec-tron-dense outer zone containing the spermatozoaand a more electron-dense central sperm-free zone.

Heterothelphusa fatum (Figs. 8 and 9D–F)

The spermatozoa of Heterothelphusa fatum arecharacterized by their prominent periopercularrim. The acrosome is nearly spherical (AL/AW 50.9 6 0.08, n 5 9), and the operculum is less bulg-ing than in Geithusa pulchra (Fig. 3, no. 19). Theacrosome ray zone is cylindrical in shape. Its gran-ular structure is clearly visible. Only in cross-section can the differentiation into a more elec-tron-dense outer and a less electron-dense middleacrosomal zone be identified (Fig. 9E). Nucleararms are present, although poorly preserved. Thespermatozoa are packed into spherical individualcleistospermic spermatophores with an inner elec-tron-lucent zone encapsulated by a thick and moreelectron-dense layer.

Siamthelphusa improvisa (Figs. 8 and 9G–I)

The spermatozoa of Siamthelphusa improvisaare spherical (AL/AW 5 1.0 6 0.07, n 5 11). Theacrosome ray zone reaches the subopercular mate-rial and is cylindrical. A middle acrosomal zone isabsent and a periopercular rim is only weaklydeveloped. Two small parallel centrioles can beidentified at the base of the perforatorium. The nu-clear arms are poorly preserved; they are situatedlaterally and at the basal side of the sperm. Likein Geithusa pulchra, the coenospermic spermato-phores are differentiated into zones. Each sperma-tozoon is situated in a granular matrix that fuseswith the matrix of the other spermatozoa withinthe spermatophore. This aggregate is embedded inmore electron-dense material (Fig. 9G). The wholespermatophore is encapsulated by a clearly visiblemembrane.

Somanniathelphusa sp. (Figs. 8 and 12A–C)and Sayamia bangkokensis (Figs. 8and 12D–F)

The spermatozoa of these two species are verysimilar as expected by the close relationship of thetwo genera (Naiyanetr, 1994). Nevertheless, asobserved within the genus Oziothelphusa, acroso-mal sizes differ, with the acrosome of Sayamia

Fig. 12. Gecarcinucid spermatozoa and spermatophores. TEM. (A–C) Somanniathelphusa species. (D–F) Sayamia bangkokensis.First column longitudinal sagittal section, second column cross section, and last column spermatophores. ar, acrosome ray zone;‘‘ma,’’ middle acrosomal zone-like layer; nu, nucleus; na, nuclear arm; oa, outer acrosomal zone; pr, periopercular rim. Scale bar 51 lm or as indicated.

188 S. KLAUS ET AL.


bangkokensis being smaller (Somanniathelphusasp.: AW 5 2.3 6 0.2, AL 5 2.2 6 0.3, n 5 12; Fig.2, no. 21; S. bangkokensis: AW 5 2.2 6 0.2 lm, AL5 1.9 6 0.2 lm, n 5 10; Fig. 2, no. 22). The shapeof the acrosome is in both species spherical ornearly spherical (Somanniathelphusa sp.: AL/AW5 1.0 6 0.07, n 5 12; S. bangkokensis: AL/AW 50.9 6 0.06, n 5 10). The thin acrosome ray zone iscylindrical. The operculum is only gently and cen-trally bulging and operculum measurements arebroadly overlapping (Fig. 3, nos. 21 and 22). Theperiopercular rim and the subopercular materialare prominent. The only structural difference is alight middle acrosomal zone in Somanniathelphusasp. that could not unequivocally be identified inSayamia bangkokensis. There are many nucleararms situated laterally and at the base of the nu-cleus. S. bangkokensis shows cleistospermia whilein Somanniathelphusa sp. no evidence of sperma-tophores could be identified, with the spermatozoafloating uncoated in the vas deferens.

