Table Of ContentTHE NAUTILUS 123(4):293-302, 2009 Page 293
Spermatozoan ultrastructure and detection of nuclear acid
phosphatase activity in spermatids o{ Anomalocardia hrasiliana
and Tivela mactroides (Bivalvia: Veneridae)
Gisele Orlandi Introini Alexandre Lobo Mario Manuel Shirlei M. Reeco-Piinentel^
Departaiiiento de Anatomia da Cunlia da Silva Leite Sousa Departamento de Anatomia
BiologiaCelulare Fisiologia Universidade do Porto Universidade do Porto Biologia Celular e Fisiologia
Instituto de Biologia Institnto de Ciencias Institnto de Ciencias Instituto de Biologia, Universidade
Universidade Estadual de Biomedicas Abel Salazar Biomedicas Abel Salazar Estadual de Campinas
Campinas (UNICAMP) Laboratorio de Biologia Laboratorio de Biologia Celular (UNICAMP)
CP 6109, 130S3-863 Celular, Largo Professor Largo Professor Abel Salazar, 2 CP 6109, 13083-S63, Campinas
Campinas, BRAZIL Abel Salazar, 2 4099-003 Porto, PORTUGAL BRAZIL
4099-003 Porto, and [email protected]
PORTUGAL Centro de Genetica da
Reprotlnyao ProfessorAlberto
Barros, Avenida do Bessa591
1° Direito Pb'ente 4100-009
PORTUGAL
Porto,
ABSTRACT and phylogenetic analyses (Bernard and Hodgson,
1985; Guerra et ah, 1994; Sousa and Oliveira, 1994;
We compared the ultrastructure of spermatozoa from the Healy, 1995a, b; Garrido and Gallardo, 1996; Komarn
biviilves Anomalocardki hrasiliana and Tivela mactroides and Konishi, 1996; Healy et ah, 2001; Erkan and Sousa,
a(Vqeunaesrpideanen),tsTpheei.nspwehrimcahtotzhoea hofeabdotchonstpaeicnisesawceurie'veodfnthuecleecuts- 2002; Gwo et ah, 2002; Introini et ah, 2004; Healy et ah,
with a short cone-shaped acrosome. An invagination penetrated 2006). In addition, these studies showed that closely
almosttheentirelengthoftlieacrosome.Themidpiececontained related species can be distinguished based on the nltra-
ap;xirofoitliogonaUy arranged eentrioles surrounded byspheri- strnctnre of their spermatozoa (Hodgson et ah, 1990;
calmitochondriaandtheIlagellumhadthetyjDical9+2structure. Gwo et ah, 2002; Introini et ah, 2004).
The spermatozoa ol A. hrasiliana had a slightly cuived nucleus Various features of spermatozoan ultrastructure have
while those of T. mactroides had a long, prominently cun’ed been associated to aspects of reproductive biology.
nucleus. The mitochondria were equally distributed around the Franzen (1955, 1956, 1977, 1983) proposed that inver-
eentrioles in the midpiece of A. hrasiliana spermatozoa, but tebrates have two tyqies of sperm, namely, primitive
asymmetrically in the midpiece of T. mactroides spermatozoa. sperm, produced by species with external fertilization,
Thereweresixmitochondriaandglycogen clustersin themiddle
pieceoi the T. mactroides spermatozoon. Thepresence of glyco- and modified sperm, produced fry species with internal
genclusters andthehighernumberof mitochondria,incompari- fertilization. Primitive sperm consists of a short, round
sonwithAnomalocardki hrasiliana, couldextemlthe longevityof or conical head, a midpiece containing 4-5 sphierical
die Tivela mactroides spennatozoa. An increase in spenn life mitochondria and a flagellum with a 9+2 microtu-
e.xpectancy implies in an increase in the probability of finding bular structure. Spermatozoa that are released directly
eggs and accomplishing fertilization. The glycogen clusters and into the surrounding water are named acpiasperm or
thehighermitochondrianumberpossiblycorrespondtoanadap- atpiatic sperm. Bouse and Jamieson (1987) introduced
tiveadvantagetothebivalvesinturbulentwaters. a new terminology and described two cupiasperm cate-
gories: (1) eet-acpiasperm referring to sperm that lerti-
lizes eggs in the ambient water; (2) ent-aipiasperm
referring to sperm that fertilizes eggs into the mantle
cavity of mollnscs or into the tube of sedeutaiy poly-
INTRODUCTION chaetes.
Sperm moqrhology has also been correlated \ritli egg
Comparative studies of the Bivalvia have confirmed the sizeandlaiwaldevelopment (Franzen, 1983; Komarn and
usefulness of spermatozoan morphology for taxonomic Konishi, 1996). These relationships amongspermatozoan
morphology and distinct reproductive patterns have
' Corresponding author strengthened the relevance of studies that investigate
Page 294 THE NAUTILUS, Vol. 123, No. 4
tlie association ofevolution, ecology, and the morpholog- Paulo, Brazil, under the accession numbers 1419 (Tivela
ical diversits'of spermatozoa. mactroides) and 1420 {Auomalocardia hrasiliana).
