Gametic cycle and gonadal maturity of Bulimulus bonariensis (Rafinesque, 1833) (Gastropoda: Bulimulidae), in a rural area of Buenos Aires, Argentina

Sampling

The study was conducted over the course of two consecutive years (March 2018 through December 2019), in a private field in the rural area of Villa San Luis belonging to the town of Florencio Varela (34°51′7.617″S, 58°15′53.932″W), province of Buenos Aires (Fig. 1). Authorization granted by note under docket number Cod 100 No 6359. The sampling was carried out in the early hours of the morning or at night in periods after rainfall or during drizzles, since the high humidity of the environment enables gastropods to be more active (Barrientos, 2003). The sampling area was delimited following De Winter & Gittenberger (1998) with modifications. Three 15 ×15 meter plots were demarcated in the field which were rotated in the different collection dates. The material was collected manually for 2 h each time, after a thorough checking of all the microhabitats in the plot. Figure 2 illustrates the sampling environment. In addition, climatological information was obtained from the National Meteorological Service (Servicio Meteorológico Nacional) available at https://www.smn.gob.ar/.

Processing

The samples obtained were transported to the laboratory following the recommendations of Barrientos (2003). The specimens were relaxed and sacrificed by placing them in airtight containers with water and Parafarm^® menthol crystals for 12–16 h. The separation of soft tissue was performed under a Leica EZ4 stereo microscope. The total length of the shell was measured with a millimetric vernier caliper (precision of 0.01 mm). After the dissection and anatomical observation, the penile complex was measured under the stereo microscope with a micrometer eyepiece from the base of the penile sheath to the end of the flagellum.

For histological analysis, the gonad was fixed in Raillet-Henry’s solution for 12 h and preserved in 70% (v/v) aqueous ethanol followed by dehydration in an increasing sequence of alcohols. For inclusion, the tissue was immersed in Histoplast^®, slices of 7 µm were made with a Minot-type microtome and stained with Mayer’s hematoxylin-eosin before mounting in synthetic balsam. The histological preparations were observed and photographed under a Leica-DMLS light microscope.

Gametogenic cycle

The establishment of the different gonadal stages as the basis of descriptive qualitative data was performed upon observation of the histological preparations. The gonadal cycle was divided into five stages: proliferation, growth, pre-evacuation, evacuation, and resorption; and in order to complement the description of the gametic cycle, a classification of the germ-cell population was made according to the criteria proposed by Maltz & Sulikowska-Drozd (2010). Accordingly, the cell types were considered as follows: 0, absence of a cell type; 0.5, with 1-2 cell clusters or 1-2 oocytes; 1, several cell clusters or 3-6 oocytes; 2, more than 10 cell clusters or more than 6 oocytes. In addition, to determine the stages of female germ-line development, the oocyte diameters, as estimated after Griffond & Bolzoni-Sungur (1986), were recorded after measurement by the digital-analysis program FIJI. (available at https://fiji.sc/). The reproductive stages were established in accordance with the terminology used in Giese & Pearse (1977) and Baqueiro Cárdenas (2014).

Size at first maturity

To determine the minimum size of the snails at initial gametogenesis, individuals were classified according to the state of the ovotestis: 0, individuals with undifferentiated or immature gonads involving the presence of undifferentiated germ cells and previtellogenic oocytes, and 1, mature individuals exhibiting the presence of vitellogenic oocytes and spermiogenesis stages. The data were then pooled every 0.5 mm and the proportion of gonadal maturity calculated. A logistic regression, through the use of the PAST 3.25 program (available at https://past.en.lo4d.com/windows), was used to calculate the body length at which 50% of the individuals were mature. Finally, the fit of the model was evaluated with SPSS Statistics version 22 (available at https://www.ibm.com/products/spss-statistics).

In addition, the relationship between total penile complex length and total body length was evaluated by a simple regression analysis to determine if the latter measurement could be considered another maturity parameter. Furthermore, the general appearance of the reproductive system was observed in order to determine if a morphologic variation occurred in any structure throughout an annual gonadal cycle.

Sampling

The study site is characterized by a temperate humid subtropical climate. Table 1 shows the environmental variables, temperature and precipitation, by season for the years studied.

Material was obtained from 15 samplings involving 168 individuals in total, which were grouped by season. In 2018, 88 individuals were collected 21 (summer), 25 (autumn), 14 (winter), 28 (spring), and in 2019, 80 individuals 24 (summer), 26 (autumn), 13 (winter), 17 (spring).

Cell types

The ovotestis in an immature (Fig. 3A) or mature (Fig. 3B) state, is formed by parallel acini or follicles grouped in lobes one after the other.