DISCUSSIONSpermatophore Morphology

There are basic differences in sperm packingbetween the investigated Potamidae (Potamis-cinae) and the Gecarcinucidae. Although in theGecarcinucidae the spermatozoa are irregularlyembedded in a mucous matrix, they are denselypacked in the Potamiscinae and surrounded by acomplex pellicle consisting of several layers. In thesecond subfamily of the Potamidae, the Potaminae,only cleistospermic spermatophores are described(Guinot et al., 1997), but also consisting of severallayers. The mucous type of spermatophore was bio-chemically analyzed by Jeyalectumie and Subra-moniam (1987) for Spiralothelphusa hydrodroma(Gecarcinucidae, their misspelled Paratelphusahydrodromous) and showed to contain protein, freecarbohydrates, and lipids. The morphological dif-ferentiation in more and less electron-dense areaswithin mucous spermatophores (identified in Gei-thusa pulchra and Siamthelphusa improvisa)points to a biochemically different composition ofthese areas. This could be due to different primaryfunctions of the matrix types, for example, nutri-tion or protection. Such a morphological differen-tiation of the spermatophore matrix was alreadydescribed in marine brachyurans like Scylla ser-rata (Portunidae, see Uma and Subramoniam,1979) and Chaceon fenneri (Geryonidae, seeHinsch, 1991). The densely packed spermatophoresof the Potamiscinae, leaving very little space notonly between the individual spermatozoa but alsobetween spermatozoa and spermatophore wall,argue for a main function as a transfer device. Atleast a nutritional function can most probably beexcluded, in contrast to the gecarcinucid spermato-phores. The potamiscine spermatophores also

question the assumption that in all brachyuransthe spermatophores consist of sperm masses thatare merely surrounded by seminal fluid (Hinsch,1991).

It is shown that the occurrence of cleistospermiais not an apomorphy uniting the genera Potamon(Potamidae: Potaminae) and Potamonautes (Pota-monautidae: Potamonautinae) as taken into con-sideration by Guinot et al. (1997). It is a veryvariable character and both cleisto- and coenosper-mia occur within the investigated potamids andgecarcinucids. Even within the monophyletic‘‘Somanniathelphusa-group’’ in the Gecarcinucidaeboth types of spermatophores occur, cleistospermicspermatophores in Sayamia bangkokensis and Het-erothelphusa fatum and coenospermic in Siamthel-phusa improvisa and Geithusa pulchra. The earlyseparation of G. pulcher from the investigated spe-cies of the ‘‘Somanniathelphusa-group’’ (Klauset al., 2009) argues for coenospermia to be the ple-siomorphic character state. The endpoint of thisreductive evolution could be the spermatozoa ofSomanniathelphusa sp., where any evidence of aspermatophore envelope is absent. The reductionof spermatophores in the ‘‘Somanniathelphusa-group’’ might be correlated with a change in themechanism of sperm transfer, as all species exceptG. pulchra have males with reduced second gono-pods. However, this possibility would not explainthe occurrence of coenospermia in S. improvisa.Based on the frequent occurrence of coenospermiain the Gecarcinucidae and Potamiscinae, this char-acter state most probably is plesiomorphic in bothgroups, although they are probably not homolo-gous. The exclusive presence of coenospermic sper-matophores containing only two spermatozoa inPotamiscus beieri (depicted in Brandis, 2000) pos-sibly represents an intermediate between coeno-and cleistospermia. Unfortunately, the phyloge-netic relationship between the potamoid families(Gecarcinucidae, Potamidae, and Potamonautidae)and the identity of their marine sister groupremains elusive, preventing an outgroup compari-son for spermatophore morphology.

In Oziothelphusa ceylonensis (Gecarcinucidae),intact spermatophores were found in the femalegonoduct that leads from the gonopore to the sper-matheca (Fig. 13A). The spermatophore massesare situated centrally in the gonoduct and are sur-rounded by an amorphous substance, probablyseminal fluids. The diameter of the sperm masses(about 80 lm; gonoduct diameter about 150 lm)can be correlated with the diameter of the grooveof the male second gonopod (about 50 lm in O. cey-lonensis, data not shown). Possibly, the spermato-phores in the female gonoduct serve together withthe hardened seminal fluids as a sperm plug pre-venting a successive fertilization by male competi-tors (Diesel, 1991). A few millimeters upward inthe corresponding spermatheca only free spermato-