The Veiieroida is considered one of the most impor-
tant orders of bivalves because it comprises several ma- Scanning Electron Microscopy: Sperm suspensions on
wriindeespfarmeialdiegseoogfraepchoincodmiisct,ribeuctoiloong,icsaulchraesletvhaencVeenearnid- pcoavrearfsolirpmsalwdeerheydfeixiend0i.n22M.5%sogdlinutmarcaalcdoedhyyldaete,anpdH2.7.02%,
dae (Gw'o et ah, 2002). Data describing spermatozoan For 1 h at room temperature. Subsequently, they w^ere
nltrastructnre of Veneridae species have supported the rinsed several times in the same buffer and post-fixed in
identification of traits shared by the majority of the 2% osmium tetroxide in the dark for 1 h. Samples were
members belonging to this family (Pochon-Masson and dehydrated in a graded series of ethanol solutions and
Gharagozlon, 1970; Gharagozlon and Pochon-Masson, critical-point dried in C02. The dried coverslips w^ere
1971; Nicotra and Zappata, 1991; Rennov and Plodgson, mountedon stubs, coatedwith gold andexaminedwith a
JSM 5800 LV microscope.
1994; Guerra et ah, 1994; Matos et ah, 1997; Gwij et ah,
2002; Erkan and Sousa, 2002; Park et ah, 2002; Guerra Transmission Electron Microscopy: Small fragments of
et ah, 2003; Yiug et ah, 2008). testis were fixed with 2.5% glntaraldehyde and 2.0%
Numerous reports have emphasized that venerid paraformaldehyde in 0.2 M sodium cacodylate, pH 7.2,
bivalves are important components of the marine ben- for 5 h at 4°C and then rinsed in the same buffer.
thos, including non-consolidated bottom communi- Samples were post-fixed in 2% osmium tetroxide in the
ties (Narchi, 1972; Etchevers, 1976; Schaeffer-Novelli, same buffer, for 1 h at 4°C, and then dehydrated in a
1980; Prieto, 1980; Soares et ah, 1982; Prieto, 1983; graded acetone series follow^ed by gradual infiltration
Mclachlan et ah, 1996; Arruda andAmaral, 2003; Arruda with EPON resin before embedding. Ultrathin sections
et ah, 2003). Anomolocardia hrasiliana (Gmelin, 1791) is w^ere stained with nranyl acetate and lead citrate, and
distributed throughout the Caribbean islands and also in examined w4th a Zeiss Leo 906 transmission electron
Suriname, Brazil and Uruguay (Amaral et ah, 2005). microscope.
This species lives buried a few centimeters below the
suiface of compact sand in the intertidal zone of calm Phosphotungstic Acid Staining: Small samples oftestis
w'aters. Adults ofA. hrasiUana rapidly buiy themselves w^ere fixed as described above, bnt wnthont post-
w'hen placed on wet mud or muddy sand. Tivelo inac- fixation, and subsequently dehydrated in a graded
twides (Born, 1778) occurs around the Ascension ethanol series. Samples were stained with a 2% phos-
Island, along the Caribbean seaboard, and in Venezuela, photnngstic acid-ethanol (E-PTA) solution at low pH, in
Suriname and Brazil (Amaral et ah, 2005). In contrast to order to detect glycoproteins and enable c\4ochemical
A. hrasiliaua, T. mactroides lives in turbulent waters in analysis ofspermatozoa. After 2 h in the E-PTA solution,
the intertidal zone. samples were rinsed with ethanol, transferred to ace-
Althongh, the spermatozoa ultrastrncture of bivalves tone, and infiltrated with EPON resin before embed-
has been largely investigated and used to solve many ding. Ultrathin sections were examined with a Zeiss Leo
tnxonomic and phylogenetic issues, immune and cyto- 906 transmission electron microscope.
chemical studies of sperm and precursoiycells are scant. Nuclear ACPase Detection During Spermiogenesis:
Biochemical features of reproductive cell lineages could
Small fragments oftestis were fixed with 2.5% glntaralde-
contribute to taxonomical descriptions andto distinguish hyde in 0.2 M sodium cacodylate, pH 7.4, for 2 h at 4°C,
some of the bivalve species. and rinsed in the same buffer. They were then washed
In thiswork, scanninOeandtransmission electron micros- with 50mM Na-acetate-HCl buffer, pH 5, at 4 °C. Subse-
copywere usedin thestudyofthe spermatozoan moiphol- quently, the tesbs fragments were incubated in 50mM
ogy of the venerids Auomalocardia hrasiliana and Tivela Na-acetate-HCl buffer, pH 5, with 5% sucrose, 13.9 inM
mactroides, w'hich w'ere compared with those of other sodium P-glycerophosphate (P-GP) and 3.6 niM lead
bixalve species, aiming at a better understanding oftheir nitrate, for 30 min at 37°C, under dark conditions with
taxonomic placement andphylogeny.
constantandgentlemixing. Sampleswerewashedtwicein
50mAI Na-acetate-HClbuffer, pH 5,with 5% sucrose, for
5 min at4°C, andtwicein0.1 M Na-cacodylatebuffer, pH
MATERIALS AND METHODS 7.2, with 5% sucrose, for 5 min at 4°C. The specimens
w'ere post-fixed in 2% osininm tetroxide in the same
Specimens ol A)iomalocardia hrasiliana were sampled in bnifer, for 2 h at 4°C, and then dehydrated in a graded
the intertidal zone ofSao Sebastiao County (23°48'57.2" ethanol series and propylene oxide. A gradual infiltration
S, 45°24'29.8" W), and Tivela mactroides sampled in with EPON resin w'as done before embedding. Ultratliin
Garaguatatuba County (23°38'51.7", 45°25'31.4" W), sections were examined with a |EOL 100 CX H-TEM,
both located on the southeastern coast of Sao Paulo
without staining.
