Histological Study of Gonadogenesis in Potamopyrgus antipodarum and Valvata piscinalis

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Introduction
he present work involves an anatomical and histological study of two freshwater gastropod prosobranch snail species: 1) Potamopyrgus antipodarum, using only parthenogenetic females (Figure 1 and 2) Valvata piscinalis hermaphrodites (Figure 2), and paying particular attention to their gonad development. hey were chosen as models to study the efects of endocrine disruptors on production.  he biological cycle of these two species depends heavily on environmental factors. he growth rate increases in spring and summer while in autumn it slows down and even halts in winter [1].
Like other gastropods, their diet varies, alternating between detritus and vegetation depending on the season. hey principally feed on epiphytic algae in summer and on detritus when living among mudded foliage during cold periods. heir dispersion has been afected generally by humans, birds, ish, and even amphibians, insects, and currents [2]. hese snails are relevant ecological indicators due to their wide distribution. hese biological models are sensitive to diferent reprotoxic substances. P. antipodarum is known as a pertinent organism for studying endocrine-disrupting substances [3]. Taking advantage of this, we took up a histological study focusing on their anatomy and gonadogenesis to examine precise biological changes, such as late gonadogenesis and the decrease in embryo reproduction, during reprotoxicity studies.

Classiication and morphology
P. antipodarum is an invasive species from New Zealand, hence its common name "New Zealand mudsnail", which over time has colonized Australia, Europe and the America.
As a general rule, this species is diploid in its native and sexual population though exclusively parthenogenetic and ovoviviparous in Europe.
hey thrive mostly in fresh and brackish waters and are remarkably resistant to harsh conditions, especially with regards to oxygen demands and thermal variations. hey are also easily transportable from natural habitats to laboratory conditions (if the temperature is kept around 18°C).
As far as reproduction characteristics are concerned, egg-laying is continuous, although reproduction may be considered as seasonal, with optimal fertility of females in spring. P. antipodarum spends most of the winter in the juvenile stage and begins reproducing in June. heir life span is up to 1 year or more.

General morphology
P. antipodarum has a sot body and a clearly distinct head. he visceral mass curls up in a helical shape and the bronchial cavity is in the front [4]. he mantel surrounds the visceral mass and forms a cavity containing feather-like gills. he tentacles are long and thin, with eyes situated on the tip ends.
Secreted by the mantel, the cone-shaped shell is thin although solid, with a yellowish-brown color, and has convex or rounded spirals [5].
he gonads are in the inferior half of the whorl, forming a spiral with a digestive gland and other individual organs [6] V. piscinalis, the European stream valvata, is an autochthonous species found throughout France [2]. Present in all types of freshwaters, they mostly colonize muddy habitats having superior vegetation. hey are abundant in sewer pipes as well in brooks and streams with slow water low ( Figure 2) [1].
his snail is a hermaphrodite with both distinct male and female organs. hey have similar biological and ecological characteristics to other gastropods. hus the characteristics discussed above for P. antipodarum can be applied to this species. he shell is subdiscoiled with a circular opening and an almost centered corneous operculum. Yellowish in color, it has three and a half whorls.
Reproduction is seasonal and takes place between April and September.

Materials and Methods
his experiment in controlled conditions was carried out in the laboratory using healthy individuals. It deined the size at which the two snail species (P. antipodarum and V. piscinalis) began sexual maturity and detected the diferent oocyte maturation stages.
Physicochemical experimental conditions for raising P. antipodarum and V. piscinalis he study was conducted from February 6 th to July 2 nd 2008. Fortyive P. antipodarum and V. piscinalis juveniles, just 48 h old, were raised in the same experimental conditions at 16°C for 6 months (ive organisms per beaker/L H 2 O for ive beakers).

Histology
Two samples for each species were examined once a week. hey were ixed to Bouin liquid for 24 h for juveniles and 48 h for adults. his step is essential in order to stabilize the tissue and cellular structures [7]. Bouin liquid was chosen because it does not disturb the morphology of the organisms, and it contains glacial acetic acid for dissolving the shell.
he material was rinsed with increasing alcohol concentrations for complete dehydration. he "lightening" step follows, which involves a solvent bath (Butanol) to replace intracellular water with parain. hen 4-µm cuts were made by the Leica® microtome, and then stained with hematoxylin-eosin stain. he hemalum stain dyes the acidophil structures (such as the nuclei) and the eosin dyes the basophile structures (such as the cytoplasm).
he Leica® microscope was used for observations.

