Different Dynamics of Reproductive Cell Development in Oviparous Clausilia bidentata and Ovoviviparous Ruthenica filograna Snails

© 2022 Academia Sinica, Taiwan Open Access Different Dynamics of Reproductive Cell Development in Oviparous Clausilia bidentata and Ovoviviparous Ruthenica filograna Snails Krystyna Szybiak1, Elżbieta Gabała2, and Zbigniew Adamski3,4,* 1Department of General Zoology, Faculty of Biology, Adam Mickiewicz University in Poznań, ul. Umultowska 89, 61-614 Poznań, Poland. E-mail: [email protected] (Szybiak) 2Institute of Plant Protection - National Research Institute, ul. Władysława Węgorka 20, 60-318 Poznań, Poland. E-mail: [email protected] (Gabała) 3Department of Animal Physiology and Developmental Biology, Faculty of Biology, Adam Mickiewicz University in Poznań, ul. Umultowska 89, 61614 Poznań, Poland. 4Laboratory of Electron and Confocal Microscopy, Faculty of Biology, Adam Mickiewicz University in Poznań, ul. Umultowska 89, 61-614 Poznań, Poland. *Correspondence: zbigniew[email protected] (Adamski) Received 22 May 2021 / Accepted 4 February 2022 / Published 10 May 2022 Communicated by Benny K.K. Chan Most terrestrial snails are oviparous and only some are ovoviviparous. The physiology of the reproductive system and its activity in these two groups have received little attention. We compared the month-tomonth dynamics of reproductive cell formation between two species of Clausiliidae snails: Ruthenica filograna (an ovoviviparous species) and Clausilia bidentata (an oviparous species). Our aim was to test the hypothesis that the seasonal activity of gametogenesis is determined by reproductive strategy (oviparity / ovoviviparity). The results showed that the frequency and number of reproductive cells in different stages varied month-to-month, within each species, and between the two species. Ruthenica filograna maintained its reproductive activity throughout the entire observation period, whereas the gonadal activity of C. bidentata changed with the seasons (it was temperature dependent). Our results are in tune with the hypothesis of Baur, that ovovivipary has some advantage over oviparity under harsher environmental conditions. Key words: Snails, Oviparity, Ovoviviparity, Evolutionary advantage, Reproductive cells. BACKGROUND There are 25–30,000 species of terrestrial snail, making them one of the largest taxa among terrestrial animals (Grosberg et al. 2012). Most of the species are oviparous. Our knowledge of the reproduction and embryonic development of Pulmonata is restricted to a few species (Tompa 1979; Tompa et al. 1984; Wirth et al. 1997; Heller et al. 1997; Heller 2001; KuźnikKowalska 1999 2005 2006; Pokryszko 2001; Maltz 2003a b). There is also little data concerning the development of reproductive z cells in other mollusks (Villalejo-Fuerte et al. 2018). Some reasearch papers, particularly by Sulikowska-Drozd and co-workers, have described the life history and population dynamics of a number of clausiliid species. However, in our opinion, the correlation between habitats and life history has still not been fully analyzed. Detailed information concerning snail reproduction is crucial for studying snail ecology, distribution and protection in the natural environment. This is especially true for species with rare developmental strategies; for example, the phylogeny and evolution of ovoviviparous snails. The family Clausiliidae contains over 1000 species, covering the entire range of reproductive strategies. Citation: Szybiak K, Gabała E, Adamski Z. 2022. Different dynamics of reproductive cell development in oviparous Clausilia bidentata and ovoviviparous