Spermatogenesis develops associated to the epithelium that limits the follicle. As spermiogenesis proside, the developing sperms become oriented towards the lumen. Quadrangular shaped spermatogonia ∼20 µm long were identified with large ∼7 to 8 µm nuclei and abundant cytoplasm, arranged in clusters (Figs. 4A; 5B: sg) or parallel to each other (Fig. 4C) in direct contact with the follicle epithelium. The primary spermatocytes are rounded (7–10 µm in diameter), and the chromatin is dispersed occupying the cell body almost completely (Figs. 4A, 4C, 4D: sc1). The secondary spermatocytes have a rounded shape and a diameter similar to the primary spermatocytes, but the chromatin is condensed in the center of the cell, and the cytoplasm is differentiated (Figs. 4C, 4D: sc2). The spermatids are present two stages: the early spermatids have an elongated cell body with a round nucleus located at the end of the cell (Figs. 4C, 4D; 5B; 6A: st1). The cells are in contact with the epithelium of the follicle or are located surrounding the Sertoli cells, which provide support and nutrition (Figs. 4A, 4D; 5A; 6A: S). In the late spermatids the nucleus becomes oval in shape and the tail begins to differentiate (Figs. 4C, 4D, 5A, 5B: st2). The mature spermatozoa have a defined head, they form clusters with their long tails or flagella directed towards the follicle lumen, and are located in contact with the follicular wall or a Sertoli cell or lie in the follicle lumen ready to descend through the ducts (Figs. 4D; 5A, 5B, 6A, 6B; 7A, 7B: sz).

Unlike the male gametogenic line, the female germ line develops at the terminal ends of the follicles or acini.

The few oogonia present are characterized by being rounded, ∼10–15 µm in length, located in contact with the follicle walls, or attached to the latter by a pedicel, (Figs. 6A, 6B: oog). Those oogonia give rise to oocytes with condensed nuclei; which begin to accumulate yolk granules in the cytoplasm and, as they develop, increase in size to over 130 µm. The oogonia are always in direct contact with the walls of the follicle; and, only when they become mature oocytes they located in the lumen of the follicle or are found descending through the ducts.

Previtellogenic oocytes are rounded with a diameter between ∼20 and 60 µm, a basophilic cytoplasm without visible granules, a large nucleus, and a nucleolus (Fig. 4B: po). As the previtellogenic stages develop into vitellogenic oocytes, they measure between ∼60 and 100 µm and accumulate yolk progressively. Thus the cytoplasm changes to become eosinophilic (Fig. 4D: vo). Mature oocytes measuring ∼100–140 µm in diameter and possessing yolk granules, are observed in follicles at the pre-evacuation stage with more than six oocytes present in the gonad and descending towards the ducts (Figs. 5A; 6A–6D: mo). During the evacuation, up to three oocytes were descending through the ducts. During resorption (Figs. 7A, 7B) the follicles contained inner spaces or were collapsed and contained amaebocytes or phagocytic cells in the lumen (Fig. 7B: am).

Gametogenic cycle

The gametogenic cycle can be divided into five stages:

Proliferation: This stage contains an abundant number of spermatogonia and some primary spermatocytes along with Sertoli cells. Follicles with oogonia and some previtellogenic oocytes (20–60 µm in length) are also growing.

Growth: This stage features the spermatogenic series with primary and secondary spermatocytes plus early and late spermatids. Few sperm cells are in contact with the epithelium of the follicle along with Sertoli cells. In the female germinal series, the oocytes (60–100 µm in length) are in vitellogenesis and with increasing amounts of yolk.

Pre-evacuation: In this stage, the lumen of the follicle contains abundant clusters of spermatozoa and late spermatids with their heads in contact with the epithelium of the follicle. Sertoli cells are present. Mature oocytes (100–140 µm in length) contain numerous yolk granules and are mostly located in the ducts of the follicles ready to be released.

Evacuation: The follicles contain few sperm clusters and Sertoli cells along with few oocytes filled with yolk. Some ovogonia begin to appear.

Resorption: The follicles become contracted or with a wide distance between them. In other instances, within the spaces in the lumen amaebocytes and developing spermatogonia are present along with some clusters of spermatozoa and unreleased oocytes.

The seasonality of gonadal development (Fig. 8) reveals a continuous proliferation throughout the year, beging accentuated in summer, remains elevated in autumn and winter and decreases towards spring. No specimens were found in proliferation during the spring of 2018. The pre-evacuation and evacuation of gametes occurred mainly in the spring: these stages did not occur in autumn. Finally, resorption was only observed during the autumn of 2018.

