Invertebrate Reproduction and Development, 53:4 (2009) 211–222 211 Balaban, Philadelphia/Rehovot 0168-8170/09/$05.00 © 2009 Balaban

Life history strategy of unilamellata (d’Orbigny, 1835) (, , Subulinidae)

CAMILLA DE MEDEIROS CARVALHO1,3*, JAIRO PINHEIRO DA SILVA4, CRISTIANE LAFETÁ FURTADO MENDONÇA5, ELISABETH CRISTINA DE ALMEIDA BESSA1, 2,3 and STHEFANE D’ÁVILA1,2,3 1Programa de Pós-Graduação em Ciências Biológicas, Comportamento e Biologia ; 2Departamento de Zoologia; 3Núcleo de Malacologia Prof. Maury Pinto de Oliveira, Instituto de Ciências Biológicas, Universidade Federal de Juiz de Fora, Campus universitário, 36036-330, Juiz de Fora, MG, Brasil Tel. +32 32293223; email: [email protected] 4Departamento de Ciências Fisiológicas, Instituto de Biologia, Universidade Federal Rural do Rio de Janeiro, Seropédica, RJ, Brasil 5Laboratório de Helmintoses Intestinais, Centro de Pesquisas René Rachou-Fiocruz, Belo Horizonte, MG, Brasil

Received 10 June 2009; Accepted 24 November 2009

Abstract Life history theory predicts that the patterns of resource allocation in are associated with different strategies, selected in the course of evolution. In the present study, the life history of was characterized under laboratory conditions. We determined the growth, reproduction, and longevity patterns of this species and elucidated the strategy related to the development of embryos, through direct observations and examination of the morphology of the gravid uterus. Furthermore, we attempted to analyze the glycogen and galactogen contents of the albumen gland, digestive gland and cephalopedal mass in order to understand energy allocation to life history traits, for three life stages. Leptinaria unilamellata’s life history is characterized by great longevity, a short juvenile phase, early sexual maturity, and repeated reproductive events, with little reproductive effort at each event and some mortality shortly after the first reproduction. In the terraria, we found juveniles but no eggs. However, the results of the anatomical study showed no morphological connection between the embryos and the parental organism. Thus, this species should be described as ovoviviparous rather than viviparous. Egg retention in the parent organism is the primary cause of the release of juveniles, instead of eggs, enabling the offspring to withstand environmental stress. The higher quantity of galactogen found in the adults’ albumen gland, as compared to juveniles and senescent individuals, as well as the ratio of glycogen to galactogen, reveal the allocation of energy to reproduction rather than to growth. The remaining energy is directed to the maintenance of homeostasis. Such pattern was confirmed by the low levels of glycogen and galactogen observed in the senescent stage, compared to the juvenile and adult stages. In the life strategy of L. unilamellata, the distribution of the reproductive effort among many events associated with ovoviviparity indicates a long-term investment in reproductive success.

Key words: Embryonic development, indeterminate growth, galactogen, glycogen , long- term investment, ovoviviparity, trade-off