Ruthenica filograna snails. Zool Stud 61:14. doi:10.6620/ZS.2022.61-14. Zoological Studies 61:14 (2022) doi:10.6620/ZS.2022.61-14 1 © 2022 Academia Sinica, Taiwan There are oviparous, egg-retaining (ovoviviparous), and viviparous species (Maltz and Sulikowska-Drozd 2013). There are Clausilid species that can reproduce both ovoviviparously and oviparously—with egg retention (Mamos et al. 2021). Some aspects of the life cycles of five of these species (Cochlodina laminata, Vestia elata, Alinda biplicata, Balea perversa and Ruthenica filograna) have been described (Bulman 1996; KuźnikKowalska 1998; Maltz and Sulikowska-Drozd 2008 2011 2012 2014; Sulikowska-Drozd 2009; SulikowskaDrozd and Maltz 2014; Szybiak 2010). Three clausilid species occur in the nature reserve “Rezerwat Dębno nad Wartą”: two oviparous species (Clausilia bidentata and C. laminate) and one ovoviviparous species (R. filograna). We decided to focus on the annual life cycle of two species with different reproductive strategies: C. bidentata, Strøm 1765 and R. filograna (Rossmässler 1836). Both are a similar size and found in the same habitat, thus at the same environmental conditions. Ruthenica filograna is a relatively abundant but not frequently discovered (i.e., appears in scattered but dense populations) species found from Southern Europe (Italy, Bulgaria) to Northern Europe (Estonia). It has been found in France, Austria, Hungary, Switzerland, Czech Republic, Slovakia, Poland and Germany. It is also present in mountains: Harz, Thuringian Forest, Carpathian Mountains, Alps, Franconian and Swabian Jura (Damjanov and Likharev 1975; Falkner 1990; Kerney et al. 1983; Manganelli et al. 1995; Wiktor 2004). It lives in broadleaf and mixed forests, and inhabits ground litter and dead wood (Szybiak 2009 2010). It does not climb plants or abiotic vertical structures, and avoids stones and rocks. R. filograna reaches altitudes up to 1500–1700 m above sea level. These snails spend most of their time in the soil and leaves if the temperature and humidity are appropriate (AnimalBase 2012; Baidashnikov 1989). Clausilia bidentata is a common, frequently discovered snail present in western, southwestern, northern and central Europe: the southern and western boundaries of southern Portugal and northern Spain, northwestern Italy (Liguria), western Switzerland, southwestern and southern central Germany, northern Bohemia, northwestern Poland and Baltic countries near to Baltic Sea (Nordsieck and Neubert 2002). It inhabits humid areas, moderately humid forests, and parks. It tolerates altitudes up to 1000 m above sea level. It can be found on and under rocks, on walls and on tree bark, as well as in litter (AnimalBase 2012). Shell sizes are similar for both species: C. bidentata—height up to 10 mm, width 2.0–2.5 mm (Wiktor 2004); R. filograna— height up to 10.2 mm, width 1.4–2.6 mm (Szybiak 2010). The hormone-regulating activity of reproductive organs in animals, including mollusks, is strongly affected by external factors, like photoperiod and temperature. Many molluscanand vertebrate-specific neuropeptides were described as being present in mollusks (Dorn 2000; Flari and Edwards 2003; Lafont and Mathieu 2007; Stange el al. 2012). The environmental stimuli, via optic tentacles and cerebral ganglia, affect androgen and oestrogen activity, cellular differentiation and growth in accessory organs (for a review, see Flari and Edwards 2003). Although comparing hormone activities between ovoviviparous and oviparous species was not the goal of the present study, we expect that the regulation of hormones and their receptors may differ between species