Copyright © 2005, Taylor & Francis.The land snail Leptaxis caldeirarum (Morelet and Drouet, 1857), a hermaphrodite Hygromiidae species, endemic from São Miguel Island (Azores), is geographically limited to a range of 31 km2, occupying an area of about 14 km2. A preliminary study confirmed the locally endangered status of the species, raising the possibility that it could reach the “critically in danger” status, the most serious category in IUCN. A population from Ramal dos Mosteiros was
studied all year round in order to elucidate its reproductive cycle and to test the validity of the
total weight of the animal and the maximum diameter of the shell as diagnostic parameters for maturation. Gonadal maturation of L. caldeirarum proceeds from late winter to early summer. Intense gametogenesis takes place from January until May and the snail is apparently in a
reproductive condition to copulate from late spring onwards. The strong correlation observed between the relative volumetric density of mature gametes and the maximum diameter of the shell suggests that the reproductive status of the species can be inferred from the latter parameter. This may be of major importance in future studies, minimizing potential damage to
already unstable populations, and facilitating the development of conservation strategies.Centro de Investigação de Recursos Naturais (CIRN)

Medeiros, Jorge

Rodrigues, Armindo

International Journal of Invertebrate Reproduction and Development

ARMINDO SANTOS RODRIGUES

JORGE MEDEIROS

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Invertebrate Reproduction & Development

Reproductive cycle of<i>Leptaxis caldeirarum</i>, a locally endangered Azorean land snail