State, Brazil.
The voucher specimens were deposited in tlie Museii 4lie experimental controls consistetl ol: (1) Omission
de Zoologia ‘ Prolessor Adao |ose Cardoso” (ZUEC) at o( p-glyceropho,sphate; (2) Addition of inhihitor fOniM
the State University ol Campinas (PJNICAAIP), Sao NaPh (3) Other snb.strates: 6.4 inM Thiamine pyro-
C;. O. Introi'ni et al„ 2009 Page 295
phosphate chloride; 2.5 niM Na-inosine- 5-diphosphate; the residual cytoplasm was progressively eliminated,
mM
0.6 Na-trimetaphosphate. splierical mitochondria assembled in tlie base of the
nnclens around the two centrioles (Figures 27 and 31).
In Anomalocardia brasdiana gonads, a nuclear acid
RESULTS phosphatase (ACPase) was detected in all spermatid
stages using the improved Gomori-chloride technicpie.
Spermatozoa: The spermatozoa of both species were In comparison, in Tivela mactroides gonads, the pres-
either oi the ect-acjuaspenn tv^re (Figures 3, 4, and 12). ence of ACPase was detected in mid and (inconspicu-
The short acrosomal complex was cone-shaped and lo- ously) late spermatids using the same methodology. In
cated anterior to the nnclens. Two components of the the sperm cell of both species, nuclear ACPase was not
acrosomal vesicle were distinguished based on their di- detected (Figures 28 and 32). The experimental controls
verse electron densities (Figures 20 and 22). The conical did not show staining.
acrosome was deeply invaginated, the snbacrosomal re-
gion was filled with a diffuse material, and there was no
axial rod (Figures 1, 2, 10, 11, and 13). DISCUSSION
PTA staining at low piI revealed no glycoproteins in
the acrosomal vesicles of Anomalocardia I)rasiliaiia and The moiphological structures of the venerid spermato-
Tivela mactwides spermatozoa (Figures 5, 6 and 14). zoa described herein agree with the ect-aqnasperm type
The nnclens was relativelv long, cnn/ed, cylinder, and proposedby Rouse and famieson (1987), exhibiting mor-
the midpiece consisted of spherical mitochondria phological characteristics described for free-spawning
grouped around a pair of short cylindrical centrioles bivalves. In a previous study ofbivalve sperm nltrastrnc-
(Figures 7, 8, 15, 16, and 17). Extensive electron-dense ture, Healyet al. (1995b) proposed five categorieswithin
grannies or grannie clusters, considered to he glycogen the order Veneroida. The spermatozoa descriptions pre-
deposits, were observed around the centrioles and sented here are in agreement with the data reported by
mitochondria of T. mactroides spermatozoa (Figures 15 Ilealy et al. (1995b) regarding members belonging to
and 16). In the region immediately posterior to the mid- Group A. Members of Group A share the following
piece, the triplet snbstrnctnre of the centrioles was traits: a randomly organized snbacrosomal material, an
replaced by a standard 9 + 2 inicrotnbnlar pattern trxo- electron-lucent area at the acrosomal apex, a relatively
neme that terminated in a long flagellnm (Figures 9 long and slender rod nnclens that slightly decreases in
and 18). Overall, the spermatozoa ofA. brasdiana and thickness toward the gamete apex, and absence of an
T. )iiactroides shared high morphological similarities, anterior nuclear fossa.
even though the nnclens was sliglitly cniwed in the As described below, previous studies described de-
A. brasdiana spermatozoa compared to the markedly tailed nltrastrnctnral patterns of venerid spermatozoa;
cniwed and long nnclens of T. mactroides (Table 1, the reported data allows for an infoianal taxonomic anal-
Figures 19 and 24). The mitochondria were equally ysis of this bivalve group.