Periods of the gonadal cycle

The gonadal activity exhibited variations during the year that were separated into three periods:

Spawn: This stage began in winter when one part of the population was in gametogenesis and the other in spawning.

• Female germ line: during the spring, the germ line evidenced a proliferation of the oogonia, whereas during the rest of the year this cell type was not detected. In addition, the gonads contained oocytes of all sizes ranging from 20 to 140 µm (Fig. 9), few previtellogenic oocytes, increasing numbers of vitellogenic oocytes of 80–100 µm in length, and oocytes reaching the mature stage with lengths ranging from 100–140 µm were observed. Up to eight mature oocytes were found only in this season ready to be evacuated with abundant yolk granules (Figs. 6B, 6D). Thereafter, the size of the oocytes became smaller, indicating that the reproductive period occurred in this season.

• Male germ line: pre-evacuation was observed with several groups of mature spermatozoa in addition to spermatocytes and spermatids (Table 2). The ovotestis contained numerous clusters of sperm ready to be evacuated, while the hermaphroditic duct was likewise found to be replete with mature sperm (Fig. 10).

Post-spawn: this stage occurs in summer, with some individuals still in evacuation.

• Female germ line: most of the oocytes measured were in the size range of 20–80 µm (Fig. 9), thus indicating the presence of a new generation of oocytes without a gonadal rest.

• Male germ line: in the spermatogenesis, the highest abundance of the five cell types was found.

Mature:

• Female germ line: the oogenic activity during the fall was observed with previtellogenic oocytes and abundant growing vitellogenic oocytes. The most frequent size range in the growing previtellogenic oocytes was 40–60 µm and in vitellogenic oocytes 60–80 µm, with the cells gradually incorporating yolk (Fig. 9).

  Resorption occurred only in the autumn along with the lysis of the oocytes and sperm that were not discharged, with gaps now being present between the follicles (Fig. 7A). In other specimens, follicles were observed with large lumens and amaebocytes (Fig. 7B). In winter, the most abundant previtellogenic oocytes were those ranging between 40 and 60 µm in length (Fig. 9).

• Male germ line: the highest abundance of primary and secondary spermatocytes was found in autumn. Although the male germ line proved have continuous spermatogenic activity throughout the year, with numerous clusters of spermatogonia to initiate the next gametic generation; all the stages were present throughout the two years of the study. In winter, as mentioned above, that part of the population evidenced a great proliferation with abundant secondary spermatocytes plus early and late spermatids.

Morphologic variation in relation to the gametic cycle

The only structure that underwent periodic anatomical variations was the spermioviduct, which passed through three phases corresponding to the successive stages of gametic development, being thin with an undeveloped appearance and not folded; during development, manifesting an intermediate diameter with slight folding; and, finally, containing a spermioviduct with an appreciable degree of development and folded back on itself (Fig. 11). When we compared these characteristics and the stage in which the gonad was found, we were able to recognize the following:

• Some specimens evidenced undeveloped or thin spermioviducts corresponding to proliferating or growing stages of gonad (Fig. 11A).

• Other specimens contained spermioviducts in the process of development with an intermediate diameter correspond to the Maturity stage of gonadal development (Fig. 11B).

• Still other specimens exhibited well developed spermioviducts corresponding to the Spawning or evacuation, Post-Spawn and reabsorption stages of gonadal development (Fig. 11C).

Therefore, the stage of development of the spermioviduct was found to be clearly an informative element in determining, a priori, the stage of development of the ovotestis.

Size at first maturity

The ovotestis was observed at different stages of development, namely, undifferentiated with no germ-cell differentiation (Fig. 3A), followed by immaturity or gametogenic activity and later maturity. We also observed that the first previtellogenic oocytes appeared from the time associated with a total shell length of 8.42 mm, while the first spermatogonia were observed at a shell length of 9.92 mm. Figure 12 illustrates the distribution of the proportion of gametic maturity that corresponds to the total shell size (TL). The data indicated a good fit to the model with the following pseudo-R squared: Cox and Snell = 0.732, Nagelkerke = 1.

The size at which 50% of the population was considered mature was approximately 12 mm TL.

When the length of the penile complex (LPEC) was plotted as a function of the TL of the specimens (Fig. 13), the correlation between both variables was positive and statistically significant (r = 0.88, p < 0.01). In addition, we observed that in individuals larger than 13 mm, when they had already reached gonadal maturity, the dispersion was greater, where 81.7% of the instances corresponded to individuals with an LPEC of four millimeters or more.