Repositório da Universidade dos Açores

*Corresponding author.Invertebrate Reproduction and Development, 47:3 (2005) 191–195 191Balaban, Philadelphia/Rehovot0168-8170/05/$05.00 © 2005 BalabanReproductive cycle of Leptaxis caldeirarum,a locally endangered Azorean land snailARMINDO SANTOS RODRIGUES* and JORGE MEDEIROSDepartamento de Biologia and CIRN (Research Centre in Natural Resources), Universidade dos Açores,9500 Ponta Delgada, PortugalTel. +351 (296) 650 109; Fax: +351 (296) 650 100; email: rodrigues@notes.uac.ptReceived 11 July 2005; Accepted 6 October 2005SummaryThe land snail Leptaxis caldeirarum (Morelet and Drouet, 1857), a hermaphrodite Hygromiidaespecies, endemic from São Miguel Island (Azores), is geographically limited to a range of31 km2, occupying an area of about 14 km2. A preliminary study confirmed the locallyendangered status of the species, raising the possibility that it could reach the “critically indanger” status, the most serious category in IUCN. A population from Ramal dos Mosteiros wasstudied all year round in order to elucidate its reproductive cycle and to test the validity of thetotal weight of the animal and the maximum diameter of the shell as diagnostic parameters formaturation. Gonadal maturation of L. caldeirarum proceeds from late winter to early summer.Intense gametogenesis takes place from January until May and the snail is apparently in areproductive condition to copulate from late spring onwards. The strong correlation observedbetween the relative volumetric density of mature gametes and the maximum diameter of theshell suggests that the reproductive status of the species can be inferred from the latterparameter. This may be of major importance in future studies, minimizing potential damage toalready unstable populations, and facilitating the development of conservation strategies.Key words: Leptaxis caldeirarum; reproduction; gametogenesis, endemic, conservationIntroductionSeveral studies have investigated the maturationprocess and the reproductive system of land snailspecies (Lusis, 1961; Smith, 1966, 1967; Runham andLaryea, 1968; Els, 1978; Cuezzo, 1990; Rodrigues etal., 1998; Rodrigues and Martins, 2003). Althoughsome studies have been based on the histology of thegonad (Runham and Laryea, 1968; Apley, 1970;Luchtel, 1972a, 1972b; Runham and Hogg, 1979;Cuezzo, 1990, 1993), other authors reported a strongrelation between gonadal maturation and the shape andsize of the reproductive tract, particularly the seminalvesicle and spermoviduct (Lusis, 1961; Runham,Bailey and Laryea, 1973; Cuezzo, 1990; Rodrigues etal., 1998). Several authors reported that maturation ofthe reproductive system is related to the growth of theanimal and Rodrigues et al. (1998) revealed a strongcorrelation between maturation and the size of the shellA. Santos Rodrigues and J. Medeiros / IRD 47 (2005) 191–195192in Oxychilus atlanticus (Pulmonata: Zonitidae). Never-theless, as pointed out by Cuezzo (1993), gonadaldevelopment depends on season, age and size of theanimal, with the expected life time of the species beingan important element in a population’s prospects. Thispresupposes knowledge of the species’ life cycle.Land snails represent an important component ofthe endemic fauna on the Azores (Backhuys, 1975),about 44% of the 107 species described in the archi-pelago being endemic. In the Azores, the genus Lep-taxis comprises five species and two subspecies:Leptaxis caldeirarum (Morelet and Drouet, 1857),Leptaxis terceirana, (Morelet, 1860), Leptaxis vetusta(Morelet and Drouet, 1857), Leptaxis drouetiana(Morelet, 1860) Leptaxis azorica azorica (Albers,1852) and Leptaxis azorica minor (Backhuys, 1975).All of these are considered as locally endangered, butL. caldeirarum, L. terceirana and L. vetusta are listedby the World Conservation Union (IUCN) red list ofthreatened species as globally threatened (Hilton-Taylor, 2000).L. caldeirarum is a hermaphrodite Hygromiidaespecies, endemic on São Miguel Island (Açores). Thespecies is confined to the Sete Cidades volcanic com-plex, in the western part of S. Miguel Island, thelargest island of the Azorean archipelago. This islandwas formed by two separate islands that coexisted forabout 0.550 My and that merged about 50,000 yearsago (Forjaz, 1997).Work from Vieira (2001) stated that this species isgeographically limited to a range of 31 km2, occupy-ing an area of about 14 km2, leading to its locallyendangered status, and drawing attention to the possi-bility that it could reach the “critically in danger”status, the most serious category according to theIUCN. Volcanic activity may be the main historicalcause of the species’ endangered status, with 17 erup-tions during the last 5,000 years, some of themproducing deposit layers more than 2 m thick. How-ever, man’s activities are probably the most significantpresent threat to the species, reducing its naturalhabitat as a result of the enlargement of the area forpastures and logging.Apart from the work of Morelet and Drouet (1857),dealing with the systematics of the species, and that ofVieira (2001), on its geographical distribution andstatus, little is known about life cycle of L. caldeira-rum, especially with reference to its reproductivebiology. Thus, the main objective of this work is tostudy the reproductive cycle of L. caldeirarum