distiibnted around the centrioles in the midpiece of Venentpisanrea (Gmehn, 1791) produces a.spermatozo-
A. brasdiana spermatozoa (Figure 21). In Tivela mac- on with a veiy slightly cnwed nnclens and electron-dense
troides spermatozoa, the mitochondrial assembly did not regions at the base ofthe acrosomal vesicle (Gharagozlon
form a ring structure but slunved a biased distrilrntion of and Pochon-AIasson, 1971). Nicotra and Zappata (1991)
the organelles around the orthogonally arranged pair of investigated the sperm cells of Callista chione (Linnaeus,
centrioles, such that they were always more numerous 1758), which also exhibit unclear cni'vatnre and the same
in one side of the sperm cell (Figure 23). Hence, the patternofacrosomalelectrondensiW Rennovand Hotlgson
midpiece of T. mactroides spermatozoa was rotationally (1994) described tire spermatozoan moiphology of tire
asymmetrical and conPiined clusters of glycogen, which venerid clanr Tivela polita (G.B. Sowerby II, 1851) from
were not seen in the midpiece ofA. brasdiana. South Africa. The head of T. polita spernratcrzoa is about
3.2 pur long and has a cylindrical, slightly cnived nnclens
Precursoiy Cells: The nomenclature used to refer to capped by a sirraU conical acrosonre. In the venerid clairrs
precursor)' cells was that of Nicotra and Zapata (1991), Protothacatliaca (Moliira, 1782) andAmeohinomi/aantuina
which is not in accordancewith the tlefinitions proposed (Kingaird Broderip, 1832) (asVemisantiqna). the reported
by Ying et al. (2008). lengdr of the .sperm head is about 7.5 pur and 5.3 pnr,
The early spermatids were rounded but irregular in re.spechvely, and the acrosonre is a snrall vesicle in the
outline and had a spherical nnclens with patches of anterioi' region of the cell (Guerra et al, 1994). Spernr
condensed chromatin in the middle and in the cell pe- nltrastructnre studies of Protothaca pecforifia (Lanrarck,
riplieiy (Figures 25 and 29). The nnclens was still 1818) from the northenr coast of Brazil show'ed that the
rounded in the mid-spermatid stage, but the chromatin male ganrete e.xhibits acni'ved nnclens (Alatos et al., 1997).
condensation was intensified (Figures 26 and 30). The species Gafrarinm tinnidnm Rikling, 1798, and Circe
The late spermatid was characterized by the elonga- scripta (Linnaeus, 1758) (Gircinae), Pilarsnlfnrenm Pilsbiy,
tion of the nnclens. Chromatin condensation has com- 1804 (Pitarinae), and Gomphiita acijiiilatera (G.B, Sowerby
pleted and only a few nuclear vacuoles remained. While I, 1825) (Tapetinae) share highly sinrilar spernratozoa
Page 296 THE NAUTILUS, Vol, 123, No. 4
Figure.s 1-9. S]H'niiato/()on o( Anoiualocimliabrasiliaiui. 1. Acrosonie, nucleus and inulpiece. 2. Acrosomal comple.xand unclear
apex. .3-4. SEAl sliowing head and nagellmn. 5-6. Idiospliotuugstic acid staining. 7. Longitudinal section ol the midpiece.
S. Ti'ansversal section of the midpiece showing lour sjiherical mitochondria grouped as a ring around the proximal centriole.
9. Longitudinal section ol the llagellnm. Scale l)ars = 0.5 pm, exc(']it lor Figure 3 = 10 pm and Figure 4=1 pm. Ahl)re\'iations:
a, acrosome: f, llagc'llmii; h, head; m, mitochondria; n, miclens.
G. O. Introini et al, 2009 Page 297
Figures 10-18. Spermatozoon of Tivcla inacfroidcs. 10. Acrosonie, nucleus and inidpiece. 11. Aerosouial complex and unclear
apex. 12. SEM slumnughead and flagellum. 13. Acrosomal complex and nuclear apex. 14. l.ackof pliosphotuugstic acid stainingin
the acrosomal vesicle. 15. Longitudinal section ol the rnidpiece, sliowing six spherical mitochondria grouped around the proximal
centriole. 16. Longitudinal section of the midpiece. 17. Spherical mitochondria grouped around the distal centriole. 18. Longitudi-
nal section of the nagelhnn. Scalebars = 0.5 pm, except forFigure 12 = 1 pm. Abhreviations: a,acrosonie; f, llagclhini; gly,ghcogen
clusters: h, head; ni, mitocliondria; n, nucleus.
Page 298 THE NAUTILUS, Vol. 123, No. 4
Table 1. Morphometric and numerical data of analyzed The elongated head of venerid spermatozoa usually
sperm structures in A. bmsiliana and T. mactroides. contains a cui'ved nucleus, which characterizes these
bivalves (Rennov and Hodgson, 1994). However, tire nu-
Acrosomal Nuclear Head
length width length Numberof cleus cuivature is not unique to venerids since it has also
been reported in galeommatoidean bivalves (Eckelbarger
Species (pm) (pm) (pm) mitochondria
et al, 1990). Nicotra and Zappata (1991) stated tirat it
A. hrasiliana 0.4 0.9 3 4 shouldbeinterestingtoaniilyzemovementandfeitilization
T. mactroides 0.4 0.5 4.5 5-6 patterns in tiiis curved sperm, in order to evtiluate the
function^ significance oftitis characteristic. However, con-
sideringtire microscopicdimensions ofthespermcells, the
moiphology and ultrastmcture, as reported by Gwo et al. nucleus shape should be irrelevant to tire movement be-
(2002). Spenn cells of tliese biviilve species consisted of cause these bodies have low Reynolds numbers, notbeing
a cim-'ed head, a short inidpiece with tightly packed mito- possible to derive anyappreciable tinnst from inertia.
chondria and a long tail. The spermatozoa of die bivalve The apical halfofthe spermatozoon acrosomal vesicle
mollusksPitarnicUs (Poh, 1795) andChomeleagallina (Lin- is stained with PTA in the species Riichtapes decussatus
naeus, 1758) (Veneridae) from Turkey were characterized (Linnaeus, 1758) (as Venenipis decussatus), Eurhomalea
by a conical and slightly cuwed nucleus about 2.6 pm and rufa (Lamarck, 1818), Protothaca thaca, and Venus anti-
3.5 pm long, respectively, and acrosomal vesicles about cjua (family Veneridae), as reported by Sousa et al.
