Table Of ContentAnnual gonadal cycle of the land snail Scutalus tupacii
(Pulmonata: Bulimulidae)
Maria Gabriela Cuezzo
FacultaddeCiencias NaturaleseInstitutoMiguel Lillo, Universidad Nacional deTucuman-CONICET,
MiguelLillo205, 4000S.M. deTucuman, Argentina
Abstract: Theannual gonadalcycleanditsrelationtotheseasons were investigated inthelandsnailScutalustupacii(d'Orbigny). Collections ofadult
snailswerecarriedoutmonthlyovertwoconsecutiveyears. The animals wereprocessedforanatomical and histologicalstudies.The ovotestis iscomposed
ofanumberoftubulesorelongatedacini,eachonecontainingboth maleandfemalesexualcellsaswell asSertoli and follicularcells. Spermatogenesisand
oogenesisoccurinalltheacini.
Theannualcycleoftheovotestiswasdividedintothreephases: 1)Pre-breeding(September-November: Spring),characterizedby thereactivationof
spermiogenesis and the subsequent increase in number of mature sperm; 2) Breeding (November-February: Spring-Summer), characterized by the great
numberofspermatozoainthegonad,andthehighestproportionsofmatureoocytes; 3) Post Breeding(March-September: Autumn-Winter,withabundance
ofspermatogonia,oogoniaandprimaryspermatocytes,andtheinterruptionofspermiogenesis.
Although theproliferation ofmaleand female germinal cells is practically coincident, male gametes attained maturity at leastone monthbeforethe
femalegametes.Thepost-breedingperiodoccursduringhibernationwhichiscoincidentwiththedryseason. Basedonthedataobtained,theannualcycleof
reproductiveactivityinthisspeciesdependsprincipallyuponthewetorrainyseason.
The annual gonadal cycle ofmany species has been tupacii were carried out from September 1988 to March
previously investigated [e.g. Lymnaea stagnalis (Linne) 1991 in "Reserva Aguas Chiquitas" (El Cadillal, Tucuman,
(Berrie, 1966); Helix pomatia (Lind, 1973); Semperula Argentina), which is part of the subtropical tucuman-boli-
maculata (Tompleton) (Nanaware and Varute, 1975); Helix vian forest. The samples were divided into two groups. One
aspersa Miiller (Gomot and Griffond, 1987)]. Related group used for histological studies was fixed in Bouin's or
works include research on the maturation of the reproduc- in Baker's fixative (Humanson, 1979). A second group was
tive tract of various species (see Lusis, 1966; Smith, 1966; used for anatomical analysis, after being relaxed in cooled
Runham and Laryea, 1968; Sokolove and McCrone, 1978; boiled water. These were then immersed in Baker's fixative.
Runham and Hogg, 1979; Cuezzo, 1990) as well as the life The ovotestis and hermaphroditic duct were dissected out,
cycles of a number of groups (see Van Der Laan, 1975, dehydrated in an ascending alcohol series, embedded in
1980; Hodasi, 1979; Bailey, 1980; Solem, 1984; Roth, Paraplast and sectioned at 6 um. Sections were stained with
1986). More recently, there have been a growing number of Ehrlich haematoxylin-eosin and Mallory (Azan) Heiden-
studies that analyse the influence of different factors on hain (Humanson, 1979).
gametogenesis under laboratory conditions (Gomot and Monthly, ovotestis and hermaphroditic ducts were
Gomot, 1988; Gomot, Gomot and Griffond, 1989; Griffond fixed in Karnovsky's solution (Karnovsky, 1965) with 0.1
and Medina, 1989). M phosphate buffer (pH 7.2), post-fixed in 2% osmium
The genus Scutalus Albers, 1850, is well represent- tetroxide and embedded in Epon-araldite. Semi-thin sec-
ed in the western region of Tucuman Province (27°S, tions of 1 um were stained with toluidine blue and exam-
66°W), Argentina. Despite the large and widespread popu- ined by light microscope. Voucher specimens have been
lations, very few data are available regarding the biology of deposited in the Fundacion Miguel Lillo's malacological
these bulimulid land snails. In the hope of filling part of collection (Catalogue No. FML001000).