zoa could be found (Fig. 13B). This argues for animmediate dissolution of the spermatophores thatenter the spermatheca as it was reported for Libi-nia emarginata (Pisidae, see Hinsch, 1991). A ‘‘dif-ferential dehiscence’’ as observed in Chionoecetesopilio (Majidae) by Beninger et al. (1993) can beexcluded, at least for O. ceylonensis. The occur-rence of free spermatozoa in the spermatheca andintact spermatophores in the vagina points to sper-mathecal fluids eliciting spermatophore disintegra-tion in O. ceylonensis, and not external triggers(Diesel, 1991).

Sperm Morphology

Sperm morphology is another character thePotamiscinae and the Gecarcinucidae can be dis-tinctly separated on. Morphometric differences aremost obvious. The spermatozoa of the potamiscinesare much larger, as indicated by measurement ofacrosome width and length. The size differencebetween gecarcinucid and potamiscid spermatozoais definitely independent of body size. Possibly,there is a relationship between sperm size (andtherefore a relationship with spermatophore size)and the diameter of the second gonopod structuresfor sperm transfer. These structures consist of asmall groove in the Gecarcinucidae and a largertube in the Potamidae. Nevertheless, this wouldnot explain size differences in cleistospermia andin gecarcinucid species that have a reduced groove.The ratio of AL to width shows that the Gecarci-nucidae have more spherical acrosomes (AL/AW 50.9–1.0), while the acrosomal shape in the Pota-miscinae is slightly depressed (AL/AW 5 0.7–0.8).In relation to operculum width, the gecarcinucidshave higher, more bulging operculae than thepotamiscines (see Fig. 3). Moreover, in the Pota-miscinae operculum height is also strongly affectedby operculum thickness and therefore by overallsperm size and not by operculum shape.

A major difference between Gecarcinucidae andPotamiscinae is the complexity of the acrosome. Inthe Potamiscinae, in addition to the acrosome rayzone, the spermatozoa can always be distinguishedby a thin middle and prominent outer acrosomalzone. This seems to be an apomorphy of the Pota-miscinae, as in the Potaminae, the Potamonauti-dae, and the Gecarcinucidae this character isabsent. The faint middle acrosomal zone of Hetero-thelphusa fatum and Somanniathelphusa sp. is nothomologous to the potamiscine character state asthese two species nest deeply within the Gecarcinu-cidae (Klaus et al., 2009). Also the ‘‘tongue andgroove’’ connection between operculum and outeracrosomal zone only occurs in the Potamiscinae andis a potential apomorphy of this group. Both char-acters, the middle acrosomal zone and the ‘‘tongueand groove’’ connection, are also absent in theinvestigated Potaminae so far (see Guinot et al.,1997) and therefore separate, as diagnostic charac-ters, the two subfamilies of the family Potamidae.

The spermatozoa of the Gecarcinucidae can becharacterized, besides their smaller size, by theoccurrence of a prominent, electron-lucent perioper-cular rim. The periopercular rim of the Gecarcinu-cidae extends beneath the rim of the operculum.Therefore, it is probably not homologous to the peri-opercular rim described in Potamonautes sidneyi(Potamonautidae: Potamonautinae) that is situatedon the shoulder of the acrosome (Jamieson, 1993).In Hydrothelphusa aff. madagascariensis (Potamo-nautidae, Deckeniinae), no equivalent structure toa periopercular rim can be found. In the Potamisci-nae and in the Potaminae (Guinot et al., 1997), akind of vestigal periopercular rim is present.

Hydrothelphusa aff. madagascariensis differsfrom all other potamoid spermatozoa investigatedin this article by its slightly elongated acrosomeand the inwardly convex outer acrosomal zone.The shape of the operculum is similar to that ofPotamonautes sidneyi, but more centrally bulging

Fig. 13. Histology of the female reproductive apparatus of Oziothelphusa ceylonensis (Goldnerstaining). (A) Section through the vagina, with intact spermatophores (arrows). vg, vagina; cu,vaginal cuticle. (B) Free sperm in the spermatheca of the same specimen.