*Corresponding author. 212 C.M. Carvalho et al. / IRD 53 (2009) 211–222

Introduction Methods Studies on the life history of land snails are funda- Rearing of the snails mental to understanding how these organisms allocate Laboratory colonies were established from speci- energy resources over the course of their lives to mens collected in the municipality of Juiz de Fora, maximise reproductive success (Cichón, 1999; Norton Minas Gerais (21E45N50OS, 43E21N0OW) (678 m & Bronson, 2005). The patterns of resource allocation altitude). The typical tropical weather of this locality is for reproduction and growth are associated with characterized by two well-defined seasons: a dry winter different life history strategies, selected in the course of with relatively low temperatures, which extends from evolution (Antkowiak & Chase, 2003). May to September, and a rainy summer with higher Most studies on the longevity of terrestrial molluscs temperatures, from October to April. The average and its relationship with life history traits involve annual temperature is 19.3EC (15–24EC) and the annual species from temperate regions (Baur & Baur, 2000; rainfall is 1627 mm (IBGE, 2008). Heller, 2001; Hommay et al., 2001; Ocaña, 2003; Newly hatched snails were kept in transparent plastic Anderson et al., 2007). Few studies concerning the life boxes (14 cm in diameter, 9 cm in length), with 30 strategies of these animals have been conducted in individuals per box in two replicates. The experimental tropical regions such as Brazil. In particular, little is groups underwent the same treatment. The bottom of known about life history of ovoviviparous pulmonate each box was lined with humus, which was moistened land snails, including well-studied species from every 2 days with 10 ml of water. The snails were fed ad temperate regions (Anderson et al., 2007). libitum with a commercial poultry feed (composed of a Leptinaria unilamellata (d’Orbigny, 1835) are small mixture of corn meal, soy meal, wheat bran, gluten bran, terrestrial , approximately 11 mm long chicken flesh and bone meal, fish meal, calcium and 5 mm wide. This species is restricted to tropical phosphate, and sodium chloride) supplemented with a South America (Simone, 2006) and inhabits the soil premix of minerals, vitamins and calcium carbonate. over and under fallen leaves and stalks, avoiding direct The snails were kept at room temperature and under sunlight and, consequently, desiccation. In Brazil, popu- natural light conditions. This study was carried out from lations of L. unilamellata are spread over Amazonas, September 17, 2004, to August 17, 2007, 1,064 days in Pará, Rondônia, Pernambuco, Bahia, Mato Grosso, total (142 weeks). Minas Gerais, Rio de Janeiro and São Paulo (Araújo, Sexual maturity was confirmed by the presence of 1982). This land snail species can be easily raised in the unborn young in the uterus, visible through the trans- laboratory and was used as model organism for biology parent shell. Afterwards, the molluscs, previously kept and morphology studies (Almeida & Bessa, 2001; in groups during the juvenile stage, were placed in Araújo, 1982; Brandolini & Gomes, 2002; Dutra, 1988). individual transparent plastic boxes (10 cm wide × 6 cm However, in all previous studies on the L. unilamellata, long), under the above described conditions. The reproduction and growth patterns were not considered in number of reproductive events and offspring produced characterization of their life history. One aspect of the per individual were recorded. biology of this species that needs to be elucidated is the relationship between the parent organism and the Observations on growth, reproduction, and survival embryos. At the end of the reproductive process, the In order to characterize life history traits of L. uni- adults release juveniles into the environment. In order to lamellata, we quantified for each replicate colony elucidate whether the embryos develop through ovo- survival, changes in mean size and the date of onset of viviparity or viviparity, a morphological approach to the reproductive activity. After the individuals reached study of the reproductive system is necessary. sexual maturity, the per-clutch reproductive output, In the present work, the life history strategy of L. longevity, and changes in size per individual were unilamellata was characterized. The growth, repro- determined. From these data, we calculated growth rate duction, and longevity patterns of this species, as well as (increment in shell height divided by the interval the interrelationships among these traits, were deter- (number of days) between each measurement) and mined. The strategy for embryonic development was reproductive output (total number of offspring × total clarified by means of direct biological observations and number of reproductive events). morphology of the gravid uterus. Moreover, the The size of each snail was determined by measure- variation in the levels of glycogen and galactogen in the ment of shell height using a Kanon caliper to 0.1 mm cephalopedal mass, digestive gland and albumen gland precision. Up tol the age of 90 days, shell height was at three life stages was assessed. measured at 15-day intervals. Thereafter, measurements were taken at 30-day intervals. C.M. Carvalho et al. / IRD 53 (2009) 211–222 213