this gap, a population of S. tupacii (d'Orbigny) was sam-
pled over two consecutive years to find out the characteris- RESULTS
tics of the gonadal cycle and the possible changes that
occurduring the different seasons. As in other pulmonate gastropods, the ovotestis or
hermaphroditic gonad of adult Scutalus tupacii is found
MATERIALS AND METHODS embedded in the digestive gland which is located in the
upper whorls of the shell. The activity and the size of the
Monthly collections of adult land snails Scutalus ovotestis depend upon the age and size of the animal as
American Malacological Bulletin,Vol. 10(2)(1993):121-127
121
122 AMER. MALAC. BULL. 10(2) (1993)
well as the season. The structure ofthe ovotestis was found with abundant interacinar space (Fig. 1). The post-breeding
to be similar to most pulmonates (Joosse and Reitz, 1969; part of the cycle is characterized by an abundance of sper-
Luchtel, 1972a, b; deJong-Brink etal, 1977). matogonia and primary spermatocyte (Figs. 2, 3). Oogonia
From November to May (end of Spring to early are present isolated or in clusters of two to four cells and
Autumn), the acini are whitish and clearly separated from are always located inside the epithelium lining the acini,
each other. At the beginning of April, a progressive reduc- remaining in contact with the basal lamina. These cells are
tion in the size of the ovotestis occurs that peaks between recognizable by a poorly contrasted cytoplasm (Fig. 4).
June and July (Winter). The decrease in length and diame- Spermatids are scarce and are present only in early stages
ter ofthe acini are accompanied by a change in colouration of differentiation. Few morphologically mature spermato-
from white to light brown. The pigmentation is more evi- zoa are present in the lumen of the acini. In the distal
dent in the distal part of the acini. In October the ovotestis region of the acini the presence of mature Sertoli cells is
progressively begins to recover its size and color. remarkable. Their abundant cytoplasm is highly vacuolated
The annual cycle of the ovotestis can be divided and extends into the lumen, contributing to the "packed'
into three phases of activity: 1) Post-breeding phase appearance ofthe acini. Male germ cells at different stages
(March-September), Fall-Winter; 2) Pre-breeding phase of differentiation are clearly embedded within the same
(September-October), Spring; 3) Breeding phase (Novem- Sertoli cell.
ber-February), Spring-Summer.
2) Pre-breeding period (Figs. 5-10):
1) Post-breeding phase (Figs. 1-4):
Externally the ovotestis maintains the same appear-
During this phase the acini are small in diameter ance as in winter. The acini have few or lack spermatozoa
Figs. 1-4: Post-breedingperiod. Fig. 1.GeneralappearanceoftheaciniduringthePost-breedingperiod(Ac,acinarwall;scalebar=20pm). Fig.2. Group
ofspermatogonia(Sg)inclosecontactwithaSertolicell (S)withdarknuclei(scalebar= 10pm). Fig.3. Groupofspermatocytes(Sc)embeddedinthecy-
toplasmofaSertolicell(S).(scalebar= 10pm). Fig.4.Threeyoungoocytesincontactwiththeacinarwall(O,oocyte;Ac;acinarwall,scalebar= 10pm).
CUEZZO: GONADAL CYCLE OF SCUTALUS TUPACII 123
*
9 ^0
fy
Figs. 5-10: Pre-breedingperiod. Fig. 5.General aspect ofan acinus duringthe pre-breeding period (Ac, acinarwall; Sc, spermatocytes; Sz, spermatozoa;
Sp, spermatids; S, Sertolicells; scalebar=40pm). Figs.6-9. Differentstagesofspermatid maturation. Fig. 6. Early spermatidstage.Thesecells possessa
roundnucleuswithanteriorandposteriorplaquealreadydifferentiated(N,nucleus;scalebar= 10pm).Figs.7-9.Midspermatidstage.Thenucleusbecomes
indented atthe posteriorplaque. An elongating axonome is visible. (N, nucleus; A. axoneme; G, Golgi apparatus; scale bar= 10 pm). Fig. 10. Late sper-
matidstage.Elongatednucleuswithcondensedchromatin.TheheadsareembeddedinthecytoplasmofaSertolicell(S).(scalebar= 10pm).