0.6 pm long (Erkan and Sousa, 2002). The sperm cell of (1998). This is in contrast to the negative PTA staining
Gomphina veneiifonnis (Lamarck, 1818) exliibited a con- pattern of Anomalocardia hrasiliana and Tivela mac-
spicuous cuwature and tiie head (8.5 pm) is considered troides spermatozoa.
veiylong(Parketal.,2002). Spermcells oftheclamMerce- Sperm moi'phological studies described non-crnwed
naiia mercenaria (Linnaeus, 1758) (from China), were nucleus in the venerid Eurhomalea nifa (Sousa et ah,
investigatedbyYingetal. (2008), sharingcommon features 1998, Guerra et ah, 2003). The nucleus of sperm cells
withotiierveneridspecies, suchastiiepresenceofacurved were cinwed in all other studied venerid species. The
nucleus andashortacrosomalvesiclewhichshowselectron ultrastructural characteristics of Ruditapes decussatus
dense regions in itsbase. (Pochon-Masson and Gharagozlou, 1970; Gharagozlou
and Pochon-Masson, 1971) were different in compari-
son to other venerid species, especially in relation to
Tivelamactroides acrosomal features. It is important to emphasize that
the sperm cell of Eurhomalea ntfa shows significant
moqjhological differences in comparisonwith other spe-
cies of the family Veneridae. These sperm cells differ-
ences have not yet been explained, representing an
invitation to a taxonomic review ofthese species.
Besides their similarities, A. hrasiliana and T. mac-
troides spermatozoa showed prominent ultrastructural
differences. The sperm nucleus was slightly cinwed
in A. hrasiliana and prominently cuiwed and long in
T. mactroides. In the A. hrasiliana sperm cell midpiece,
there were four mitochondria uniformly distributed
around the centrioles. As for T. mactroides, there were
SLx asymmetrically distributed mitochondria. Finally, the
midpiece of T. mactroides contained glycogen deposits
whereas that ofA. hrasiliana did not. These moipholog-
ical patterns suggest that the T. mactroides spermatozoa
could be adapted to turbulent en\4ronments.
Narchi (1972) compared structural and functional
morphologies as well as adaptations ofA. hrasiliana and
T. mactroides, whicli are species living close to the sur-
face in soft substrata with suspension-feeding habits.
Their most prominent anatomical feariires were related
0
I ' to burrowing behavior and suspension-feeding. Aiioma-
locardia hrasiliana, which lives in calm waters ofmuddy
beaches, does not have tentacles along the mantle edge
whereas the inhalant siphon and mantle edge of T. mac-
troides have several ramified tentacles. The siphonal
Figure 19. Diagrammatic representation of A. hrasiUatw tentacles prevent the penetration of large particles into
and T. mactroides spermatozoa. Scale bar = 1 pm. the mantle cavity while tlie ramified tentacles along the
C,. (). Introini et ;il., 2009 F'd^v 299
Figures 20-24. Illiislvations ol spei'iiiatozoaii leatnres ol Aiiomaloranlia hmsiliaua and 'I'iichi nuuiroitics based on SFM and
TEM images processed tlirongli 3D animation rendering and modeling soltware. 20. Apex ol the lieail ol tlie spermatozoon ol
Ijrasilianii Acrosomal length = 0.4 pm. 21. Micklle piece ol the spermatozoon ol A. Ijmsiliann Nuclearwidth = 0.9 pm; mitoclion-
. .
drion = 0.55 pm. 22. Ajrex ol the head ol the spermatozoon ol 71 matiroidcs. Acrosomal length = 0.5 pm. 2.3. Middle piece ol the
spermatozoon ol T. tiuictroidc.s. Mitochondiion = 0.5 pm. 24. (ieneral \isnalization ol the spermato/.oan leatnres ol both spennato-
zoa. Mitochondrion = 0.55 pm (in sjrermatozoan rc'presentation at right side ot the lignre.) ,Abbre\iations: a, acrosonie; dc, distal
centriole; f, llagellnm; gly, glycogen clusters; h, head; ni, mitochondria; n, nnclens, pc, proximal centriok-.
Page 300 THE NAUTILUS, Vol. 123, No. 4
m
•
n
t‘
f
25
/
1 - '
V- .
• ' n
u.-'
W:
;;A a vd ^ i
iLl .. 26
• -sat-
m
^
:
n
* ;? /
T /
,
27
i
C '
L,.
\
- .