Snails were observed weekly from the first embryos Glycogen and galactogen levels at three life stages in the uterus to record the deaths, the number of To assess glycogen and galactogen levels in the reproductive events, and the number of offspring per cephalopedal mass, digestive gland and albumen gland individual. at three life stages of L. unilamellata, 65-day-old Data for juveniles from the start of the experiment juveniles (18 individuals), 145 day-old adults (24 indi- until sexual maturity were expressed as means per viduals) and 1064 day-old senescent individuals (9 indi- replicate. Data on the adults were expressed as means viduals) were used. The juveniles and adults were per individual. These data were analyzed through obtained from laboratory colonies and the senescent Student’s t-test to detect differences in snail growth individuals were those used for observations on growth, during the juvenile (before the first reproductive event) reproduction and survival. and adult phases of the life cycle. The Pearson cor- The individuals were dissected under stereoscopic relation coefficient (p<0.05) was used to investigate the microscope. The digestive gland and cephalopedal relationships between adult growth rate and the total mass, as well the albumen gland, were removed and number of reproductive events, and adult growth rate stored at !20EC until processed for the determination of and the total offspring produced in the course of adult glycogen and galactogen contents, respectively. The life. collected tissues were pooled by life stage and organ. They were kept on ice at all times to avoid the meta- Morphological study bolizing of carbohydrates through enzymatic action. The tissues were processed according to Pinheiro & In order to characterize snail life-history strategy in Gomes (1994) in order to obtain the precipitation with relation to embryonic development, we investigated the glycogen and galactogen. The precipitation was sub- morphology of the gravid uterus for the presence of any mitted to acid hydrolysis and subsequent spectrophoto- anatomical interaction between the parent snail and its metric quantification through 3.5-dinitrosalicylic acid offspring. technique (3.5 DNS) (Sumner, 1924), with an absor- bance at 535 nm. The final values were calculated through Beer law with at least three coherent readings, Histology of the gravid uterus and expressed in mg of glucose.g!1 of tissue or mg of For the histological study, six adult individuals from galactose/g of tissue (fresh weight). The chromato- the laboratory colonies were fixed in Carson’s formalin, graphic analysis was performed aiming to confirm that Milloning (Carson et al., 1973) and processed following the samples contained only glycogen and galactogen. the steps of conventional histological technique. The This is necessary because it is a general method for 5-mm cuts were hematoxylin-eosin stained and ob- extracting and quantifying polysaccharides. The pre- served under an Olympus (BX 51) differential inter- sence of polysaccharides other than glycogen in diges- ferential contrast microscope. The images were captured tive gland and cephalopodal mass, or galactogen, in through a digital camera coupled to the microscope and albumen gland, may interfere with the quantification then analyzed with the software Image Pro Plus 5.0. process since the 3.5 DNS method is used in the quantitative determination of reducing sugars. Anatomy of the gravid uterus and relationship between size and reproductive traits Results For the anatomical study, 60 specimens from the laboratory colonies (30 individuals aged 80 days and 30 Growth adult individuals with different ages) were fixed in 70% Snail growth was monitored during 1050 days post- ethanol and dissected under stereoscopic microscope. In hatching (140 weeks), although the total life span order to verify the relationship between size and observed for the group was 1064 days (142 weeks). The reproductive traits, a morphometric study of 30 adult snails continued to grow after reaching sexual maturity, individuals used previously in the anatomical study was which occurred between Day 96 and Day 195 (between performed. Shell height and diameter measurements, Week 12 and Week 25) (Fig. 1). There was a significant and the albumen gland and uterus length and diameter difference (p = 0.0343) between the mean increase in measurements were made with a Kanon caliper to shell height during the juvenile stage and during the 0.1 mm precision. In addition, the number of whorls in adult stage, between the two replicates. However, the the shell and the number of embryos inside the uterus growth rate was less pronounced in adults (between were counted. 210- and 1050-day-old) (0.024 mm.day!1) than juveniles 214 C.M. Carvalho et al. / IRD 53 (2009) 211–222

Fig. 1. Temporal trend in shell height of 60 individuals of Leptinaria unilamellata. The white portion of columns represent mean values of shell height during juvenile stage and the black portion of columns represent mean values of shell height during adult stage. which grows on average 0.044 mm.day-1 from 1 and There was no relationship (r = !0.1; n = 36; p = 180 days old. A considerable increase in growth rate of 0.96) between adult growth rate and total number of 0.08 mm.day!1occurred between Day 30 (Week 4) and reproductive events performed by the individuals Day 45 (Week 6), during the juvenile stage. However, throughout the adult life span. Moreover, there was a the growth rate declined progressively through the adult relationship between adult growth rate and total off- lifespan, mainly after Day 450 (Week 60). The greatest spring produced in the adult stage (r = !0.1; n = 36; p = mean length, 18.1 mm and the maximum shell height, 0.034). 20.8 mm, were registered on Day 1050 (Week 140) at the end of the snails’ life span. The mean shell height of Mortality newly hatched snails corresponds to 22% of mean adult shell height at sexual maturity. The mean shell height There were no high levels of mortality at the juvenile when snails reached sexual maturity was 8.8 mm (6.3– stage (percentage of mortality 33%), and 67% of the 13.6 mm) and the mean growth rate in the adult stage individuals reached sexual maturity. Shortly after reach- was 0.009 mm.day!1 (0.002–0.034 mm.day!1). ing sexual maturity, 24% of the individuals died. Afterwards, the number of living adults decreased gradually during the adult lifespan (Fig. 2). The juvenile Reproduction stage lasted in average 180 days (6 months) (24 weeks), The snails reached sexual maturity between 96 and and the adult stage 884 days (2 years and 4 months) 195 days post-hatching. A reproductive output of 5.880 (118 weeks). The mean longevity observed was juveniles in 813 reproductive events was accomplished 558 days (between 15 and 1064 days), approximately by 36 reproductive adults. The snails each performed 1 year and 5 months. The life span observed for the last between 1 and 49 reproductive events and produced individual to die was 1064 days (142 weeks), approxi- between 5 and 376 juveniles. There was a significant mately 3 years. positive relationship (r = 0.89; n = 36; p<0.001) between the number of offspring and the number of reproductive events of each individual during its life- Morphology of the gravid uterus time. The snails performed an average of 22.6 repro- The first embryos found in the uterus of the ductive events, producing 163.3 juveniles throughout molluscs, which were visible through the transparent their life. The reproductive output observed was shell, indicated snail sexual maturity (Fig. 3A). The pre- between 3.27 and 11.4, with a mean of 6.72 offspring sence of zygotic cells in the uterus of some individuals per reproductive event per snail. with different ages was observed. During the snails’ 80- C.M. Carvalho et al. / IRD 53 (2009) 211–222 215