124 AMER. MALAC. BULL. 10(2) (1993)
(Fig. 5). Toward the end ofSeptember and the beginning of In longitudinal sections of ovotestis (Figs. 11, 12),
October spermatids proliferate and different stages start to no interacinar space is observed and the layers of the adja-
become apparent (Figs. 6-10). Spermatids become more cent acini are in contact. The ovotestis of individuals that
abundant than spermatocytes. Also a growing number of hadjust copulated were almost empty, lacking mature male
spermatozoa can be observed in the lumen ofthe acinus. At gametes.
the bottom ofthe acini vitellogenic oocytes are present but The male phase in the ovotestis ofScutalus tupacii
never more than one peracinus. starts during the post-breeding period with an active prolif-
eration of spermatogonia and spermatocytes, coincident
with hibernation (end of autumn and during winter).
3) Breeding period (Figs. 11-14):
Although mature spermatozoa are present in the ovotestis
During this part of the cycle all the spermatogenic in every month of the year it is important to remark that
stages, from spermatogonia to spermatozoa are present in spermiogenesis is inactive during winter. Therefore the
the ovotestis (Figs. 11, 12). The lumen ofthe acini are filled mature gametes are less abundant in the acini while the
with spermatozoa. Concerning the female line, previtel- snails are hibernating. As soon as spermiogenesis begins,
logenic and vitellogenic oocytes (Figs. 13, 14) (Griffond toward the end ofOctober and beginning ofNovember, the
and Bolzoni-Sungur, 1986) are distributed along the acini lumen ofthe acini fdls with spermatozoa.
with a tendency towards maturation near the bottom of the Mature oocytes are present throughout the year
acinus. being especially abundant during the breeding period.
Figs. 11-14: Breeding-period. Fig. 11. Longitudinal sectionoftheproximal partofseveral acini. The lumensare saturatedwithspermatozoa(Sz) (Ac, aci-
narwall; scalebar=40pm). Fig. 12. Longitudinal section ofthedistal portionofseveral acini. The moreadvanced stages, spermatocytes (Sc), spermatids
(Sp) and mature oocytes (O) are observed in this region (scale bar- 50pjn). Fig. 13. Young premeiotic oocyte restingon the acinarwall (Ac) (O, oocyte;
scale bar= 10pm). Fig. 14. Vitellogenic oocyte. Notethe presenceoffollicularcells (Fc)thatenvelope theoocyte(O, oocyte; Ac, acinarwall;scalebar=
20pm).
CUEZZO: GONADAL CYCLE OFSCUTALUS TUPACII 125
Female cell proliferation begins in autumn, during May and selves under a thick layer of dry leaves and secrete a thin,
June, but the number of mature oocytes grow between transparent epiphragm. No mucus tracks were observed
November and December in the pre-breeding and breeding during these months (temperature range: 0 - 20°C). Another
periods. noticeable change is the retraction ofthe animal's body into
The seminal vesicle stores spermatozoa throughout the shell as aconsequenceofweight and waterloss.
the year. In the summer months, however, a noticeable Reproductive activity begins between October and
increase in both the numberofstored male gametes and the November when the first rains can deeply moisten the soil.
diameter of the hermaphroditic duct was noted. Although From this moment it is possible to observe copulating
rather constant patterns were recorded in the three repro- snails, especially nocturnally, but also during certain days,
ductive periods, individual variation in the lengths of the in particular the rainy ones. Mating can last several hours.
periods existed. The first clutch ofeggs, both in 1988 and 1989, were found
in November, generally buried in moist soil at the base of
large trees. The eggs have an opaque-white albuminous
SEASONALITY IN THE REPRODUCTION
substance within a nearly transparent membrane. The num-
According to W. Koppen's climatic classification ber of eggs per clutch ranged from 70 to 120 with diame-
ters from 2.5 to 3.0 mm. Under laboratory conditions the
(Torres Bruchman, 1976), the region that this population
duration ofdevelopment ranged from 15 to 18 days.