V*
/ n-
/
^
^ ‘ rn\
. 28
Figures 25-28. Prccurson' cells ol Aiuwitilocardia hrasili-
duti. 2.5. Parly spermatids with spherical mulens with patches
ol condensed chromatin in the middle and in the cdl jreriph- Figures 29-32. 1’reciirson' cells ol TivcI/i madroides.
eiA'. Note staining indicating the presence ol nuclear AClhise. 29. Parly spermatiils with spherical lindens witli ]xitches ol
Nuclear diamet<n' = 4.2 pm. 20. NirIcils still ronndetl in niid- condensed chromatin in the middle ainl in the cell [leriphen'.
spci'inatid stage, hnt chromatin condensation was intensilied Mitochondrion = 0.0 pm. .30. Nucleus was still rounded
(|iresence ol unclear ACddrse). Nuclear width = 3 pm and in mid-spermatid stage, hnt chromatin condensation was in-
unclear length = 4 pm. 27. Late sjiermatid diaracteri/i'd hv tensilied (presence ol unclear ACPase). Nuclear diameter =
dongation ol lindens (jirescnce ol nuclear ACPase). Mito- 2.25 pin. .31. Latesjierniatid characterized hytheelongation ol
chondrion = 0.0 pm. 28. Nndear AtiPase not detected in the the imcleiis. Mitochondrion = 0.0pm. .32. NuclearACPase not
sperm cdl. Mitodiondrion = 0.0 pm. Alihre\'iations: a, acro- detected in thesperm ct>ll. Mitochondrion = 0.0 pm. Ahhrevia-
some: ni, mitochondria; n, lindens. tions: a, acrosome; iii, mitochondria: ii, nnclens.
G. O. Intromi et al„ 2009 Pa^e 301
mantle edge prevent large particles from entering the LITERATURE CITED
pallial cavih'. These adaptations allow T. mactwides to
live on open sandy shores where large quantities ol ma- Amaral, C., E.R Arnula, and A.E. Rizzo, 200.5. Manual dc
terial are kept in suspension bv constant wave move- Ideiitifica^'ao dos Iiu'citebrados Marinhos da regiao
ments. Tivela mactwides also has the most ellicient Sudeste-Sul do Brasil. Vol. 1. Uni\’ersidade de Sao Paulo
form ol particle transport among demihranchs, which AuderPsroesus,, VSVa.oA.Paaulnod, 2P.88Peprps.onne. 1970. The localization ol
partly rellects adaptation to a spc-cific liabitat. In agree- glvcogen in the spermatozoa of various invertebrate and
ment with this characteristic, the stomach of T. mac- vertebrate species. The |ournal of Cell Biology44: 29-51.
twides is more complex than that ol A. hrasiliana, Anderson, W.iK. and P. Personne. 1976. Molluscan spermato-
which is related to the large number ol particles pi'esent zoon - dynamic aspects of its structure and function.
in this organ. Onr obsemitions on the spennatt)zoan American Zoologist 16: 29.3-313.
moqrholog)' of A. })rasiliana and T. mactwides mostly Arrnda, E.R and C.Z. Amaral. 2003. Spatial distribution <4
agree with Narchi (1972), who concluded that anatomi- mollnsks in the intertidal zone ol sheltered beaches in
southeastern ol Brazil. Bevista Brasileira de Zoologia 20:
cal \ariations in these species rellect adaptations to di-
291-.300.
verse environments. According to Andei'son and
Arrnda, E. P, (). Dornaneschi, andC.Z. Amaral. 2003. Mollnsk
Personne (1970; 1976) and Introhh et al. (2009), glyco- feedingguilds on sandybeaches in Sao Paulo State, Brazil.
gen storage in the middle piece ol bivalve sperm cells Mai'ine Biolog)' 143: 691-701.
has an important meaning in the spermatozoon physio- Bernard, R.T.Ft andA.N. Hodgson. 1985.Thefinestructureof
logical metabolism. The presence ofglycogen clusters in thespermandspermatiddifferentiationinthebrown mus-
the mid-piece could extend the longeviU' ol the sperm sel Pcrnapcrna. South African |onrnal ofZoology20: ,5-9.
cells. Any increase in the life expectancy ofsperm cells Eckelharger, K.J., R. Bieler, and P..VI. Mikkelsen. 1990. Ultra-
implies in an increase in the proliability of finding eggs structure of sperm development and mature sperm mor-
and increase in opportunities for fertilization. Tins could pholog)'in threespeciesofcommensal hi\al\es (Mollusca:
Galeommatoidea). Journal of Moiphohyg)'205: 6.3-75.
be an adaptive advantage in turbulent waters.
Erkan, M. and M. Sousa. 2002. Fine structural stuch' of the
Spermiogenesis in Veneroida species has been de- spermatogenic cycle in Pitar nidis and Cdunnelca gcdlina
scribed wath great accuracy emphasizing architectural (Mollusca, Bixalvia, \'eneridae). Tissue & Cell 34: 262-
details of cells (Nicotra and Zapata, 1991; Johnson 272,
et ah, 1996, Ying et ah, 200S). In the present work, the Etchevers, S.L, 1976. Notas ecologicas y cuantilicacidn de la
relatively low fixation shown in the electron micrographs pohlacion de gnacuco, Tivela mactroides (Born, 1778)
ofprecursory cells was carried out intentionally, in order (Biwilria-Veneridae), en la ensenada de La Cnardia, Isla
to avoid masking detection of Nuclear ACPase activity. de Margarita, Venezuela. Boletin del InstitutoOceanogra-
Considerable nltrastrnctnral modifications take place fico de la Universidad de Oriente 15: 57-64.