displaying different reproductive strategies. Little information is available on the life cycle of C. bidentata (Frömming 1954; Likharev 1962; Nordsieck 2005; Szybiak et al. 2015), whereas the lifecycle of R. filograna was described in detail by Szybiak (2010). Laboratory observations revealed that R. filograna litters range from one to four offspring, and 80% are two to three offspring (Szybiak et al. 2015). They produce offspring from April to November, peaking in April and May (75% of individuals laid eggs in those months). In many cases, the individuals reproduced twice a year. C. bidentata reproduces from April to October, but these data come from stable, laboratory conditions. However, only 58% of individuals laid eggs, and egg laying peaked in June (47% of individuals laid eggs) (Szybiak et al. 2015). One interesting aspect of the two species is the limitations and possible benefits of each’s reproductive strategy. The differences in geographical range, altitude and habitats raise the question whether (and to what extent) ovoviviparity has benefits over oviparity in terrestrial snails in harsher environments. Perhaps, as suggested by Baur (1994), ovoviviparity minimizes mortality within eggs and protects them from predators or unfavorable environmental conditions. If this is true, then ovoviviparous species would be found more frequently or at higher numbers in areas and habitats unavailable for oviparous species, like in some reported cases (Heller et al. 1997; Shine 2004; Maltz and Sulikowska-Drozd 2013). In terms of habitat, C. bidentata and R. filograna’s geographical range and— primarily—altitude above sea level may support this statement. This suggests that developmental strategy may correlate with gonad activity, reproductive behavior and environmental conditions. More extreme habitats— e.g., higher altitudes, borders of the geographical range or harsh seasons—enforce the reproductive strategies that involve protecting the developing juvenile stages. This, in turn, allows the mature snail to reproduce page 2 of 13Zoological Studies 61:14 (2022) © 2022 Academia Sinica, Taiwan during the short periods of favorable conditions or in the conditions when oviparity is not possible. This may be an important reason why egg-retaining species are found in such habitats, while the distribution of oviparous species is limited to the more hospitable areas. In the present study, we investigated the seasonal dynamics of reproductive cell production in the gonads of two syntopic clausilid species in a nature reserve in western Poland. By comparing the seasonal dynamics and distribution ranges between the oviparous (C. bidentata) and the ovoviviparous species (R. filograna), we hoped to find indications for a correlation between reproductive strategies and ecology. MATERIALS AND METHODS Collection of snails Adult specimens of R. filograna and C. bidentata were collected at random from the nature reserve “Rezerwat Dębno nad Wartą” along the river Warta in western Poland (92 m above sea level, 52°05'N, 17°28'E, c. 20 ha); the reserve is in a riparian forest with oaks, alders, ashes and elms and is periodically flooded. The research was carried out from August 2011 to October 2012, with samples collected roughly every four–six weeks, with the exception of January 2011, when the weather conditions were poor and samples could not be collected. On each sampling occasion, five specimens with a fully developed shell were collected from each species by chance and taken for histological examinations. It is also worth mentioning that parts of the nature reserve periodically flood in spring, which may