190 S. KLAUS ET AL.


(Jamieson, 1993). Acrosomal proportions and theshape of the acrosome ray zone are distinctly dif-ferent from Potamonautes, although both specieswere assigned to the same family (Cumberlidgeet al., 2008). The absence of a periopercular rim isin contrast to Potamonautes sidneyi (see Jamieson,1993). These differences probably reflect a long in-dependent evolutionary history and may justifythe assignment to different families (but see Cum-berlidge et al., 2008). A close relationship of theDeckeniinae and the Gecarcinucidae (the ‘‘Gecarci-nucoidea’’ sensu Klaus et al., 2006) is definitely notsupported by spermatozoan morphology, which isconsistent with the studies of Daniels et al. (2006),the molecular data in Klaus et al. (2006), and thetaxonomic reappraisal of Cumberlidge et al.(2008). Further investigations are necessary toevaluate probable synapomorphies of the Deckenii-nae, especially concerning the dubious absence ofnuclear arms (that would be unique within theBrachyura) and spermatophore structure. Moreinsight into sperm morphology of the Potamonauti-nae would be preferable, too.

Three spermatozoal characters were claimed tobe synapomorphies uniting Potamonautes andPotamon by Guinot et al. (1997): the elongation ofthe two centrioles, their parallel disposition, andthe reduction of the thickened ring that surroundsthe basal opening of the perforatorial chamber (thelatter occurring also in the Grapsidae and theGecarcinidae). Unfortunately, the centrioles arehardly visible in the presently investigated species,probably due to fixation problems in tropical envi-ronments. Only in Thaiphusa sirikit, Siamthel-phusa improvisa, and Sartoriana spinigera cantheir parallel arrangement be identified. It cannotbe excluded, that this character state also occursin the other gecarcinucid and potamid species andtherefore might represent a synapomorphy for thePotamoidea. The thickened ring is reduced in allinvestigated species. A vestigal thickened ring canbe identified in T. sirikit, Pudaengon thatphanom,Johora singaporensis, S. spinigera, and Sayamiabangkokensis. This character could be a secondspermatological synapomorphy uniting the OldWorld freshwater crabs.

Furthermore, Guinot et al. (1997) proposed twopotentially synapomorphic characters of Potamo-nautes and Potamon (besides the occurrence of coe-nospermia that we contested as a synapomorphyabove): a wide inner acrosomal zone and the absenceof a definite acrosome ray zone. We disagree withthe interpretation of these characters. We recognizetheir ‘‘inner acrosomal zone’’ as the acrosome rayzone because of its distinct granular or tubuliformpattern. This zone can be identified so far in allfreshwater crabs including the genera Potamon andPotamonautes. Here we follow Jamieson (1993),who already described this zone in Potamonautessidneyi as the acrosome ray zone. It can also be con-

firmed, that the nuclear arms are wrapped aroundthe spermatozoon in freshwater crabs (with thedoubtful exception of Hydrothelphusa aff. madagas-cariensis) and that the nuclear membrane is simpleand not multilamellar (see Guinot et al., 1997).

