Gametogenic processes in the pearl oyster, Pteria penguin (Roding, 1798) (Bivalvia, Mollusca).

ABSTRACT The aim of the study is to reveal the gametogenic cycle in the pearl oyster, Pteria penguin. The specimens were collected from Si-chang Island (Thailand) from January 2000 to January 2001. Gametogenesis was observed in histological preparations of gonads from 142 adults sampled monthly. Spermatogenesis was classified into 10 stages: spermatogonia, 5 stages of primary spermatocyte, secondary spermatocyte, 2 stages of spermatid, and spermatozoa. Oogenesis was comprised of 6 stages: oogonia and 5 stages of oocyte. Three stages of gonad development were identified in the male: developing, mature, and partial spawning. In the female, there were 4 stages of gonad development: developing, mature, partial spawning, and spent. The spawning cycle of the male was observed throughout the year with a peak of partial spawning in March to June (60%) and in December to January (40%). Females spawned in June and July (50%). The water temperature and the salinity were not correlated to the reproductive cycle of P. penguin. The results of this study can be applied to the induction of spawning and the production of seed in the aquaculture system of this pearl oyster species.

KEY WORDS: Pteria penguin, gametogenic cycle, pearl oyster

INTRODUCTION

There are many species of pearl oyster in Thailand. Among these species only 3 species, Pinctada maxima, Pinctada fucata, and Pteria penguin, are widely cultivated in Phuket Island, Phuket Province, and Samed Island, Rayong Province. P. maxima produce large and good quality pearls. They are widely cultured to produce round pearls, which are quite expensive. P. fucata are smaller in size and they are cultured to produce small round pearls. P. penguin are medium in terms of size and are cultured to produce hemispherical pearls.

Pearl culture presents a significant potential for economic development in coastal village communities throughout the range of the more valuable species. The industry requires minimal capital input, yet has wide ranging benefits to farmers, coastal communities, and national economies. Pearls are the ideal export commodity; they are nonperishable, shipping costs are negligible, and lucrative markets are already established.

The biology of pearl oysters is poorly understood considering the importance of pearl culture and shell fisheries (Chellem 1987, Gervis & Sims 1992). Research and development priorities in developing countries include the assessment and protection of remaining stocks, evaluation of culture potential, and definition of management strategies for disease prevention. Improvements in spat collection methods, recent hatchery culture successes, selective breeding and genetic manipulation, and advances in pearl implantation techniques all have potential applications in village-based production (Alagarswami et al. 1987).

Among pearl cultures in Thailand, only that of P. maxima has been extensively studied, especially in regard to obtaining spat. They are the most important species in the pearling industry of Thailand. For the other two species, they are either collected from the sea or their spats are imported from Japan. There has been an attempt to culture P. penguin at Phuket Island but it is still at the early stage. In addition, information on the reproductive biology such as gametogenesis and reproductive cycles of these three species of pearl oyster is still lacking in Thailand. Because P. penguin can be easily collected by SCUBA diving, this species was chosen to study gametogenesis and the reproductive cycle.

MATERIALS AND METHODS

Ten to 15 P. penguin pearl oysters were collected monthly between January 2000 and January 2001 from natural stock in the Si-chang Islands, Chonburi Province. Specimens were collected by SCUBA diving at 15-18 m depth. The pearl oysters were measured for shell length and shell width with Vernier calipers and the soft body weights were recorded. Water temperature and salinity at the collecting site were recorded.

A total of 142 P. penguin pearl oysters (72 males and 70 females) were examined histologically to determine the gametogenic cycle of both sexes. Gonad tissue between the proximal end of the gut loop and the base of the foot was excised to obtain the largest sections possible (Rose et al. 1990, Wada et al. 1995). Samples were fixed in Bouin's solution overnight. The tissues were then washed in 70% ethyl alcohol, dehydrated with a graded series of ethyl alcohol (70% to 100%) for 30 minute each, embedded in paraffin and sectioned and stained with Harris hematoxylineosin or PAS hematoxylin. Sections were observed and photographed under an Olympus CH40 light microscope.

In addition, samples of gonads were fixed in 3% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.8) for 24 hours. The tissues were then washed in the buffer solution and postfixed for 1 hour in 1% osmium tetroxide in 0.1 M buffer. After being dehydrated in a graded series of ethyl alcohol, the tissues were embedded in Araldite 502 resin. Sections (1 [micro]m) were stained with l% methylene blue and observed under an Olympus CH40 light microscope.