significantly affect the survival of eggs, embryonic development and hatching success of the early-laid eggs. Histological observations The specimens were anesthetized under CO2, sectioned and then investigated under a light microscope. The gonads were dissected in Ringer’s solution (5.87 g of NaCl, 0.73 g of KCl, 1.99 g of CaCl2, 1.87 g of NaHCO3, 2.63 g of MgCl2, 0.22 g of KHCO3 in 1000 ml of distilled water) under a stereomicroscope. They were fixed in Bouin’s fluid for 24 h. Then they were rinsed in 70% ethanol several times. Following dehydration, samples were embedded in paramat (paraffin wax blended with synthetic polymers (BDH)) and serially sectioned at 5–6 μm. Tissue sections were stained with hematoxylin and eosin. All the serial sections were examined using a light microscope. The sections are currently being stored at the Department of General Zoology of the Adam Mickiewicz University in Poznań. The following stages of spermatogenesis were described: sc1 - primary spermatocytes in meiotic I (chiefly in prophase): the most prominent spheroidal cells 10–12 μm in diameter with a large nucleus surrounded by a narrow band of cytoplasm. sc2 - secondary spermatocytes in meiosis II: cells 7–8 μm in diameter with a more abundant quantity of cytoplasm. st1 - early spermatids: small cells 4–5 μm in diameter with no flagellum. st2 - late spermatids: with flagella. sp - mature sperm cells forming bundles. The stages of oocyte development were distinguished during oogenesis (Griffond and BolzoniSungur 1986; Maltz and Sulikowska-Drozd 2010): po - previotellogenic oocytes: the lowest cells (30–40 μm in diameter), with basophil cytoplasm with no visible lipid droplets. vo - vitellogenic oocyte: extensive cells (90– 120 μm in diameter) with numerous lipid droplets. mo - mature oocyte: rounded cells (about 27 μm in diameter, cytoplasm filled with numerous inclusions and lipid droplets. Processing of data The stages of individual reproductive cells were estimated based on the method of Maltz and Sulikowska-Drozd (2010). The number of the cells in a particular stage were classified into the following ranks: 0 - no cells found. 1 - occasional cells in 1–2 lobules. 2 - cells were numerous in 3–5 lobules, but the number of cells does not exceed 50% of the lobule. 3 - cells were very numerous (more than 50% of the lobule) in more than five lobules. The mean number of the rank and the mode for each sectioned gonad were calculated. Then, the mean of these values for the gonads collected on the same day was calculated. Since the charts for means and modes looked very similar, we decided to use means to avoid potentially underestimating the presence of low numbers of cells within a particular point—in the case of low values, the mode may suggest there were no cells present. The mean values for adult gonads were analyzed, as indicators of the months when the fertilization might take place. When all stages were present, the rank was defined as > 1.5. The environmental temperatures were obtained from the webpage of the Polish Institute of Meteorology and Water Management - National Research Institute (2019). Table 1 shows the mean, maximum and minimum temperatures for the closest measurement geographical point at the day the sample was collected. In addition, the temperature change tendencies in the page 3 of 13Zoological Studies 61:14 (2022) © 2022 Academia Sinica, Taiwan days prior to the collection of snails were analyzed (Table 1). RESULTS The frequencies and number of particular stages of reproductive cells varied among months, both within each species and between the two species. The stages of reproductive