Within the Potamiscinae, Johora singaporensisand Thaiphusa sirikit have very similar spermato-zoa, due to the situation of the nuclear arms, theshape of the acrosome and operculum, the zonationof the acrosome, and the occurrence of cleistosper-mia. The only differences are in size, the ray zonein T. sirikit attaching to the perforatorial chamberapically and the operculum in J. singaporensisbeing perforate. Also the spermatozoa of Geothel-phusa albogilva, Pudaengon thatphanom, andMalayopotamon cf. brevimarginatum show distinctsimilarities. The acrosomal ray zone is muchbroader in these species, the outer acrosomal zoneis outwardly convex, the operculum bulges cen-trally and they always have coenospermic sperma-tophores. Larnaudia beusekomae shows an inter-mediate morphology between J. singaporensis–T.sirikit and the other investigated Potamiscinae.The acrosome shows Johora-like features, as theprominent outer acrosomal zone and the middleacrosomal zone and acrosome ray zone are both cy-lindrical and thin. In contrast to J. singaporensisand T. sirikit, L. beusekomae has densely packedcoenospermia, a centrally bulging operculum thatis not planar but curved downward laterally. Asdetailed phylogenetic data on the Potamiscinae, ei-ther morphological or molecular, are still lacking, itis difficult to evaluate the phylogenetic informationof these spermatological similarities concerningtheir apo- or plesiomorphic character. Most prob-ably, J. singaporensis and T. sirikit are more closelyrelated than to the other investigated Potamisci-nae. The spermatozoa of Potamiscus beieri (seeBrandis, 2000) seem to resemble closely those ofPudaengon thatphanom in acrosome morphologyespecially concerning the shape of operculum andperforatorial chamber. The spermatozoa of Sinopo-tamon yangtsekiense show potamiscine characterslike the depressed acrosomal shape, the middleacrosomal zone, and a shallow ‘‘tongue and groove’’connection (see Du et al., 1999). The wide acrosomeray zone attaching the operculum and the bulgingoperculum of S. yangtsekiense resemble stronglythe morphology of Geothelphusa albogilva.

The investigated spermatozoa of the Gecarcinuci-dae are very similar. Apart from their compara-tively small size, the bulging operculum, the perio-percular rim, the relatively broad outwardly convexray zone, and the absence of a middle acrosomalzone belong to their ground pattern. The acrosomeray zone repeatedly changes from outwardly convexto cylindrical shape (in Sartoriana spinigera andthe ‘‘Somanniathelphusa-group’’ excluding Gei-thusa pulchra). Also the periopercular rim isreduced several times. It is present but weak in



Phricotelphusa gracilipes, belonging to the sistergroup of all other gecarcinucids (Klaus et al., 2009),but absent in the genus Oziothelphusa, and in Ter-rathelphusa kuhli and Austrothelphusa transversa(see Jamieson and Tudge, 2000). As T. kuhli and A.transversa belong to the same phylogenetic lineagewithin the Gecarcinucidae (see Klaus et al., 2009),the reduction of the periopercular rim could repre-sent an apomorphy for this group.

The spermatozoa of the investigated congenersare of very similar shape. Surprisingly, both in Ozio-thelphusa and Parathelphusa spermatozoa of conge-ners differ profoundly in size. In Parathelphusa, thedifferences in the opercular structure are also dis-tinct. Moreover, the electron density of the ray zoneand the overlying outer acrosomal zone varies inlongitudinal sagittal section in both species.

In contrast to the Potamidae and the Potamo-nautidae, a differentiation of the Gecarcinucidae isnot supported by spermatology. This applies bothfor the approach of Klaus et al. (2006) based onthe second gonopod (two subfamilies: Gecarcinuci-nae and Parathelphusinae) and on approachesbased on character states of the frontal triangle(Bott, 1970, three families: Gecarcinucidae, Para-thelphusidae, and Sundathelphusidae).

Spermatozoal morphology clearly carries phylo-genetic information within the Old World fresh-water crabs. This is evident especially for thePotamiscinae, which show several character traitsthat are probably apomorphic. Acrosomal size andshape differences are also of phylogenetic signifi-cance, as shown by the differences between theGecarcinucidae and the Potamidae. In contrast,within these groups, acrosomal size does notreflect phylogenetic relationship. The Potamonau-tinae and Potamidae seem to overlap in acrosomalsize, while within the Potamonautidae the twosubfamilies Deckeniinae and Potamonautinae canpreliminarily (as we investigated only one speciesof the Deckeniinae) be separated.

A prerequisite for spermatological studies infreshwater crabs, as probably for studies in Bra-chyura in general, is a larger sample size. Investi-gating just one species per group highly increasesthe probability of false assumptions on the homol-ogy for spermatozoal character states.

ACKNOWLEDGMENTS

The authors thank Prof. P.K.L. Ng, NationalUniversity of Singapore, C. Lukhaup, Stuttgartand Aquarium Glaser, Offenbach for providingspecimens. C. Kempendorf and A. Lautenschlager(Heidelberg) gave laboratory support and G. Adam(Heidelberg) assisted with the photographic work.They are also grateful to Dr. C.C. Tudge, AmericanUniversity, Washington D.C., and to an anony-mous reviewer, who kindly improved their Englishand gave valuable comments on the manuscript.