RESULTS

The water temperature and salinity at the collecting site of pearl oyster, P. penguin were measured every month. The water temperature was not much different all year round being in the range of 26.8-30[degrees]C. The salinity at the collecting site was quite stable all year round (30-32 ppm).

In general, the female P. penguin shells were slightly larger than those of males. The mean shell width and length were 14.59 [+ or -] 1.69 cm and 11.32 [+ or -] 1.34 cm, respectively. Male and female P. penguin could not be distinguished by their shell morphology. Gonad color was also an unreliable indicator of sex. The gonads of P. penguin were not discrete organs, but were a greater proliferation of gonad follicles in the area between the byssal gland and gut loop. Gonad follicles proliferate within the connective tissue between the epithelium and the visceral mass.

Classification of Germ Cells

Germ cells appearing in the histologic sections of gonads of P. penguin, as observed by light microscope in paraffin and plastic-embedded semithin sections, could be classified as follows:

1. Spermatogenesis

The testis of P. penguin is composed of several acini, each surrounded by connective tissue. Based on nuclear characteristic and cell size, the male germ cells of P. penguin can be classified into 10 stages. They are spermatogonium, 5 stages of primary spermatocyte, secondary spermatocyte, 2 stages of spermatid and spermatozoa (Fig. 1).

[FIGURE 1 OMITTED]

Spermatogonium (Sg). The spermatogonia are attached largely to the acinus wall. The cell is spherical or oval shaped with nuclear size about 5 [micro]m in diameter (Fig. 1A). The nucleus contains mostly euchromatin and small blocks of heterochromatin dispersed throughout (Fig. 1A). The thin cytoplasm was stained light blue with methylene blue and the nucleolus is distinguishable within the nucleus.

Primary spermatocyte (PrSc). The primary spermatocyte consists of 5 stages: leptotene (LSc), zygotene (ZSc), pachytene (PSc), diplotene (DSc) and metaphase (MSc) (Figs. 1 A, B, C). The distinctive differences among various PrSc are the chromatin condensation pattern and the amount of euchromatin and heterochromatin.

Leptotene spermatocyte (LSc). The spherical-shaped cell of LSc is larger than Sg with a nuclear size approximately 5 [micro]m in diameter (Fig. 1B). The chromosome begins to condense into small blocks of heterochromatin and are scattered throughout the nucleus (Fig. 1B). The nucleolus is still present.

Zygotene spermatocyte (ZSc). The cell in this stage is approximately the same size as LSc (nuclear diameter of 5 [micro]m) (Fig. 1C). The heterochromatin blocks become larger and increase in thickness (Fig. 1C), The nucleolus is no longer distinct.

Pachytene spermatocyte (PSc). The PSc cell is smaller than ZSc (Figs, 1A, C). The nucleus contains mostly heterochromatin which are long thick fibers and are arranged in bouquet like pattern (Figs. 1A, C). The nucleolus is not visible.

Diplotene spermatocyte (DSc). The nuclear size of DSc is about 4 [micro]m in diameter. The chromatin strands become increasingly condensed and are more visible than those of the earlier stages (Fig. 1A).

Metaphase spermatocyte (MSc). The cell in this stage displays the condensed chromatin (Fig. 1A). The nuclear size is similar to those of the previous stages, The thick chromatin strands move to align along the center of the cell (Fig. 1A).

Secondary spermatocyte (SSc). SSc is a round cell. The thick chromatin blocks are clumped along the cell periphery (Fig. 1C).

Spermatid. Spermatid consists of spermatid I and II.

Spermatid I ([St.sub.1]). The cell becomes smaller than SSc (Fig. 1D). Its nucleus is spherical and about 1.5-2 [micro]m in diameter and is located close to the lumen. The chromatin appears as small, thin clumps throughout the cell (Fig. 1D).

Spermatid H ([St.sub.2]). The general features of [St.sub.2] are similar to those of [St.sub.1] but the cell size is decreased and the chromatin becomes completely condensed (Fig. 1D).

Spermatozoa (Sz). The cell is spherical with 1 [micro]m in diameter. The nucleus contains dense chromatin (Fig. 1D). The acrosome is not visible in semithin section. The heads of spermatozoa are separated from trabeculae and aligned in rows (Fig. 1D).