Fig. 2. Survival of a cohort of 60 individuals of Leptinaria unilamellata kept isolated under laboratory conditions. The vertical line indicates a range of age at maturity.

Fig. 3. Embryos in the uterus of Leptinaria unilamellata. A. Asterisks indicate the presence of embryos in the uterus. B. Embryo presenting shell completely developed, the arrows indicate the lamellae. Note the thin membrane (m) that enveloped the embryo. C. Embryos at different stages of development, (1e) embryos at initial developmental stage; (2e–4e) embryos at more advanced developmental stage. D. Non-viable eggs (zygotic cells with a gelatinous mass around it). Scale bar = 1 mm. 216 C.M. Carvalho et al. / IRD 53 (2009) 211–222 day reproductive period, all adults dissected had embryos at different developmental stages in the uterus, from zygotic cells to juveniles. The juveniles had fully developed shells whose morphological features, e.g. the shape of shell aperture and presence of lamellae, were similar to adult shells (Fig. 3B). No anatomical structure that might indicate inter- action between the embryos and parent organism was detected in the present study. As mentioned by Tompa (1979), the embryos were located in the uterus like a sequential assembly line (Fig. 3C), in which the embryos at more advanced developmental stages were located proximal to the genital aperture. The embryos are enclosed in a thin membrane, which is breached before the juveniles are released into the environment (Fig. 3B). In the terraria, we found only hatchlings at the substrate. The “eggs” found (Fig. 3D) were zygotic cells surrounded by a gelatinous mass, showing incomplete development of the embryo, thus being unviable. Therefore, this species should be described as ovovivi- parous rather than viviparous. The uterus is elastic and may be expanded to accommodate more embryos (Fig. 4). The uterine wall is non-muscular and formed by epithelial cells on a thin layer of connective tissue, the basement membrane (Fig. 5). The uterine epithelium has two cell types, and, Fig. 4. Reproductive system of Leptinaria unilamellata. The consequently, two functions. In the ventral region, the uterus is flexible and its size may be expanded to epithelium has ciliated cubic cells, interspaced by accommodate more embryos. a: genital atrium; pc: penial glandular cells. In the dorsal region, the epithelium complex; dd: deferent duct; v: vagina; o: oviduct; bcd: bursa lining is formed by pavement cells only (Fig. 5). copulatrix duct; bc: bursa copulatrix; pr: prostate; e: embryo; u: uterus; ag: albumen gland; hd: hermaphrodite duct; sc: spermathecal complex; c: carrefour. Scale bar = 500 µm. Adult morphology and its relation to reproductive traits Significant positive relationships were found between the number of embryos contained in the uterus and shell size (r = 0.84; n = 30; p <0.0001); the number of embryos and the number of whorls in the shell (r = 0.73; n = 30; p <0.0001) and the number of embryos and uterus size (r = 0.83; n = 30; p <0.0001). Moreover, there was a positive relationship between shell and uterus size (r = 0.77; n = 30; p <0.0001). As expected, a strong positive relationship between shell size and the number of whorls in the shell was found (r = 0.81; n = 30; p <0.001). We also found a positive relationship between albumen gland and shell size (r = 0.58; n = 30; p = 0.007).