cells found in both species are presented in figures 1 and 2. The development of mature reproductive cells in R. filograna is shown in figure 3. Neither sperm cells nor oocytes showed prominent seasonal peaks. One can observe that there were fewer mature cells in autumn and winter, but these mature cell numbers also differed among individuals. An interesting observation is that the curves for both male and female cells followed the same pattern, but sperm cells matured about one month ahead of oocytes. In addition, the majority of immature stages are present at almost every sampling date. In the case of both male and female reproductive cells, the number of particular stages resembles pyramids, with the youngest stages being the wide base of the pyramid. The premature stages were numerous (prevalence > 1.5) in more months than the mature oocytes and sperm cells (Fig. 4). In fact, the mature sperm cells never reached a prevalence > 1.5. The detailed dynamics of all tested types of reproductive cells are presented in table 2. Seasonal changes in C. bidentata reproductive cells showed a different pattern than in R. filograna. During autumn, winter and spring, the number of mature cells was relatively low (Fig. 5). However, as temperatures increased, the number of these cells increased, too, peaking in summer. Like in the case of R. filograna, we noted that the oocyte numbers increased one month after sperm cells. Also, the prevalence of immature cells was significantly higher in the summer months than winter and spring ones. The prevalence chart does not resemble a pyramid, as the production intensity for all stages stayed more or less constant across the months. Also of note is the difference in the number of the adult reproductive cells between August and September 2011 and 2012 (Fig. 6). Details on the dynamics of all tested types of reproductive cells are presented in table 3. DISCUSSION The main finding of our research is that the two snail species with different reproductive modes show temporally different patterns of reproductive cell development. Previously, we had not found any significant anatomical and histological changes in reproductive organs differences in the structure of reproductive tracts between C. bidentata and R. filograna, except for a periodic transformation of the oviduct’s distal part in R. filograna, which folds due to muscle contractions, and may widen to form a kind of a brood bag (Szybiak et al. 2013). In addition, Maltz and Sulikowska-Drozd (2013) did not find significant differences in the anatomy or histology of the spermoviduct and free oviduct sections between oviparous and viviparous species of snails from the clausilid subfamily Baleinae. Therefore, the dynamics of sperm and egg production seem to be particularly interesting, and important for comparing reproductive strategies. Brooding in ovoviviparous and oviparous snails has already been studied. For example, A. biplicata was found to switch its reproductive strategy towards oviparity under the influence of an oceanic climate (Sulikowska-Drozd et al. 2013). Incidental cases of A. biplicata laying eggs, as reported by Table 1. Official environmental temperatures at the measurement point closest to the Dębno Date 11.08 15.09 15.10 12.11 07.12 08.02 20.03 Mean temperature [°C] 14.5 11.9 2.2 -3.3 0.3 -13.5 5.5 Max temperature [°C] 21.2 20.2 7.2 4.7 3.2 -8.5 12.3 Min temperature [°C] 6.7 5.6 -1.5 -9.1 -1.5 -17.4 0.4 Tendency ↓↓↓↓↓↑↕ Date 30.04 02.06 12.07 23.08 20.09 18.10 Mean temperature [°C] 19.7 11.7 19.1 20.8 9.7 7.6 Max temperature [°C] 29.6 16.8 25.1 27.1 12.3 17.8 Min temperature [°C] 8.2 7 