and toassess the validity of two morphometric parameters;namely, the fresh weight and the maximum diameter ofthe shell, respectively, as being diagnostic for repro-ductive maturation, in order to minimize the ecologicalimpact of future studies and to underpin conservationmeasures.Materials and MethodsTo perform this study we choose a site (Ramal dosMosteiros: latitude 37E52N26O N; longitude 25E49N30O W) where the human influence is already veryevident — a small valley near to a national road andalmost without native vegetation — in order to mini-mize our impact on the habitat of the species. Every2 months, from January to November 2003, 7–10 ofthe largest individuals found during a collecting periodlasting 2 h were transported alive to the laboratory(January, n = 10; March, n = 8; May, n = 10; July, n =10; September, n = 9; November, n = 7). The remain-ing individuals were returned to the environment. Inthe laboratory, the maximum diameter of the shell andthe total fresh weight were measured before excision ofthe gonad for histology.In order to determine the state of gonadal matu-ration, the ovotestis were fixed in 10% formalin andembedded in paraffin. Serial sections, 7-µm thick, werestained with Mayer’s haemalum and eosin (Martojaand Martoja-Pierson, 1970). The relative volumetricdensity of the gametes was estimated using the M168Weibel Multipurpose Test System (Weibel, 1979).Five stages of spermatogenesis were identified onthe basis of the classification of Griffond et al. (1991):(1) spermatogonia, small spheroidal cells (8–10 µmdiameter), with a relatively large nucleus and scantycytoplasm; (2) spermatocytes, larger than spermato-gonia (11–17 µm in diameter), with more abundant andeosinophilic cytoplasm; (3) early spermatids, small insize (8–10 µm diameter), spheroidal in shape, butfrequently showing basophilic condensations at theirpoles; (4) late spermatids, with the whole cell elon-gated and with small tails; (5) spermatozoa, withstrongly basophilic head and long eosinophilic tails. Aswith Oxychilus atlanticus (Pulmonata: Zonitidae)(Rodrigues et al. 1998), no unambiguous distinctioncould be made between spermatocytes I and II, norbetween early- and mid-spermatids, with lightmicroscopy.In accordance with Hill and Bowen (1976) andRodrigues et al. (1998), three developmental stages ofoogenesis were identified: (1) previtellogenic oocytes,small, rounded cells with strongly basophilic cyto-plasm; (2) vitellogenic oocytes, larger than the pre-vious stage, more flattened and lightly basophilic; and(3) maturing oocytes, larger than the previous, roundedin shape and with an eosinophilic and granular cyto-plasm. This last stage also includes the fully matureoocytes.A. Santos Rodrigues and J. Medeiros / IRD 47 (2005) 191–195 193In order to identify the gonadal maturation state,scores for volumetric density were summed for eachspecimen and converted to percentages. The data weresubject to statistical analysis using SPSS 11.5 forWindows (SPSS Inc., 2002). Pearson’s correlationswere determined between the following variables:maximum diameter of the shell, total fresh weight andrelative volumetric density of each gametogenic stage.One-way analysis of variance (ANOVA) was per-formed to compare morphometric variables and rela-tive volumetric density of each stage of oogenesis andspermatogenesis during the collection period. Gameto-genic data was arcsin-transformed prior to analysis,while morphometric data were transformed into. When significant differences were observed,the least significant difference (LSD) test was used forpair-wise comparisons among samples.ResultsThe profiles of changes in the maximum diameterof the shell and the total fresh weight, respectively, arevery similar during the sampling period, showing somegrowth between January and May, with larger speci-mens occurring at the end of spring (May) and duringthe summer (July) (Fig. 1). In September a significantdecrease was observed in total fresh weight comparedto May (P = 0.008, LSD test) and in the maximumdiameter of the shell compared to May (P = 0.024,LSD test) and July (P = 0.037, LSD test).Stereological study showed that the gonadal vol-ume occupied by maturing oocytes increased fromJanuary to July, when it reached a relative volumetricdensity over 10%, significantly higher than in JanuaryP = 0.01, LSD test) and September (P = 0.045, LSDtest) (Fig. 2A). Individuals from September revealedthe lowest relative volumetric density of maturingoocytes, a situation similar to that of January. Apartfrom January and September, maturing oocytes occu-pied more than 5% of the gonadal volume throughoutthe year (Fig. 2A).The relative volumetric density of spermatozoashows that they occupy more than 30% of the gonadalvolume throughout the year, reaching the highestvalues in May, July and September (Fig. 2B). Indi-viduals from September show a relative volumetricdensity of spermatozoa which is significantly higher(F = 3.382; df = 5, 48; P = 0.011) than those fromJanuary, March, May and November; and they havethe lowest volume of the gonad occupied by the imma-ture stages of the spermatogenesis, as well as thelowest volumetric density of maturing oocytes(Fig. 2B).The morphometric variables of shell diameter andfresh weight are strongly correlated (n = 54, r = 0.917;P <0.001), and the maximum