Variation in glycogen and galactogen levels in three Fig. 5. Uterus wall of Leptinaria unilamellata is non- life cycle stages muscular and is formed by ciliated cubic cells (arrow) inter- spaced by glandular cells in the ventral region. Embryos (*) The glycogen levels in the digestive gland were are seen in the uterus lumen (u). Lining pavement epithelium 14 mg of glucose.g!1 of tissue in the juveniles, 9 mg in the dorsal region of the uterus (arrow head). Scale bar = of glucose.g!1 of tissue in the adults and 1 mg of 50 µm. C.M. Carvalho et al. / IRD 53 (2009) 211–222 217

Beese & Baur, 2006). The snails need several partners to breed. They mate at short intervals, frequently digesting a significant amount of the donor’s sperm, before laying the eggs (Beese et al., 2006). The long term-sperm storage ability has been described for several land snail species (Vianey-Liaud et al., 1996). Arianta arbustorum mate repeatedly with different partners and store viable sperm for more than 1 year (Baur, 2007). The repro- ductive behavior of Helix aspersa involves multiple mating and long-term sperm storage (Evanno et al., Fig. 6. Life history strategy of Leptinaria unilamellata char- 2005). The snails in our study mated for the first time acterized by great longevity, short juvenile lifespan, early because, otherwise, juveniles would have been detected sexual maturity, and a great number of reproductive events in the terraria. We never observed the snails during over life, with a small reproductive effort in each event. courtship or mating and this is probably due to the small These traits are associated to K-strategist species. The size of the animals or their nocturnal habits. Also, we illustration is a proportional scale of life history traits know little about these behaviors (whether or nor they observed during the experiment period. Scale bar (= 2 cm) concerns 112 days of the lifetime. are usual) and about the proportion of self-fertilization or cross-fertilization even among the snails kept in groups. Thus, in our study, the only way to ascertain glucose.g!1 of tissue in the senescent individuals. The sexual maturity and transition to adult stage was the glycogen levels in the cephalopedal mass were 4 mg of detection of embryos in the uterus. In addition, since the glucose.g!1 of tissue in the juveniles, 3 mg of glucose.g!1 L. unilamellata has indeterminate growth, shell lip of tissue in the adults and 4 mg of glucose.g!1 of tissue appearance, a feature commonly used to identify mature in the senescent individuals. The total glycogen levels individuals of other pulmonate species, could not be were 18 mg of glucose.g!1 of tissue in the juveniles, used. 12 mg of glucose.g!1 of tissue in the adults and 5 mg of Leptinaria unilamellata can produce offspring by glucose.g!1 of tissue in the senescent individuals. The self-fertilization (Almeida & Bessa, 2001). In our galactogen levels in the albumen gland were 7 mg of laboratory, we raised newly hatched virgin snails galactose.g!1 of tissue in the juveniles, 148 mg of galac- individually, and they were found to produce viable tose.g!1 of tissue in the adults and 5 mg of galactose.g!1 offspring (unpublished data). of tissue in the senescent individuals. In the present study, the molluscs previously kept in Chromatographic analysis of samples of carbo- group for the duration of the juvenile stage were placed hydrates from the digestive and albumen glands showed in individual boxes to assess the number of reproductive only the expected glucose or galactose spot. No events and offspring produced per individual. Although intermediate spots were formed attesting that the extrac- the snails were kept in groups until they reached sexual tion and quantification procedures provided a highly maturity and produced the first offspring, we cannot pure material, with no contaminants that could interfere assert whether the first reproduction occurred by cross- fertilization or self-fertilization. Individuals of the same with the analysis. offspring generated by cross-fertilization and self- fertilization have been observed for many species of land snails. It is possible that L. unilamellata reproduces Discussion by the combination of these two strategies, even in the In the present study, the first embryos found in the same reproductive event. Other Subulinidae land snails, uterus of the molluscs, which were visible through the as Rumina decollata, (Heller, 1993) and Subulina transparent shell, provided the best indication of sexual octona (Bessa & Araújo, 1995) reproduces by a breed- maturity. However, this assumption arises from a ing system of facultative self-fertilization. However, methodological convenience, and it is important to there are no studies on the frequency of selfing and consider that these molluscs had a functional ovotestis outcrossing per reproductive event of Subulinidae and a fully developed reproductive system prior to the mollusks so far. Parent-offspring analyses revealed that formation of the embryos. Moreover, we considered the ninety percent of the progeny of paired snails were of first offspring production, rather than the first mate, as uniparental origin and no pair reproduced exclusively by being the first reproductive event because multiple outcrossing in Bulinus forskalii (Gow et al., 2005). paternities are very common among pulmonate molluscs Biomphalaria glabrata use allosperm during the first (Baur, 1994; Bojat et al., 2001; Evanno et al., 2005; day after copulation. Thereafter, self fertilization occurs 218 C.M. Carvalho et al. / IRD 53 (2009) 211–222 again, and the egg masses contain a mixture of crossed organ, may influence the