Potamopyrgus antipodarum
he dissections show that the gonad overlaps with the digestive gland taking up the basal half ( Figures 5A, 6A,6B). Evolving near the foot, the albumen gland, whose role is to secret albumen which envelops the oocytes, can be observed (Figures 6A,6B). Only female tissues were observed from the parthenogenetic specimens.
he histological study of gonad development for P. antipodarum showed early gonadogenesis when they reached 2 mm, at which point the primary oocytes increased in size (2.5 mm) (Figures 5A,5B) as well as the 1 st order secondary oocytes ( Figure 6A). At 3.5 mm, we observed a complete maturation of the gonad with a presence of all maturity stages ( Figure 6B) and at 4.5 mm the embryos in the embryonic sac ( Figure 7A). he maturation stages were still present at this size for other individuals ( Figure 7B).

he diferent oocyte maturity stages
Oocytes in primary vitellogenesis: During primary vitellogenesis, the cells were small and star-shaped. he central, nucleolated nucleus was hardly diferentiated and the basophile cytoplasm was inely granulated (Figures 5A,5B).

Oocytes in secondary vitellogenesis:
Two types of oocytes were diferentiated: immature oocytes and mature oocytes (Figures 6A,6B). Immature Oocytes: hese were medium-sized cells with a central, round, nucleolated, basophile nucleus and a cytoplasm that was very receptive to hematoxylin: oocytes in irst-order, secondary vitellogenesis ( Figure 6A).
Mature Oocytes: hese are large cells with a basophile nucleus (germinating center) with a half-moon shape. Its movement from the center toward the periphery is an indicator of its maturity. At this stage, the cells were illed with lipid droplets and homogenous vitellus eosinophil globules with a half-moon diameter, which accumulate in the oocyte cytoplasm during the maturation phase: oocytes in secondary mature vitellogenesis ( Figure 6B).