diameter of the shellshows a significant correlation (n = 54, r = 0.307; P =0.024) with the gonadal volume occupied by maturegametes (spermatozoa and maturing oocytes con-sidered together). The relative volumetric density ofspermatocytes is negatively correlated with the maxi-mum diameter of the shell (n = 54, r = !0.323; P =0.017) and total fresh weight (n = 54, r = !0.312; P =0.022).Fig. 1. Total fresh weight (mean ±SE) and maximum diameter of the shell (mean ± SE) of Leptaxis caldeirarum, from Januaryto November 2003. Different lower cases indicate significantly different values, ANOVA (P <0.05).A. Santos Rodrigues and J. Medeiros / IRD 47 (2005) 191–195194Fig. 2. Relative volumetric density (mean ±SE) of the gametogenic stages in Leptaxis caldeirarum, from January to November2003. A. Oogenesis: pre-vitellogenic (PV), vitellogenic (V) and maturing ocytes (M). B. Spermatogenesis: spermatogonia(Sg), spermatocytes (Sc), early spermatids (Ste), late spermatids (Stl) and spermatozoa (Sz). Different lower cases abovecolumns, within a stage of reproductive maturation between sampling dates, indicate significantly different values, ANOVA(P <0.05).DiscussionMorphometric data indicate a single generationeach year with mature snails reproducing and dying insummer, and juveniles growing in the autumn, winterand spring. The occurrence of many large empty shellsin September is a result of the post-reproductive deathand supports this hypothesis.Our observations on gonadal maturation show thatL. caldeirarum matures from late winter to earlysummer, accumulating gametic resources to copulatefrom late spring onwards, with more than a half thegonadal volume in summer being occupied by sperma-tozoa, and with a relative volumetric density of matur-ing oocytes above 10% (Fig. 2).A protandric tendency of the species is supportedby the fact that spermatozoa occupy more than 35% ofthe gonadal volume throughout the year, even inMarch and November, when spermatocytes reach thehighest relative volumetric densities and maturingoocytes are uncommon. Whilst considering simul-taneous hermaphroditism as characteristic of pul-monates, Gómez (2001) noted the existence of aninitial, predominantly masculine phase, followed byone where female function is more evident. Morton(1967) also drew attention to the protandric tendencyof molluscs, suggesting that male gametes will beproduced at the beginning of the reproductive season,or when the animal is younger, and this is inaccordance with the size advantage model of Ghiselin(1969). According to the latter, large body size isadaptive for female function, given the fact that eggsare larger than the sperm.A. Santos Rodrigues and J. Medeiros / IRD 47 (2005) 191–195 195There is a positive correlation between sexualmaturity, as expressed by the volume occupied by bothspermatozoa and maturing oocytes combined, and themaximum diameter of the shell. These results are inaccordance with those of Cuezzo (1990, 1993) andRodrigues et al. (1998). In late spring and in summer,we categorize all L. caldeirarum individuals with ashell diameter larger than 11.5 mm as being fullymature, since more than 50% of their gonadal volumeis occupied by mature gametes. Shell diameter couldthus be important in assessing the reproductive statusof this endangered pulmonate, since it minimizesdamage to a vulnerable population.AcknowledgmentsThe authors thank Dr. André Amaral and PauloMelo for their support in field and laboratory essays.The present work was supported by Centro de Inves-tigação de Recursos Naturais (CIRN).ReferencesApley, M., Field studies on life history, gonadal cycle andreproductive periodicity in Melampus bidentatus (Pul-monata: Ellobiidae). Malacologia, 10 (1970) 381–397.Backhuys, W., Zoogeography and Taxonomy of the Land &Fresh Water Molluscs of the Azores. Backhuys &Meesters, Amsterdan, 1975.Cuezzo, M.G., Maturation of the reproductive tract ofNeohelix major (Binney) (Gastropoda: Polygyridae).Amer. Malacol. Bull., 8 (1990) 19–24.Cuezzo, M.G., Annual gonadal cycle of the land snailScutalus tupacii (Pulmonata: Bulimulidae). Amer.Malacol. Bull., 10 (1993) 121–127.Els, W.J., Histochemical studies on the maturation of thegenital system of the slug Deroceras laeve (Pulmonata:Limacidae) with special reference to the identification ofmucosubstances secreted by the genital tract. AnnaleUniversiteit Stellenbosch, 1, A, 3 (1978).Forjaz, V.H., Alguns Vulcões da Ilha de São Miguel. I Parte.Observatório Vulcanológico e Geotérmico dos Açores,Ponta Delgada, Açores, Portugal, 1997.Ghiselin, M., The evolution of hermaphroditism amonganimals. Q. Rev. Biol., 44 (1969) 189–208.Gómez, B. J., Structure and functioning of the reproductivesystem. In: The Biology of Terrestrial Molluscs, BarkerG.M. (ed.), CABI, Wallingford, UK, 2001, pp. 307–330.Griffond, B., Dadkhan-Teherain, Z., Medina, A. and Bride,M., 1991. Ultrastructure of Helix aspersa spermato-genesis: scanning and transmission electron micro-scopical contributions. J. Moll. 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Reproductive cycle of Leptaxis caldeirarum, a locally endangered Azorean land snail

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Reproductive cycle of Leptaxis caldeirarum, a locally endangered Azorean land snail

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