reproductive output. Beese & and selfed egg-cells (Vianey-Liaud et al., 1996). Baur (2006) observed that the numerical aspects of Jordaens et al. (2000) observed a geographical pattern in sperm transfer are less important in influencing fertili- the prevalence of outcrossing in Arion (Carinarion) zation success of sperm in A. arbustorum. fasciatus, in which the molluscs of central Europe The use of molecular or genetic tools that may allow reproduce by a mixed breeding system (selfing and the identification of offspring paternity and, thus, outcrossing) and those from northern and north-eastern determine the relative contributions of selfing and cross- Europe reproduce preferentially by selfing. fertilization is essential to elucidate the mating system of Thus, in the present study, from the moment the L. unilamellata. snails were kept alone in separate terraria, the suc- Patterns of resource allocation for reproduction and ceeding reproductive events might have occurred by growth in land snails are associated with different life cross-fertilization with the use of stored spermatozoa, or history strategies (Antkowiak & Chase, 2003). There are else by self-fertilization, or even by both strategies. two kinds of resource allocation during lifespan. In the Assuming that cross fertilization occurred, we have not most common pattern, all the excess energy after birth is been able to assert that repeated mating did not occur directed to growth until the individual reaches sexual nor that all snails have stored and used the same content maturity. After maturity, growth may be interrupted, and of spermatozoa to fertilize the oocytes through the all the energy is directed to reproduction, which charac- reproductive life. On the other hand, we cannot ignore terizes determinate growth. The second pattern, termed that those snails that were given only one opportunity to indeterminate growth, is characterized by continued mate and those which mated less frequently than usual, growth after maturity and terminated only by death did not try to compensate their reduced level of stored (Cichón, 1999). sperm by using their own sperm to fertilize the oocytes, The results of the present study showed that L. in all the succeeding reproductive events. unilamellata has indeterminate growth, continuing to Baur (1998) investigated the influence of multiple grow after sexual maturity. It implies that, in adult mating on the fecundity of Arianta arbustorum. The phases, energy resources were allocated not only to the author used individuals that had mated and laid eggs in reproductive effort. but also to growth and survival, as the preceding year. The number of egg clutches laid did confirmed by glycogen levels only slightly higher in not change for molluscs kept alone, in pairs or in groups, juveniles than in adults. However, growth rate was but isolated individuals laid smaller egg clutches per lower in the adult stage than in the juvenile stage, reproductive season, indicating that the snails prevented indicating that there is a trade off between reproduction from remating had reduced reproductive success. and growth, as confirmed by the negative relationship In the present study, the snails reached maturity at between growth rate and the total number of repro- three different ages, as follows: 96, 130 and 195 days of ductive events throughout the adult lifespan. The higher life. Those individuals that reached maturity later in life quantity of galactogen found in the adults’ albumen were kept longer in the common terrarium together with gland, compared to juveniles and senescent individuals, other individuals. We can expect that those molluscs are as well as the ratio of glycogen to galactogen indicate more likely to mate with several partners and, conse- the allocation of energy to reproduction in detriment of quently, are able to store more spermatozoa and obtain growth. In adult individuals, energy through nourish- greater reproductive success, as expressed by offspring ment is mostly converted into an energetic substrate, numbers. However, we observed that the reproductive which is not available to any other biological activity output was very similar among the individuals, and no but reproduction, especially egg formation. The ratio of significant difference was found between the mean glycogen to galactogen at juvenile, adult and senescent values of offspring per reproductive event (ANOVA, stages illustrates energy allocation throughout the p = 0.078). Accordingly, we concluded that even if lifetime of L. unilamellata. multiple mating occurred, with differential frequency Juveniles accumulate energy reserves as galactogen, between the individuals, this fact has not biased their which are allocated to reproduction during the adult reproductive output. The influence of multiple mating stage, but they also store energy as glycogen, that is on the reproductive output should be more significant in directed to survival after the high energetic effort self-incompatible species such as the A. arbustorum demanded by reproduction. Such prediction was con- than in self-fertilizing snails as the L. unilamellata, firmed by the low mortality observed shortly after the which possibly have a mixed mating system. Otherwise, onset of sexual maturity. Indeterminate growth has also adaptations of the sperm receiver as the selective storage been observed in land snails Achatina fulica Bowdich, of allosperm and the selective digestion of sperm, 1822 () by Tomiyama (1993), Archachatina reducing the amount of sperm that reaches the storage marginata (Swainson, 1821) (Achatinidae) by Plummer C.M. Carvalho et al. / IRD 53 (2009) 211–222 219