2. Oogenesis

The ovary of P. penguin is composed of several acini enclosed by trabeculae or connective tissue. Early staged oocytes are attached to the trabeculae and mature oocytes are located in the lumens of acini (Fig. 2B). There are 6 stages of female germ cells of P. penguin based on cell size and shape. They are the oogonia and 5 stages of oocyte (Fig. 2).

[FIGURE 2 OMITTED]

Oogonium (Og). The oogonium is round in shape (Figs. 2A, B). The nuclear envelope is not distinct. Og is about 3.5 [micro]m in size and contains mostly euchromatin (Fig. 2A). The cells are attached to the inner side of trabeculae (Fig. 2A).

Oocyte 1 ([Oc.sub.1]). [Oc.sub.1] is about 14 [micro]m in size. The nucleus is round and about 8 [micro]m in diameter (Figs. 2A, B). It contains mostly euchromatin, which is dispersed throughout the nucleus. The nucleolus is clearly present (Figs. 2A, B). The [Oc.sub.1] cells are attached to the trabeculae (Fig. 2B).

Oocyte 11 ([Oc.sub.2]). [Oc.sub.2] contains a large nucleus and a thin cytoplasm (Fig. 2B). The cell size is about 18 x 22 [micro]m, with the nuclear size about 11 x 12 [micro]m. The nucleus contains a distinct nucleolus (Fig. 2B). Lipid droplets are present in the cytoplasm. [Oc.sub.2] are still attached to the trabeculae (Fig. 2B).

Oocyte III ([Oc.sub.3]). The cell size is about 25 x 31 [micro]m, with the nuclear size being about 15 x 17 [micro]m. The cell is larger in size and the nucleus contains mostly euchromatin (Figs. 2B, D). There are numerous yolk granules, which are dispersed throughout the cytoplasm. [Oc.sub.3] cells still remain attached to the trabeculae at the base of the cell (Figs. 2B, D).

Oocyte IV ([Oc.sub.4]). The cell is large and it assumes the droplet shape (Figs. 2C, D). The cell size is about 31 x 51 [micro]m, with the nuclear size being about 22 x 26 [micro]m. The nucleolus is clearly visible and the nucleus occupies most of the cell area (Fig. 2C). The ratio of cytoplasm to nucleus is about 0.44. Yolk granules are numerous. The [Oc.sub.4] cell is covered with a very thin jelly coat on the outer cell membrane (Figs. 2C, D). The cells are still attached to the trabeculae (Fig. 2D).

Oocyte V ([Oc.sub.5]). This is the mature oocyte before spawning. [Oc.sub.5] is the largest cell and has a polygonal or round shape (Fig. 2D). The ceil size is about 42 [micro]m in diameter and the nuclear size is about 27 [micro]m in diameter. The nucleus is very large when compared with that of [Oc.sub.4]. The ratio of cytoplasm to nucleus is 0.56. Yolk granules are abundant. All [Oc.sub.5] cells are completely detached from the trabeculae but remain in the capsule (Fig. 2D).

Reproductive Cycle

The reproductive cycle of P. penguin was studied by observing the changes in the gonad histology. The stages of gonad maturation could be classified into 3 stages in the male (developing, mature, partial spawning) and 4 stages in the female (developing, mature, partial spawning, spent).

1. Male

Developing stage. The gonad was characterized by the expansion of the follicle and the appearance of spermatogonia along the follicular wall (Fig. 3A). Primary spermatocytes proliferate rapidly and move to the center of the follicular lumen (Fig. 3B). The spermatids and spermatozoa are present in the lumen but they are not abundant (Fig. 3B). The developing stage of the male occurred all year round but production of spermatocytes was highest in August and November (Fig. 5).

[FIGURE 3 OMITTED]

[FIGURE 5 OMITTED]

Mature stage. The gonad still contains early staged cells but they are attached to the wall like a narrow band. The spermatid and spermatozoa, which occupy most areas of the follicle, are radiating toward the center of the follicle. They arrange themselves in radial rows (Fig. 3C). The mature stage of male P. penguin occurred from February to March during the annual spermatogenic cycle (Fig. 5).