13.3 16.9 8.4 1.5 Tendency ↑↓↓↕↓↕ ↓ - decreasing tendency of temperature changes in the days before collection of snails; ↑ - increasing tendency of temperature changes in the days before collection of snails; ↕ - no clear tendency of temperature changes in the days before collection of snails. page 4 of 13Zoological Studies 61:14 (2022) © 2022 Academia Sinica, Taiwan Frömming (1954) and Fechter and Falkner (1990), might be triggered by some short term environmental cue influencing parturition. For the genus Vestia, interspecific variation and between-season variation were observed. The largest number of eggs were observed in the species that retain eggs for a shortest time, and the major diameter of eggs was about 0.25 mm longer in the longest-retaining species than in Fig. 1. Reproductive cells of R. filograna. 1°spermatocytes (sc1, a–d), 2° spermatocytes (sc2, a, b), 1°spermatids (st1, a), 2° spermatids (st2, b), mature sperm cells (sp, e), previtellogenic oocytes (po, b), early vitellogenic oocytes (ev, a, c), vitellogenic oocyte (vo, d), mature oocyte (mo, a, f). page 5 of 13Zoological Studies 61:14 (2022) © 2022 Academia Sinica, Taiwan the shortest-retaining one. (Sulikowska-Drozd 2009). Vestia turgida, which retains eggs for a the longest time, occurs at higher altitudes than Vestia gulo, which shows shorter egg-retention time. Our study is, to the best of our knowledge, the first to report on the seasonal dynamics in the formation of reproductive cells of the two studied species. The seasonal dynamics and development of Fig. 2. Reproductive cells of Clausilia bidentata. 1° spermatocytes (sc1, a, d), 2° spermatocytes (sc2), 1° spermatids (st1, a, b), 2° spermatids (st2, c), mature sperm cells (sp, e), previtellogenic oocytes (po, c), early vitellogenic oocytes (ev, d), vitellogenic oocyte (vo, d), mature oocyte (mo, c). page 6 of 13 Zoological Studies 61:14 (2022) © 2022 Academia Sinica, Taiwan reproductive cells are closely linked, and reproductive cell development is a basis for continuous or seasonal reproduction. The continuous process, in which extrauterine eggs are gradually released, was described for A. biplicata by Sulikowska-Drozd et al. (2013). These authors stated that this phenomenon is closely related to demanding, harsh climatic conditions— like higher altitudes, colder climates or unpredictable Fig. 4. Prevalence (value > 1.5) of all stages of R. filograna reproductive cells for different collection months. Mean temperatures and tendencies of temperature changes in the days before the collection of snails are shown at the top of the picture ↓ - decreasing tendency; ↑ - increasing tendency; ↕ - no clear tendency. Fig. 3. Dynamics of appearance of mature reproductive cells in R. filograna. Data are means. See table 2 for values and SD for other stages of cells. page 7 of 13Zoological Studies 61:14 (2022) © 2022 Academia Sinica, Taiwan changes in the seasons—and gives an advantage over oviparous species. Also, non-oviparous Baleinae species are found in mountain areas (Maltz and SulikowskaDrozd 2013). On the other hand, Mamos et al. (2021) state that, although the egg-retaining strategy may not have a clear advantage at the macroclimatic scale, but may affect the distribution within microhabitats. Environmental factors, like photoperiod, humidity Fig. 5. Dynamics of when mature Clausilia bidentata reproductive cells appear. Data are means. See table 3 for values and SD for other stages of cells. Table 2. Dynamics of production for all tested stages of reproductive cells within the R. filograna reproductive system Date Stage 01 Aug. 15 Sept. 15 Oct. 12 Nov. 07 Dec. 08 