(1975), Subulina octona (Brugüière, 1789) (Subuli- Such pattern was confirmed by the low levels of glyco- nidae) by D’ávila & Bessa (2005) and Bulimulus gen and galactogen observed in the senescent stage, tenuissimus (d’Orbigny, 1835) (Bulimulidae) by Silva et compared to the juvenile and adult stages. al. (2008). Conversely, studying the life history of the Several studies on biology and reproductive beha- land snail Bradybaena similaris Fèrrusac (1821) vior of land snails examined how life history patterns (Bradybaenidae), we observed determinate growth and are associated with energy allocation (Raut & Panigrahi, short lifetime associated with great reproductive effort 1988; Reise, 1995; Antkowiak & Chase, 2003; Evanno, over a few events (Carvalho et al., 2008). et al., 2005; Carvalho et al., 2008). There are limited When the reproductive effort is below maximum resources for the different biological processes. Thus, shortly after reaching sexual maturity, the remaining trade-offs between life history traits may exist (Krebs, energy is sufficient to meet other demands, such as 1994; Zera & Harshman, 2001). Earlier sexual maturity growth and homeostasis. Thus, the snail is able to can lead to increased longevity, and a long life span may survive for a long time, growing and accumulating be associated with a reproductive effort distributed energy reserves. The energy accumulated during life as among many reproductive events over the lifetime, as body mass is distributed among several reproductive observed in the present study. events, with few juveniles produced in each event. The In a long-lived, iteroparous species, it can be pos- continuous growth, associated with the distribution of sible to examine the contributions made during different the reproductive effort during life, optimizes the snails’ life stages to lifetime reproductive success, survival and reproductive success. The distribution of the repro- fecundity (Brown, 1988). Leptinaria unilamellata has ductive effort over many events compensates, in the an extended life span, reaching sexual maturity early. long term, the disadvantages caused by the trade-off The hatchlings are large, and the juveniles have short between growth and reproduction in the current lifespan and high survival, and, consequently, the reproductive event. Later in life, the snails grow and can majority of the individuals reach sexual maturity. The produce more offspring. Such prediction was confirmed adults have a long lifetime, with low mortality and by the positive relationship found between adult growth continuous growth. They perform numerous repro- rate and total offspring produced during the adult stage, ductive events, with little reproductive effort concerning and also by the positive relationships between the offspring number in each event, and low mortality after number of embryos contained in the uterus and shell the first reproduction. Reproduction is not limited to a size, the number of embryos and the number of whorls reproductive season, but is continuous throughout the in the shell, and between albumen gland and shell size. year. It can be concluded from these traits that L. In their study of the life history of B. tenuissimus, Silva unilamellata is a K-strategist species (Fig. 6). et al (2008) found that this species has a long lifespan An internally fertilized egg can be provided with and delays egg production until reaching a larger size. nutritive yolk, as well as with protective egg shells or The authors argued that larger snails gain reproductive egg capsules, which ensures the greater safety of the success by producing more offspring. In their study of offspring (Egonmwan, 2007). Calcified egg shells offer ovoviviparous pulmonate land snails Oreohelix cooperi, mechanical support to the embryo in the environment. Anderson et al. (2007) observed that adult size strongly After the hatching of the young, the egg shell serves as influenced per-clutch reproductive output, with larger the source of the first alimentary calcium uptake snails having larger and more offspring. The authors (Tompa, 1979; Baur & Baur, 2000). In L. unilamellata, argued that brooding organisms such as the O. cooperi the production of non-calcified eggs may be a conse- generally produce fewer offspring and the maternal quence of the evolution of ovoviviparity and potentially body size relates both to available brood volume and to provides an economy of micronutrients destined to the reproductive allocation per unit of time. production of offspring. Moreover, the embryos develop We observed a temporal trend in which the number inside the parental organism, were they are protected of individuals that produced offspring in each repro- against mechanical injury and desiccation. The release ductive event, as well as the number of living indi- of juveniles, instead of eggs, enables the offspring to viduals diminishes. According to Zera & Harshman resist environmental stress and increases their prob- (2001), mortality becomes sensitive to age because of ability to survive and to reach sexual maturity. The shell the dynamic aspects in the allocation of resources. At morphological features of the newly hatched juveniles, first, there is an investment in productivity and main- such as the shape of shell aperture and the presence of tenance of homeostasis; afterwards, in the senescent lamellae similar to adult shells, were also observed by stage, there is a decrease in reproduction, growth rate, Tryon & Pilsbry (1906). Brooding, defined by Heller activity and an increase in mortality. The remaining (1993) as the release of young that resemble small energy is directed to the maintenance of homeostasis. adults, is widespread among freshwater prosobranchs. 220 C.M. Carvalho et al. / IRD 53 (2009) 211–222