Partial spawning stage. This is the stage when the pearl oysters release mature gametes into the water. The gonad contains a few spermatozoa in the lumen and the follicular wall is wrinkled and partially collapsed (Fig. 3D). Partial spawning stage occurred twice a year, in January and April (Fig. 5).

2. Female

Developing stage. The ovary consisted of capsule-like structures, which mostly contained early stage oocytes, Og, [Oc.sub.1], [Oc.sub.2], and [Oc.sub.3] (Fig. 4A). These oocytes were attached to the trabeculae (Fig. 4B). The developing stage occurred from August to December (Fig. 6).

[FIGURE 4 OMITTED]

Mature stage. In the mature ovary, there are abundant [Oc.sub.4] and [Oc.sub.5], but only a few remaining early staged oocytes. Most oocytes have a polygonal or a drop shape, and display both nucleolus and nucleus (Fig, 4B). Some are attached to the trabeculae but most oocytes are free in the center of the acinus (Fig. 4B). The mature stage of the female occurred in January, August to September, and December (Fig. 6).

[FIGURE 6 OMITTED]

Partial spawning stage. The follicles of the ovary appear partially empty, indicating that some mature oocytes have been released (Fig. 4C). The follicles contain mostly [Oc.sub.4] and [Oc.sub.5] that have become rounded, polygonal, or pear shaped (Fig. 4C). The early-stage oocytes are still attached to the trabeculae (Fig. 4C). Partial spawning occurred in March and July (Fig. 6).

Spent stage. Mature oocytes are completely discharged and the follicles are almost empty (Fig. 4D). The follicular wall be comes wrinkled or degenerates and the gonads are greatly decreased in size (Fig. 4D). The spent stage of female P. penguin occurred in November and December (Fig. 6).

DISCUSSION

The characteristics of gametogenesis and stages of gonad development in P. penguin are similar to those described for the pearl oysters P. albina (Tranter 1958a), P. margaritifera (Tranter 1958c), P. mazatlanica (Garcia-Dominguez et al. 1996), P. fucata (Tranter 1959, Wada et al. 1995), and P. maxima (Rose et al. 1990). However, previous authors did not emphasize the classification of germ cells. In P. maxima and P. fucata fucata, spermatogenic cells were classified into 4 stages: spermatogonia, spermatocyte, spermatid, and spermatozoa (Rose et al. 1990, Wada et al. 1995). In P. albina, Tranter (1958b) categorized male germ cells into 5 stages. Thielley et al. (1993) studied the ultrastructure of spermatogenic cells in P. margaritifera and classified them into 10 stages. Based on cell size and nuclear characteristics, the present study revealed 10 stages of spermatogenic cells: spermatogonia, 5 stages of primary spermatocyte, secondary spermatocyte, 2 stages of spermatid, and spermatozoa. However, in this study the acrosome could not be seen on the anterior end at high magnifications, which was different from that reported for P. albina (Tranter 1958b) and the primary and secondary spermatogonia could not be distinguished like those found in P. margaritifera (Thielley et al. 1993). In addition, diakinetic primary spermatocytes were not observed; only metaphase primary spermatocytes were found. This study identified 2 stages of spermatid based oil nuclear size and chromatin condensation, but in P. margaritifera, only one stage of spermatid was found.

The spermatogonium is the cell whose nucleus contains a single nucleolus and small clumps of dense chromatin. The spermatogonium divides into the first meiotic division to give rise to the primary spermatocyte. Prophase cells exhibit a different form of chromatin condensation. The chromatin appears as a very dense cluster in the leptotene spermatocyte. The zygotene spermatocyte is characterized by the presence of a synaptonemal complex in P. margaritifera (Thielley et al. 1993). However, a synaptonemal complex could not be identified in the zygotene spermatocyte of P. penguin in this study. In the secondary spermatocyte, the thick chromatids are clumped along the cell periphery. Spermatids are classified into spermatid I and spermatid II, whose chromatins become condensed throughout spermatogenesis. During the process. the few clear areas between chromatin masses are reduced in size (Thielley et al. 1993). Baccetti and Afzelius (1976) described 4 main types of sperm: primitive, modified, biflagellate, and aflagellate. Most studies have shown that bivalves have "primitive spermatozoa" (Dohmen 1983). The spermatozoa of P. penguin is of the primitive type, similar to those of other bivalves. Primitive sperms are typical of animals that spawn their gametes into surrounding water.