during spermiogenesis. The chi'omatin filaments aggre- Franzen, A. 1955. Comparative morphological investigations
gate into lamellar stnicriires and finally into a homoge- intothe spermiogenesis among Mollusca. Zoologische Bij-
neous andcompact DNAarrangement. h\Anomalocardia F^randzerna,geAn. U19p5p6s.alOan.3s0:pe3r9m9i—o4g5e6n.esis, moipholog)'ol the sper-
hrasiJiana and Tivela mactwides gonads, a nuclear acid matozoon, ami hiolog)' ol fertilization among inverte-
pho.sphatase (ACPase) was detected iii spermatids using brates. Zoologische Bijdragen Uppsala31: .3.5.5-482.
the improved Comori-chloride technicjne. In the present Franzen, A. 1977. Sperm structure with regartl to fertilization
analysis, the unclear acid phosphatase actixlh' in both biologyandph\logenetics. Verhamlhmgen der Uentschen
bivalve species follows a specific time and spatial-course Zoologischen Gesellschaft: 12.3-1.38.
pattern duringspermahd chromatin condensation. A con- Franzen, A. 1983. Ultrastrnctural studies of spermatozoa in
trolledand comparative studysuggests that this pattern ol tlii'ee bivalve species\rith notes on e\olntion of elongated
unclear acid phosphatase activityis specific ami related to spenn nucleus in primitive spermatozoa. Gamete Re-
chromatin compaction. search 7: 199-214.
Garrido, O. and G.S. Gallardo. 1996. Ultrastrnctnre ol sperms
In conclusion, onr results suggest that detailed analy-
in bivalve mollnsks of the Vlvtilidae farnilv. Invertebrate
ses of bivah'e spennatozoan ultrastrnctnre can be useful Reproduction & Development 29: 9,5-102.
tools in the investigation ol interspecific taxonomic relat- Gharagozlon, l.D.and Pochon-Masson, 1971. Etudecompar-
edness and adaptation to a given environment. Further ativeinfrastrnctnrale Duspermatozoidechezlespalourdes
studies on male gametes of venerid mollnsks are needed lie France. Archix'esdeZoologie Experimentaleet Generale
to verifv the taxonomic relevance of sperm morpliologi- 112:80.5-817.
cal and nltrastrnctnral characteristics. Guerra, R., B. Campos, and P. Espimda. 1994. Analysis of the-
spermatozoa of lour bi\alves with particular reference to
ACKNOWLEDGMENTS the acrosome and plasma membi'ane glycoproteins. Jour-
nal of Submicroscopic Cytology and Patholog)' 26: 489-
495.
The authors thank Dr. Alexander Turra and Dr. M;ircia
Guerra, R., M. Sousa, A. Torres, E. Oliveira, and L. Baldaia.
Denadai for helping with Anomalocardia brasilicnia and 200.3. Fine .stnicture oi acrosome biogenesis and ol mature
Tivela mactroides samplings. This work was supported sperm in the biialve mollu.sks Cdijci/meris sp. (Pteilomor-
by FAPESP (Grant no. 04/13887-4). APS. was supported phia) and Eiirlumialea nifa (Heterodonta). Helgoland Ma-
by POCI/SAU-MM()/6()7()9/60555/59994/()4; UMIB. rine Research 57: 7-12.
Page 302 THE NAUTILUS, Vol. 123, No. 4
Gwo, j.C., \\’.T. Yang, Y.T. Sheii, and II.Y. Cheng. 2002. Sper- Narchi, W. 1972. Comparative study of the functional mor-
niotozoan inorphologN' of four species of hi\alve (Iletero- phology oi'Anomalocardia brasilUma (Cmelin, 1791) and
donta, N'eneridae) from Taiwan. Tissue & Cell .34: 39—43. Tivcla mactroides (Born, 1778) (Bix-alxla, V'eneridae). Bul-
Ilealy, J.M. 199.5a. Sperm nltrastrnctnre in the marine bixalve letin ofMarine Science 22: 64,3-670.
lamilies Carditidae and Crassatellidae and its hearing on Nicotra, A. and S. Zappata. 1991 Ultrastrnctnre ofthe mature
nnification of the Crassatelloidea with tlie Carditoidea. sperm and spermiogenesis in CnUisfa cJdone (Mollnsca,
Zoologica Scripta24: 21-2S. Bixalxia). Inx'ertebrate Reproduction & Development 20:
Healy, J.M. 199.5b. Comparative spermatozoal nltrastrnctnre 21,3-218.
and its taxonomic and phylogenetic significance in the Park, C.K., J.J. Park, J.Y. Lee, and J.S. Lee. 2002. Spermato-
hivah'e order \Yneroida. In: Jamieson, B.C.M., Ansio, genesis and sperm nltrastructure of the equilateral x'enus
J.
and J.-L. JUSTINE (eds.) Advances in Spermatozoal Phy- Gomphina oeneiiformis (Biwilxla: Veneridae). Korean
logeny and Taxonomy. Memoires dn Mnsemn National d' Journal ofElectron Microscopy.32: 30.3-310.
Ilistoire Natnrelle 166, pp. 15.5-166. Pochon-Masson, J. and l.D. Charagozlon. 1970. Particnlarite
Healv,J.M., ).A. Bnckland-Nicks, and B.C.M. Jamieson. 2001. morphologiqne de I'aci'osome dans le speianatozoide de
Spermatozoal nltrastrnctnre of spiny oysters (Spondyli- Tapes decussatus L. (Mollnstpie lamellibranche). Annales
dae, Bixahia) inclnding a comparison with other bivalves. des Sciences Naturelles, Zoologie, 12: 171-180.