LITERATURE CITED

Beninger PG, Lanteigne C, Elner RW. 1993. Reproductive proc-esses revealed by spermatophore dehiscence experiments andby histology, ultrastructure and histochemistry of the femalereproductive system in the snow crab Chionoecetes opilio (O.Fabricius). J Crust Biol 13:1–16.

Bott R. 1970. Die Sußwasserkrabben von Europa, Asien, Aus-tralien und ihre Stammesgeschichte. Abh Senckenb Natur-forsch Ges 526:1–338.

Brandis D. 2000. The taxonomical status of the freshwater crabgenus Potamiscus Alcock 1909 (Decapoda, Brachyura, Pota-midae). Senckenb Biol 80:57–100.

Brown GG. 1966. Ultrastructural studies of sperm morphologyand sperm-egg interaction in the decapod Callinectes sap-idus.. J Ultrastruct Res 14:425–440.

Cumberlidge N, von Sternberg R, Daniels SR. 2008. A revisionof the higher taxonomy of the Afrotropical freshwater crabs(Decapoda: Brachyura) with a discussion of their biogeogra-phy. Biol J Linn Soc Lond 93:399–413.

Daniels SR, Cumberlidge N, Perez-Losada M, Marijnissen SAE,Crandall KA. 2006. Evolution of Afrotropical freshwater crablineages obscured by morphological convergence. Mol Phylo-genet Evol 40:227–235.

Diesel R. 1991. Sperm competition and the evolution of matingbehavior in Brachyura with special reference to spider crabs(Decapoda, Majidae). In: Bauer RT, Martin JW, editors. Crus-tacean Sexual Biology. New York: Columbia University Press.pp 145–163.

Du NS, Lei W, Chen LQ, Xue LZ, Li TW, Wang L. 1999. Studieson the comparative ultrastructure of crab spermatozoa (Crus-tacea, Decapoda, Reptantia, Brachyura). Zool Res 20:372–378[In Chinese].

Felgenhauer BE, Abele LG. 1991. Morphological diversity ofdecapod spermatozoa. In: Bauer RT, Martin JW, editors.Crustacean Sexual Biology. New York: Columbia UniversityPress. pp 322–341.

Goldner J. 1938. A modification of the Masson trichrome tech-nique for routine laboratory purpose. Am J Pathol 14:237–243.

Guinot D. 1978. Principe d’une classification evolutive des Crus-taces Decapodes Brachyoures. Bull Biol Fr Belg 112:211–292.

Guinot D, Jamieson BGM, Tudge CC. 1997. Ultrastructure andrelationships of spermatozoa of the freshwater crabs Potamonfluviatile and Potamon ibericum (Crustacea, Brachyura, Pota-midae). J Zool Lond 241:229–244.

Guinot D, Jamieson BGM, Richer de Forges B, Tudge CC. 1998.Comparative ultrastructure of the three dromiacean familiesexemplified by Homolodromia kai (Homolodromiidae),Sphaerodromia lamellata (Dromiidae), and Dynomene tanen-sis (Dynomenidae) (Podotremata: Brachyura). J Crust Biol18:78–94.

Heidenhain M. 1917. Uber neuere Sublimatgemische. Z WissMikrosk 33:232–234.

Hinsch GW. 1986. A comparison of sperm morphologies, trans-fer and sperm mass storage between two species of crab, Ova-lipes ocellatus and Libinia emarginata. Int J Invert ReprodDev 10:79–87.

Hinsch GW. 1988. Ultrastructure of the sperm and spermato-phores of the golden crab Geryon fenneri and a closely relatedspecies, the red crab G. quinquedens, from the eastern Gulfof Mexico. J Crust Biol 8:340–345.

Hinsch GW. 1991. Structure and chemical content of the sper-matophores and seminal fluid of reptantian decapods. In:Bauer RT, Martin JW, editors. Crustacean Sexual Biology.New York: Columbia University Press. pp 290–307.