According to this author, in freshwater habitat brooding potentially promote a trade off between the size and may be an adaptation to protect the developing embryo the total number of hatchlings per reproductive event. against osmotic stress. Heller et al. (1997) suggested Interspecific comparison demonstrates that egg-retain- that for snails of the infraorder Orthurethra, which live ing and ovoviviparous species produce smaller clutches in an excess water environment, ovoviviparity may be than oviparous species (Baur, 1994). However, even advantageous because the eggs may be retained within though the volume capacity of the uterus and the energy the adult until full embryonic development of the costs of nourishment of large-sized juveniles reduce the embryos’ kidney is achieved, which is more primitive current reproductive output of ovoviviparous snails, its than in other land snails. In the case of L. unillamellata, reproductive success may be improved as a result of the which belongs to the infraorder , ovovivi- high survival rate of the hatchlings and the production of parity may be potentially advantageous because the numerous clutches throughout the reproductive life. embryos are protected against desiccation. In the present study, we found a relationship between In their study of the Clausiliidae of the genus Vestia, life history traits of L. unilamellata due to a differential Sulikowska-Drozd (2009) obtained results similar to allocation of energy. In their life strategy, the distri- those we found for the L. unilamellata in the present bution of the reproductive effort among many events, study. In Vestia turgida (Rossmässler, 1836), eggs are with the production of a small number of large juveniles not frequently released, and those that are released associated with egg retention, characterizes a long-term enclose hatchlings with the shell completely formed, investment in reproductive success. thus, viable. The egg membrane is very tiny and does not offer significant protection for the embryos outside the parent organism. Acknowledgments There is no agreement among authors regarding the The authors are grateful to Dr. Juliane Floriano S. concepts of ovoviviparity and viviparity in molluscs Lopes, Department of Zoology of Juiz de Fora Federal (Dutra, 1988; Tompa, 1979). In the present study, we University, for her invaluable help in statistical analysis, adopted the proposal of Mackie (1978) and Tompa and to Lidiane Cristina da Silva, Malacology Museum, (1979), which consider the site of development of the and Prof. Maury Pinto de Oliveira of Juiz de Fora embryo (either within or outside the reproductive duct), Federal University, for their significant assistance to be the source of nutrients, and the way that the during the biochemical analysis. This work was partially embryo obtains them. Most land gastropods are ovi- supported by the National Research Council (CNPq) parous and release eggs formed by a single zygotic cell. and FAPEMIG. In species that have an anatomical/physiological link between the parent organism and the embryo, the viviparity concept is applied. Some species can retain References eggs within the reproductive duct and, when released, Almeida, M.N. & Bessa, E.C.A. 2001. Estudo do cresci- these eggs may contain embryos in an advanced stage of mento e da reprodução de Bradybaena similaris development. Anderson et al. (2007) observed that the (Mollusca, Xanthonychidae) em laboratório. Revista embryos of O. Cooperi are retained over winter and Brasileira de Zoologia, 18, 1115–1122. released in late spring, so that they can grow oppor- Anderson, T.K., Weaver, K.F. & Guralnick, R.P. 2007. tunistically. The ovoviviparity is defined by Tompa Variation in adult shell morphology and life-history traits (1979) as an extreme case of egg retention, in which all in the land snail Oreohelix cooperi in relation to biotic embryo nutrients come from eggs. The eggs actually and abiotic factors. Journal of Molluscan Studies, 73, 129–137. hatch inside the uterus and the neonate is expelled. In Antkowiak, T. & Chase, R. 2003. Sensory innervation of the ovoviparous species, there are juveniles within the ovotestis in the snail Helix aspersa. 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