Invertebrate Beprodnction & Development 40: 27-37. Prieto, A.S. 1980. Contrihuicion a la ecologia de Tivela mac-
Healy, J.M., P.M. Mikkelsen, and R. Bieler. 2006. Sperm troides, Born, 1778: aspectos reprodntix'os. Boletim do
nltrastrnctnre in Glanconomc planktd and its relevance Instituto Oceanografico 29: 32.3-328.
to the affinities (4 the Clanconomidae (Bixalvia: lletero- Prieto, A.S. 1983. Ecologia de Tivela mactroides Born, 1778
donta). Invertebrate Beprodnction &: Development 49: (Mollnsca, Bix’alxla) en playa Ciiiria (Sucre, Venezuela).
29-39. Boletin del Instituto Oceanografico de Venezuela de la
Hodgson, A.N., R.T.F. Bernard, and C. Van der Horst, 1990. Phiiversidad de Oriente 22: 7-19.
Comparatixe spermatologx' of three species of Doiuix Reunox', A.A. and A.N. Hodgson. 1994. Ultrastructure ofthe
(BivaK'ia) from South Afilca. JournalofMolhiscan Studies spermatozoa of lix'e species of South African bixalx'es
56: 257-265. (Mollnsca), and an examination ofearly spermatogenesis.
Introhh, C.O., C.A. Magalhaes, O. Aguiar-Jr, A,J.C. Qnar- Journal of Moiphologx'219: 27.5-283.
esma, Lino-Neto, and S.M. Recco-Pimentel. 2004. Rouse, C.W. and B.C.M. Jamieson. 1987. An ultrastructural
J.
Spermatozoan moipholog)? of Brachidoiites dancinianns study of the spermatozoa of the polychaetes Eunithoe
and B. solisianti.s (Bixalxia, Mxtilidae) from the southern complanata (Amphinomidae), ChpneneJki laseroni and
Brazilian coast. Inx-ertehrate Reproduction & Develojr- Micromaldane laseroni (Maldanidae) xvith definition of
ment 46: 149-15S. sperm txqies in relation to reproductix'e biology. Journal
Introini, C.O., C.A. Magalhaes, H. Fortimato, and S.M. Recco- ofSubmicroscopic CxYology 19: 57,3-584.
Pimentel. 2009. Comparisonofthespermatozoan moiphol- Schaelfer-Novelli, Y. 1980. Analise populacional de Anomalo-
ogyoi Isog)ioinoii bicolorand Isoajiomon alatii.s (Mollnsca, cardia brasiliana (Cmelin, 1791), na Praia do Saco da
Biv;rlx’ia, Lsognomonidae). Tissue & Cell 41: 67—74. Ribeira, Ubatnba, Estado de Sao Paulo. Boletim do Insti-
Johnson, M.J., N. Casse, and M. Le Pennec, 1996. Spermato- tuto Oceanografico29: .351-.355.
genesis in the endosymhiont-hearing hixalve Loripes luci- Soares, 11.A,, Y. Schaeffer-Nox'elli, and J.R. Mandelli. 1982.
nalis (\'eneroida: Lncinidae). Molecular Repi'odnction &: Berbigiio Anomalocardia brasiliana (Cmelin, 1791), bi-
Dex'elopment 45: 476-484. xalxe comestiVel da regiao da Ilha do Cardoso, Estado de
Komaru, A. and K. Konishi. 1996. Ultrastrnctnreofhiflagellate Sao Paulo, Brasil: aspectos biologicos de interesse para a
spermatozoa in the freshw'ater clam, Corbiciila leaiia pesca comercial. Boletim do Instituto de Pesca9: 21-38.
(Prime). Inx'ertebrate Reproduction & Development 29: Sousa, M. and E. Olix'eira. 1994. An ultrastructural study of
19,3-197, Crassostrea angulata (Mollnsca, BivaK'ia) spermatogene-
Matos, E.. P. Matos, C. Casal. and C. Azevedo. 1997. Ultra- sis. Marine Biologx' 120: .54,5-551.
estrntnra do espermatozoide de Protothaca pectorina Sousa, M., R. Cuerra, E. Olixeira, and A. Torres. 1998.
(Lamark) (Mollnsca, Bixalxia) do litoral norte do Brasil. Comparative PTA stainingofmollnscan spermatozoa. Jour-
Rexlsta Brasileirade Zoologia 14: 779-783. nalof SubmicroscopicCxtologx'and Patholog)'.30: 183-187.
Mclachlan,A., J.E. Dugan,O. Defeo,A.D.An.sell,D.M. Ilnhha7'd, Ying, X.L., WX. Yang, II.U. Dahms, Z.H. Lin, andX.L. Chai.
E. Jaramillo, and PE. Penclurszadeh. 1996. Beach clam Spermatozoa and spermatogenesis in the northern qna-
fisheries. Oceanographyand Marine Biologx': An Annual Re- hang Merccnaria merccnaria (Mollnsca, Bix'alx'ia). 2008.
xlexv34: 16,'3-232. Helgoland Marine Research 62 (4):321-,329.