inhabits has a mesothermal climate with a dry winter (tem-
perature ofthe warmest month higher than 22°C and ofthe
coldest month lower than 18°C). The vegetation corre- DISCUSSION
sponds to a basal subtropical forest which is characterized
by the presence ofbig trees [Phoebeporphyria (Grisebach) Three different regions in gonadal acini of Helix
Mez, Jacaranda mimosifolia D. Don, Juglans australis aspersa juveniles (Griffond and Bride, 1987) and in
Grisebach and Tipuana tipu (Benth.) O. Kuntze] associated Biomphalaria glabrata (Say) (de Jong Brink, 1976) have
with epiphytplants like Phlebodium aureum (L.). Abundant been described. In adult Scutalus tupacii these regions are
ferns with wide front fronds cover the ground. The pres- notclearly identifiable. A trend toward a compartmentaliza-
ence ofa wet season in the region is the key to understand tion in male medullar and female cortical regions occurs.
the pattern of activity of these land snails. The length of Also there is a higher frequency of vitellogenic oocytes,
this period is approximately 6 months with the heaviest mature follicular and Sertoli cells at the bottom of the aci-
rains between January and March (Fig. 15). Winter is the nus. Morphologically mature spermatozoa are found in the
dry season when the snails are most of the time dormant. acinar lumen with their nuclei oriented toward the bottom
In1989, the dry season was longer than in 1990 and conse- and the flagellae toward the neck ofthe acinus.
quently the hibernation period was prolonged. At the begin- Despite the lack of strict regionalization, most of
ning ofthe dry period most ofthe animals bury themselves the mature gametes are typically found at the bottom of
vertically with the aperture of the body whorl below the each acinus. Young germinal cells are distributed randomly
soil and the spire above. Other snails simply protect them- along the acini. Coincidently, Runham and Hogg (1979)
have found in Deroceras reticulatus (Muller) that there is a
gradient of oocyte size being largest at the acinar base.
PreciPitation (mm) Temperature (°C) However, they also noted that the great enlargement of the
acini could, by itself, completely explain the observed
oocyte distribution: largest oocytes arise first from the base,
the oldest part of the acinus and remain there without any
active movement.
Although proliferation ofmale and female germinal
cells is practically coincident, male gametes reach maturity
at least one month before the female ones. A number of
studies, carried out in several species (Lusis, 1966; Runham
and Laryea, 1968; Luchtel, 1972; Runham, 1978) support
Sept Dec March Jun Sept Dec March Jun Sept Dec March the hypothesis of a clear separation in time of male and
MONTHS
female phases without superposition in the time ofprolifer-
~~*—Precipitation w Temperature ation of the germinal cells. During the breeding period of
t—
(Hibernating Period Scutalus tupacii both mature spermatozoa and oocytes are
Fig.15.Dryandwetseasonsfrom 1989to 1991. present in the ovotestis, coincidently with the summer
126 AMER. MALAC. BULL. 10(2) (1993)
months and wet season. During winter, the dry season, major (Binney) (Gastropoda: Polygyridae). American Malacolog-
hibernation takes place and gonadal activity is minimal icalBulletin 8(1):19-24.
with predominating male and female juvenile germinal de Jong-Brink, M., H. Boer, T. Hommes and A. Kodde. 1977. Spermato-
genesis and the role of Sertoli Cells in the freshwater snail
cells. Biomphalariaglabrata. CellandTissueResearch 181:37-58.
Luchtel (1972) suggests that Arion circumscriptus Gomot, P.and B. Griffond. 1987. Repercussion de la duree d'eclairement
L. would be defined as a protandrous hermaphrodite con- journalier sur revolution des cellules nourricieres et de la lignee
sidering the time at which the male and female gametes male dans l'ovotestis Helix aspera. Reproduction, Nutrition and
Development!!(1A):95-108.
reach maturity. However, in terms oftime ofdifferentiation
Gomot, P. and L. Gomot. 1989. Etude exploratoire de la spermatogenese
ofthe spermatogonia and oogonia the species could be con-
induite par la chaleurchez l'escargot Helix aspersa en hibernation
sidered a simultaneous hermaphrodite and finally in terms Role du cerveau. Invertebrate Reproduction and Development
of appearance of primary spermatocytes and oocytes Arion 16:23-32.