Feb. sc1 3.0 ± 0.0 3.0 ± 0.0 2.6 ± 0.49 2.8 ± 0.40 2.6 ± 0.48 2.4 ± 0.80 sc2 2.6 ± 0.49 1.5 ± 1.12 1.0 ± 0.89 0.4 ± 0.49 2.6 ± 0.80 2.2 ± 0.39 st1 1.6 ± 0.80 1.75 ± 4.43 0.2 ± 0.40 0.0 ± 0.0 2.4 ± 0.79 1.4 ± 1.02 st2 2.6 ± 0.49 2.25 ± 0.43 0.0 ± 0.0 0.0 ± 0.0 1.0 ± 1.27 0.8 ± 1.17 sp 1.4 ± 1.02 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.2 ± 0.40 1.4 ± 1.02 po 2.2 ± 0.75 2.5 ± 0.50 3.0 ± 0.0 1.4 ± 0.49 2.6 ± 0.80 2.6 ± 0.48 vo 3.0 ± 0.0 2.75 ± 0.43 2.2 ± 0.4 2.6 ± 0.51 2.2 ± 0.75 3.0 ± 0.0 mo 1.2 ± 1.47 0.75 ± 0.43 0.4 ± 0.49 0.6 ± 0.81 0.0 ± 0.0 0.8 ± 0.75 Date Stage 20 Mar. 30 Apr. 02 June 12 July 23 Aug. 20 Sept. 18 Oct. sc1 3.0 ± 0.0 3.0 ± 0.0 3.0 ± 0.0 3.0 ± 0.0 1.2 ± 1.47 2.8 ± 0.40 2.8 ± 0.40 sc2 2.4 ± 0.50 2.6 ± 0.78 2.7 ± 0.47 3.0 ± 0.0 1.4 ± 1.21 2.0 ± 0.63 2.6 ± 0.78 st1 1.0 ± 1.09 1.6 ± 0.47 2.7 ± 0.47 2.8 ± 0.40 1.4 ± 1.12 2.0 ± 0.63 1.6 ± 0.81 st2 0.6 ± 1.20 0.4 ± 0.82 2.3 ± 0.48 2.8 ± 0.40 1.4 ± 1.0 2.8 ± 0.38 2.4 ± 0.90 sp 0.2 ± 0.40 0.0 ± 0.0 0.33 ± 0.47 0.8 ± 0.75 0.47 ± 0.29 0.4 ± 0.80 0.0 ± 0.0 po 2.6 ± 0.80 1.8 ± 0.40 2.33 ± 0.49 2.2 ± 0.70 1.2 ± 0.85 1.8 ± 0.74 2.4 ± 0.49 vo 3.0 ± 0.0 2.8 ± 0.38 2.7 ± 0.42 3.0 ± 0.0 1.3 ± 1.26 3.0 ± 0.0 3.0 ± 0.0 mo 0.6 ± 0.49 2.2 ± 1.17 0.33 ± 0.46 0.6 ± 0.50 0.6 ± 0.29 1.6 ± 0.49 0.4 ± 0.45 Data are means ± SD. page 8 of 13Zoological Studies 61:14 (2022) © 2022 Academia Sinica, Taiwan and temperature, affect the activity of reproductionregulating hormones, e.g., the egg-laying hormone, male tentacular factor, and maturation hormone, and sex hormones (Sokolova et al. 1984; Takeda 1989; Flari and Edwards 2003; Ter Maat et al. 2012). The activity of these hormones must differ between R. filograna and C. bidentata; this is certainly worth studying in the future. The two different reproductive strategies of the Fig. 6. Prevalence (value > 1.5) of all stages of Clausilia bidentata reproductive cells for different collection months. Mean temperatures and temperature change tendencies in the days before the snails were collected are shown at the top of the picture. ↓ - decreasing tendency; ↑ - increasing tendency; ↕ - no clear tendency. Table 3. Dynamics of production for all tested stages of reproductive cells within the C. bidentata reproductive system Date Stage 01 Aug. 15 Sept. 15 Oct. 12 Nov. 07 Dec. 08 Feb. sc1 2.5 ± 0.65 1.5 ± 0.77 1.2 ± 0.3 0.62 ± 0.518 1.1 ± 0.30 1.18 ± 0.34 sc2 2.7 ± 0.55 1.7 ± 0.90 0.9 ± 0.25 0.68 ± 0.519 1.4 ± 0.56 1.09 ± 0.398 st1 1.0 ± 1.03 0.8 ± 0.33 0.4 ± 0.35 0.0 ± 0.0 0.3 ± 0.69 0.1 ± 0.233 st2 1.2 ± 0.12 0.9 ± 0.76 0.5 ± 0.36 0.06 ± 0.124 0.4 ± 0.58 0.19 ± 0.467 sp 1.7 ± 1.21 1.1 ± 1.07 0.5 ± 0.69 0.0 ± 0.0 0.6 ± 0.7 0.19 ± 0.467 po 1.5 ± 0.25 0.7 ± 0.28 0.9 ± 0.28 0.87 ± 0.56 0.9 ± 0.69 1.02 ± 0.43 vo 1.3 ± 0.49 0.3 ± 0.23 0.3 ± 0.15 0.69 ± 0.65 0.4 ± 0.34 0.77 ± 0.44 mo 1.2 ± 0.51 0.2 ± 0.11 0.3 ± 0.35 0.11 ± 0.16 0.1 ± 0.23 0.8 ± 0.83 Date Stage 20 Mar. 30 Apr. 02 June 12 July 23 Aug. 20 Sept. 18 Oct. sc1 1.0 ± 0.19 2.23 ± 0.2 1.7 ± 0.67 2.4 ± 0.39 1.8 ± 0.50 2.5 ± 0.65 2.1 ± 0.57 sc2 1.1 ± 0.32 2.43 ± 0.33 2.3 ± 0.49 2.33 ± 0.3 1.8 ± 0.47 1.0 ± 0.56 2.1 ± 0.57 st1 0.0 ± 0.0 0.0 ± 0.0 2.4 ± 0.35 1.53 ± 0.27 0.6 ± 0.39 0.6 ± 0.39 0.9 ± 1.0 st2 0.0 ± 0.0 0.0 ± 0.0 2.5 ± 0.31 2.3 ± 0.37 1.1 ± 0.36 1.1 ± 0.13 0.7 ± 0.70 sp 0.0 ± 0.0 0.0 ± 0.0 1.9 ± 0.43 2.93 ± 0.13 2.9 ± 0.13 2.5 ± 0.34 1.9 ± 0.79 po 0.8 ± 0.32 0.93 ± 0.25 1.0 ± 0.63 1.03 ± 0.43 1.0 ± 0.67 1.6 ± 0.39 1.6 ± 0.63 vo 0.8 ± 0.33 1.1 ± 0.49 0.9 ± 0.38 1.0 ± 0.31 1.4 ± 0.38 1.9 ± 0.40 1.9 ± 0.34 mo 1.2 ± 0.44 1.3 ± 0.57 0.7 ± 0.43 1.6 ± 0.39 2.4 ± 0.65 1.7 ± 0.57 1.7 ± 0.27 Data are means ± SD. page 9 of 13Zoological Studies 61:14 (2022)