Literature on ongenesis of pearl oysters in the Family Pteriidae is very scarce, especially on Pteria species. Various stages of oogenesis have been described along with reproductive cycles in P. maxima and P. fucata fucata (Rose et al. 1990, Wada et al. 1995). P. maxima has 4 stages of oogenesis (Rose et al. 1990), whereas 3 stages were identified in P. fucata fucata (Wada et al. 1995). According to Tranter (1958b), oogenesis of P. albina occurred in 6 stages. This study identified 6 stages of oogenesis in P. penguin (oogonia, 5 stages of oocytes [Oc.sub.1-5]) based on cell size, nuclear size, basophilic cytoplasm, presence of yolk granules, and jelly coat. These oogonia are in the previtellogenic phase, which is characterized by the growth of oocytes and an increase in volume of the nucleus and cytoplasm (Dohmen 1983).

[Oc.sub.3-5] in P. penguin could be classified into the vitellogenic phase. During this period the oocytes grow rapidly, mainly because of the accumulation of yolk and other nutrient substances such as glycogen and lipid (Dohmen 1983). The cytoplasm in creased in size due to the presence of yolk granules. This study did not identify glycogen or lipid droplets, but the yolk granules were quite distinct.

Similar to P. albino (Tranter 1958b), [Oc.sub.4] are larger in size and the cytoplasm stains more heavily as yolk accumulates. Besides the presence of yolk granules, both [Oc.sub.4] and [Oc.sub.5] are surrounded by a thin jelly coat, which stained positively with PAS, indicating that it is mucopolysaccharide in nature. [Oc.sub.5] could be distinguished from [Oc.sub.4] by the larger nucleus and detachment from the trabeculae (Tranter 1958b).

Histological examinations of the ovarian and testicular tissues of P. penguin showed a definite annual reproductive cycle. Previous studies simplified the gametogenic cycle of family Pteriidae into 5 to 7 stages, which are clearly defined in females (Rose et al. 1990, Wada et al. 1995, Pouvreau et al. 2000). In this study, histological examination determined 3 stages in the male and 4 stages in the female. In the male, they are the developing, mature, and partial spawning stages. Spent or completely empty follicles of the testis were not observed in the male P. penguin. In the female, gonad development stages are developing, mature, partial spawning, and spent stages. In general, the gametogenic cycle is clearly defined in female P. penguin. The present study could only identify the main stages as described previously.

The histological examination of P. penguin showed that there were peaks of partial spawning in the male that occurred in March to June (summer) and December to January (winter). In the female, partial spawning occurred in January to May (winter to summer). A similar reproductive cycle has been reported in P. fucata fucata in the Gulf of Mannar, Sri Lanka. The spawning season was reported to be in the midsummer and midwinter (May to July, November to January) with continuous breeding (Pearson et al. 1929). But the breeding seasons of each of these species also differ.

Most studies reported a wide range of water temperature all year round, e.g., 18[degrees]C to 32[degrees]C in Western Australia (Rose et al. 1990), and 11[degrees]C to 29[degrees]C in Japan (Wada et al. 1995). Rose et al. (1990) considered variations in reproductive cycle to be due to the temperature range in different locations as influenced by latitudinal position. Other bivalve species exhibit distinct seasonal reproductive cycle, which usually are related to temporal variations of environmental factors such as food availability, water temperature, and/or photoperiod. In this study, the water temperature and salinity at the collecting site were quite similar all year round. It is possible that the lack of a highly defined reproductive or gametogenic cycle in P. penguin is related to the lack of observed temperature and salinity fluctuations.

ACKNOWLEDGMENTS

This investigation was supported by the Post Graduate Education, Training and Research Program in Environmental Science, Technology, and Management under Higher Education Development Project of the Ministry of University Affairs. Bangkok, Thailand.

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U. ARJARASIRIKOON, (1) M. KRUATRACHUE, (1) * P. SRETARUGSA, (2) Y. CHITRAMVONG, (1) S. JANTATAEME (1) AND E. S. UPATHAM (3)

(1) Department of Biology, Faculty of Science, Mahidol University Bangkok 10400, Thailand; (2) Department of Anatomy, Faculty of Science, Mahidol University Bangkok 10400, Thailand: (3) Burapha University, Chonburi 20131, Thailand

* Corresponding author. E-mail: [e-mail]