Jamieson BGM. 1991. Ultrastructure and phylogeny of crusta-cean spermatozoa. Mem Queensl Mus 31:109–142.

Jamieson BGM. 1993. Ultrastructure of the spermatozoon ofPotamonautes perlatus sidneyi (Heterotremata, Brachyura,Crustacea). S Afr J Zool 28:40–45.

Jamieson BGM. 1994. Phylogeny of the Brachyura with particu-lar reference to the Podotremata: Evidence from a review of

192 S. KLAUS ET AL.


spermatozoal ultrastructure (Crustacea, Decapoda). PhilosTrans R Soc Lond B Biol Sci 345:373–393.

Jamieson BGM, Tudge CC. 2000. Crustacea–Decapoda, Part 1.In: Jamieson BGM, editor. Progress in Male Gamete Ultra-structure and Phylogeny, Vol. 9, Part C of Adiyodi KG,Adiyodi RG, series editors. Reproductive Biology of the Inver-tebrates. Chichester: Wiley and Sons. pp 1–95.

Jamieson BGM, Guinot D, Richer de Forges B. 1995. Phylogenyof the Brachyura: Evidence from spermatozoal ultrastructure(Crustacea, Decapoda). In: Jamieson BGM, Ausio J, JustineJ-L, editors. Advances in Spermatozoal Phylogeny and Taxon-omy. Mem Mus Nat Hist Nat, Ser A Zool 166:265–283.

Jeyalectumie C, Subramoniam T. 1987. Biochemical compositionof seminal secretions with special reference to LDH activityin the reproductive tissues of the field crab, Paratelphusahydrodromous (Herbst). Exp Biol 46:231–236.

Klaus S, Schubart CD, Brandis D. 2006. Phylogeny, biogeogra-phy and a new taxonomy for the Gecarcinucoidea Rathbun,1904 (Decapoda: Brachyura). Org Divers Evol 6:199–217.

Klaus S, Brandis D, Ng PKL, Yeo DCJ, Schubart CD. 2009.Phylogeny and biogeography of Asian freshwater crabs of thefamily Gecarcinucidae (Brachyura: Potamoidea). In: MartinJW, Crandall KA, Felder DL, editors. Decapod CrustaceanPhylogenetics. Crustacean Issues (in press).

Matos E, Matos P, Corral L, Azevedo C. 1996. Ultrastructuraland morphological aspects of the spermatozoon of Dilocarci-nus septemdentatus Herbst, 1783 (Crustacea, Decapoda, Tri-chodactylidae) of the northern sea-coast of Brazil. Braz JMorphol Sci 13:31–35.

Naiyanetr P. 1994. On three new genera of Thai ricefield crabsallied to Somanniathelphusa Bott, 1968 (Crustacea: Decap-oda: Brachyura: Parathelphusidae). Raffles Bull Zool 42:695–700.

Subramoniam T. 1991. Chemical composition of spermatophoresin decapod crustaceans. In: Bauer RT, Martin JW, editors.Crustacean Sexual Biology. New York: Columbia UniversityPress. pp 308–321.

Tudge CC, Justine J-L. 1994. The cytoskeletal proteins actinand tubulin in the spermatozoa of four decapod crabs (Crus-tacea, Decapoda). Acta Zool 75:277–285.

Uma K, Subramoniam T. 1979. Histochemical characteristics ofspermatophore layers of Scylla serrata (Forskal) (Decapoda:Portunidae). Int J Invert Reprod Dev 1:31–40.

von Sternberg R, Cumberlidge N, Rodrıguez G. 1999. On themarine sister groups of the freshwater crabs (Crustacea:Decapoda: Brachyura). J Zoolog Syst Evol Res 37:19–38.

Wang L, Du NS, Lai W. 1999. Studies on spermiogenesis of afreshwater crab Sinopotamon yangtsekiense (CrustaceaDecapoda). Acta Hydrobiol Sin 23:31–33 [In Chinese].



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Ultrastructure of Spermatozoa and Spermatophores of Old World

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