could be aprotogynous hermaphrodite. Gomot, P.,L.Gomotand B. Griffond. 1989. Evidenceforalightcompen-
Scutalus tupacii could be considered a protandrous sation ofthe inhibition ofreproduction by low temperatures in the
snail Helix aspersa. Ovotestis and Albumen Gland responsiveness
hermaphrodite if we define "protandry" as the maturation todifferentconditionsofphotoperiodsandtemperatures.Biologyof
of male gametes earlier than the female. However the pres- Reproduction40:1237-1245.
ence of mature oocytes and spermatozoa is coincident dur- Griffond, B. and J. Bride. 1987. Germinal and non-germinal lines in the
ing the breeding period without a great separation in time ovotestis of Helix aspersa: a survey. Roux's Archives ofDevelop-
of both phases. Among the pulmonates already studied, mentalBiology 196:113-118.
Griffond, B. and A. Medina. 1989.Timing ofspermatogenesis and sper-
most are true protandric hermaphrodites (Lusis, 1966; miation in snails Helix aspersa bred under short photoperiods: A
Smith, 1966; Runham and Hunter, 1970; Parivar, 1978) histologic and quantitative autoradiographic study. Journal of
except for the Achatinellidae which are registered as pro- ExperimentalZoology250:87-92.
togynous. Griffond, B. andJ. Bride. 1981. Etudehistologiqueetultrastructuraledela
Based upon the analysis of the climatic data (Fig. gonaded'Helixaspersa Mulleral'eclosion. Reproduction, Nutrition
15) and mainly upon observations in the field, the annual Hodasia,nJd.DKe.ve1l9o7p9m.enLitf2e1-h:i1s4t9o-r1y61s.tudies ofAchatina achatina (Linne).
cycle of activity in Scutalus tupacii depends principally JournalofMolluscanStudies45:328-339.
upon precipitation. With the beginning of the wet season in Joosse, J. and D. Reitz. 1969. Functional anatomical aspects ofthe
the summer the snails start their reproductive activity, copu- ovotestisofLymnaeastagnalis. Malacologia9(1):101-109.
lating and laying eggs during these months. The duration of Karnovsky, M. J. 1965. A formaldehyde-glutaraldehyde fixative ofhigh
osmolality for use in electron microscopy. JournalofCellBiology
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season. The other factors such as the temperature and pho- Luchtel, D. 1972a. Gonadal development and sex determination in
toperiod, are not as important as precipitation and humidity Pulmonate Molluscs: I Arion circumscriptus. Zeitschriftfur
ofthe habitat in the actvitity cycle ofS. tupacii. ZellforschungundMikroskopischeAnatomie 130:279-301.
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PulmonateMolluscsII:ArionaterrufusandDerocerasreticulatum.
ACKNOWLEDGMENTS Zeitschriftfiir Zellforschung und Mikroskopische Anatomie
130:302-311.
My sincere thanks are due to Drs. Marcelo O. Cabada and Lind, H. 1973. The functional significance ofthe spermatophore and the
ErnestinaTeisaire fortheirpermanentadviceandencouragementthrough- fate of spermatozoa in the genital tract of Helix pomatia
out this work. I am indebted to Dr. A. Medina ofUniversidad de Cadiz, (Gastropoda: Stylommatophora). Journal ofZoology, London
Spain, who reviewed the manuscript. I also thank Dr. Robert Prezant, 169:39-64.
Indiana University ofPennsylvania, USA, forrevising the language. This Lusis, O. 1961. Postembryonic changes in the reproductive system ofthe
research was partially supported by Grants PID 3055500/88 Consejo slugArion ater rufus L. Proceedings ofthe Malacological Society
Nacional de Investigaciones Cientificas y Tecnicas (CONICET, ofLondon 137:433-468.
Argentina) to Marcelo O. Cabada and Project 224/90 Consejo de Nanaware,S.andA. Varute. 1975. Histochemicalstudiesonthemucosub-
Investigacion de la Universidad Nacional de Tucuman (CIUNT) to stances in the ovotestis of a hermaphrodite land pulmonate
ErnestinaTeisaire. Semperula maculata in seasonal breeding-aestivation cycle. Acta
Histochemistry 54:107-124.
Roth, B. 1986. Observations on the range and natural history ofMona-
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