Valvata piscinalis
he V. piscinalis gonad begins at the coiling extremity where the irst male cells were observed. Maturity of the hermaphrodite glands occurs near the foot of the individual. Male cells can be observed in the center of the gland and female cells near the periphery ( Figures  8A,8B, 9). he hermaphrodite gland overlaps with the digestive organ. Continuing on the foot, the atrium and the ventricle can be observed just above the embryonic sac near where the mature female cells are channeled through the follicular cord ( Figures 10C,11A,11B). he kidney is against the albumen gland, which itself touches the mucus gland ( Figures 12A,12B,12C).
Histology of the V. piscinalis gonad makes it possible to detect early gonadogenesis at week 7 (1.9-2 mm), associated to the proliferation of male tissue. At the extremity of the coil, early acini formation can be observed in the middle of a thick eosinophilic (conjunctive) basal layer, surrounded by primary oocytes, in turn surrounded by albumen gland cells (Figures 8A,8B). he empty follicular cord in the embryonic sac is visible ( Figure 5B).
At week 11 (2.5 -3 mm) the number of acini garnished with oogonia ( Figure 8A) are visible near the inferior extremity of the gonad increases.
At week 16 (4 -4.5 mm) complete gonad maturation can be observed, with perfect arborization of seminal receptacles, as well as the diferent oocyte maturity stages (Figure 9).
Male tissue: he histological cuts observed at high magniication ( Figure 10A) revealed a tubular structure resembling seminiferous tubes separated by an amorphous tissue composed mostly of isolated b f a c e PLATES 2 and 3 (Figures 8-12): Different gonad maturity stages and internal organization of different organs observed in Valvata piscinalis a) acini; b) albumen gland; c) digestive tube; d) light of digestive tube; e) digestive gland; f) primary oocytes; g) acini covered with spermatogonia; h) 1st order secondary oocytes; i) secondary mature oocytes; j) germinating center; k) efferent canal covered with spermatozoids; l) germinating spermatogonia; m) spermatozoids; n) different maturity stages of spermatogonia, with light increasing along with maturity; o) cells resembling Leydig cells; oc: Leydig cells with Reinke crystalloids; p) intestine; q) ventricle; q1) atrium; r) embryonic sac; s) nephridial gland; t) mantle; u) columella muscle; v) follicular cord; w) oviduct; x) albumen gland; y) nerve cord; z) mucus gland; arrow: cells resembling Sertoli cells; Lu: light from the seminal receptacle; *: kidney.  cells or regrouped ones surrounding blood vessels. hese are known as Leydig cells recognizable by their stick-like, crystalloid inclusions known as Reinke crystalloids [8] ( Figure 10B). he latter feature plays an important role in gonad mechanics.
Maturity of male gonads begins with spermatogenesis, a process by which the spermatids, as a result of meiotic division, go through the diferent maturation stages before being transformed into mature spermatozoids.
he transversal cut ( Figure 10A) represents spermatogenesis forming where the arborization takes on a grape-like shape progressing in the light.
he seminiferous tubes are disposed in the center of the hermaphrodite gonad ( Figure 8B) surrounded by female cells (oocyte). On the peripheral, they are surrounded by a stratiied epithelium constituted of two cellular types.
Germinal cells: he type A spermatogonia in the basal compartment was undergoing several mitotic divisions and going on to form type B spermatogonia (future spermatozoid). Type A spermatogonia may be characterized by their voluminous nucleus and condensed chromatin, where as the type B spermatogonia have dispersed chromatin and dull colored cytoplasm. hese latter were undergoing meiotic division leading to spermatozoid formation, which is recognized by their heads anchoring in the nourishing cells' cytoplasm with a lagellum extending towards the light of the seminiferous tube ( Figure 10A).
At 14 weeks (size 3.4 mm), diferentiated gametes were observed and three cellular types were taken into consideration: type A and type B spermatogonia, and then spermatids due to their anchoring in the cytoplasm of Sertoli cells ( Figure 10A).
It's rare and oten impossible to observe all the developmental stages on the same cut as they usually develop in clusters from the same cellular stages.
Non germinal: Cells alternating with type A germinal cells, Sertoli cells can be observed. hese are voluminous cells with a pyramid shape and a cytoplasm blocking light due to their apical pole, which are recognized when anchoring with spermatids ( Figure 10A). heir essential function is for nurturing young spermatozoids. Female tissue: Once the male tissue was formed, the female tissue formed with the development of the irst oocytes ( Figure 8B) whose maturity stages are identical to those described for P. antipodarum. hey have a follicular cord resting against the lower part of the gonad and pushed toward the oviduct throughout the maturation course in order to begin the fertilization chamber where their cells are produced. hey start in the embryonic sac where embryo formation is completed ( Figure 10C,11A,11B).

Discussion
In our experimental conditions, primary oocytes from individuals measuring less than 2 mm were not observed, the minimum size at which we could count oocytes for individuals 19 and 20 weeks old. We can also assume that at 2 mm or more, oocytes in 13-and 14-week-old individuals were not observed (Figure 3). he mature gonad is only visible in individuals with a minimal size of 2 mm in smaller individuals, in our laboratory conditions, primary oocytes were not observed. Nevertheless, this observation does not seem conclusive since certain individuals with this size limit and younger than 15 weeks did not reach the irst observable oocyte maturity stage. Rapid growth of juveniles does not appear beneicial for oocyte maturation and reproduction.
hese results observed in laboratory conditions should be conirmed on individuals in the ield where trophic conditions may be more optimal for reproductive activities.
For V. piscinalis, sexual maturity seems to begin when individuals reach 1 mm and 6-8 weeks old, with ovogonia appearing. Primary oocytes were not observed among individuals smaller than 2.5 mm and 12 weeks old. All individuals exceeding these limits appeared to have advanced stages of oocyte maturation.
For the males, we observed an arborization of spermatogonia in the seminal receptacles of individuals between 2.6 mm and 3.8 mm in size and full oocyte maturation for those reaching 4 mm ( Figure 4). he lack of references is explained by the fact that our laboratory is the irst to report a study of the histological anatomy of these two gastropod species.

Conclusion
hese results brought out the gonad maturity stages in the mud snail species P. antipodarum and V. piscinalis. We were able to suggest a size limit around 2.5 mm at which point all individuals reach advanced gonadogenesis. Sizes below 1 mm are too small to detect gonad maturation activity (oogonia and oocytes absent).
With these results, we may now proceed with the second level of research which will involve reprotoxic stress studies.