Annual gametogenesis of the Chinese anapella clam Coecella chinensis (Deshayes 1855) at an upper intertidal sandy beach on the east coast of Jeju, Korea.

ABSTRACT We report for the first time annual gametogenesis in the Chinese anapella clam Coecella chinensis (Deshayes 1855) living on an upper intertidal sandy beach on the eastern coast of Jeju, Korea. Using histological methods, gonadal maturation, oocyte area in the follicle (follicle index, FI), and oocyte diameter were investigated. Gametogenesis began in the middle of April, when the sediment temperature was 13[degrees]C. Annual maximum oocyte size and FI peaked in July, when water temperature reached 20[degrees]C and sediment temperature reached 35[degrees]C. Subsequent spawning of C. chinensis occurred in August and continued until the end of September. Mean ripe oocyte diameter was 31.2 [micro]m, which is comparatively smaller than that of other bivalves. Clams over 15.7 mm in shell length had mature gonads, indicating that this size is a biological minimum for reproduction. Annual sediment temperature fluctuation was positively correlated with condition and gonadal development (P < 0.01), indicating that cyclic changes in gametogenesis are in part governed by substrate temperature. The relatively smaller eggs observed here are believed to be a reproductive strategy of C. chinensis, an adaptive trade-off between reproduction and growth in the food-poor upper intertidal environment.

KEY WORDS: gametogenesis, Chinese anapella clam, Coecella chinensis, egg size, mesodesmatidae

INTRODUCTION

The Chinese anapella clam Coecella chinensis (Deshayes 1855) belongs to the family Mesodesmatidae, order Veneroida. This small clam (adult shell length: 2-4 cm) is commonly found in the middle to upper intertidal zone of sandy gravel beaches along the west and south coasts of Korea, as well as in Japan, China, and the Philippines (Bernard et al. 1993, Goto & Poppe 1996, Okutani 2004, Min 2004, Qi 2004). On Jeju Island (33[degrees]10'-33[degrees]50'N, 126[degrees]10'-127[degrees]0'E), C. chinensis populations are commonly found in upper intertidal sandy beaches or small tidal pools, where they burrow 2-4 cm below the sediment surface. Unlike other marine bivalves, the anapella clam is not commercially exploited on Jeju, possibly because of its size and taste. Basic ecological information on this clam, such as skeletal and somatic growth, reproduction, or ecological role in the community, is unknown, despite their unique ecological role in upper intertidal communities.

Numerous studies have reported that gonadal development of marine bivalves follows an annual gametogenic cycle associated with environmental factors such as water temperature, salinity, or food availability. Accordingly, gonadal development is often investigated using histology and subsequent analysis of the microscopic gonadal features. In practice, microscopic images of histologically prepared gonads are often taken using a video or digital camera and sent to a computer. The digitized gonadal image is then analyzed with respect to overall level of gonadal maturation, oocyte size, or the area of eggs or sperm within a follicle using image analysis software (Morvan & Ansell 1988, Kang et al. 2003, Morsan & Kroeck 2005).

We report for the first time histological features of the gonad and annual gametogenesis of the Chinese anapella clam collected from a sandy beach on the east coast of Jeju Island, Korea.

MATERIALS AND METHODS

Study Area and Sample Collection

Chinese anapella clams were collected from the upper intertidal sand-flat at Siheung-ri Beach, located on the east coast of Jeju Island (33[degrees]26'N, 126[degrees]55'E; Fig. 1). The substrate consisted of small pebbles and coarse sand. The clams usually occur 2-4 cm below the substrate surface. To assess the annual reproductive cycle, adult clams from 2-2.5 cm in shell length were collected monthly from July 1999 to July 2000. Surface seawater temperature and salinity were recorded when the monthly clam samples were taken. Sediment temperature was also recorded 5 cm below the surface, which is considered the maximum depth of clam inhabitation.

Gonadal Analysis

On arrival at the laboratory, shell length (SL), height (SH), and width (SW) were recorded to the nearest 0.01 mm. The shell was opened and the soft body removed and weighed for the total weight of wet tissue (TWT) to the nearest 0.01 g. Condition index (CI), a ratio of TWT to shell volume (SL x SH x SW) calculated as CI = TWT/shell volume, was used to examine seasonal variation in weight gain and loss before and after the spawning. For histology, the soft body was fixed in Bouin fixative and a longitudinal section was cut in the middle of the fixed clam body for histology. After embedding the section in paraffin, a 5-[micro]m-thin section was sliced and stained with Harris hematoxylin and eosin Y. To evaluate gonadal maturation, four microscopic fields were randomly selected from each histological slide and gonadal development phase was graded into six categories (1 = resting, 2 = early active, 3 = late active, 4 = mature, 5 = spawning or partially spawned, and 6 = spent). Oocyte diameter (OD) and the area of the eggs within the follicle were measured from digitized images of gonads using image analysis software. The percentage area of egg within a follicle estimated from the image was then expressed as the follicle index (FI).

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RESULTS AND DISCUSSION

Temperature and Salinity

Figure 2 shows seasonal fluctuations in salinity, sediment temperature, and water temperature at the study area. Salinity ranged from 16.2-28.2 ppt; the lowest salinity, recorded in August, was possibly associated with a regional mass introduction of freshwater to the upper intertidal area because of heavy rain. The highest sediment temperature was 35.6[degrees]C in July 2000, whereas the lowest temperature was 10.9[degrees]C in January 2000. Surface seawater temperature ranged from 12.3[degrees]C (February) to 23.4[degrees]C (September).

Gonadal Characteristics

Figure 3 shows microscopic features of clam ovaries observed in the annual gametogenic cycle. In the early active stage, many small early vitellogenic oocytes were attached to the thickened follicle walls (Fig. 3A). In the late active stage, the follicle wall became thinner relative to the early active stage and most of the vitellogenic oocytes increased in size to become mature ova (Fig. 3B). Mature oocytes ranged from 21-30 [micro]m in diameter, with a maximum size of 31.23 [micro]m (Fig. 3C-a). During the late active phase, the nucleolus was visible within the nucleus (Fig. 3C-b). It was also noticeable that the ovary expanded into the foot area during the late active phase. During the spawning stage, the follicle walls and connective fibromuscular tissues became thinner. Partly spawned ovaries were loosely vacated and free ripe ova remained in the lumen (Fig. 3D). In the spent stage, relict ova and eggs degenerating via phagocytosis were observed along the follicle walls (Fig. 3E).

Figure 4 illustrates gonadal development in the testis. In the early active stage, columnar epithelial tissue with spermatogonia was thin and the spermatocytes began to proliferate into the center of the lumen (Fig. 4A). During the late active stage, the testis expanded extensively. Spermatogonia, spermatocytes, and spermatzoa were observed along the follicle wails and maturation of the spermatozoa rapidly progressed into the center of follicles during the late active stage (Fig. 4B). During the spawning or partially spawned stage, the lumens were vacant, with several scattered masses of spermatozoa (Fig. 4D). In the spent testis, follicle walls were thinner and the lumens were empty, except for relict spermatogenic tissues (Fig. 4E).

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Figure 5 shows the resting stage of a C. chinensis gonad. No gametogenic cells were present along the follicle wall (Fig. 5A). On some occasions, degenerating gonadal cells were observed along the follicle and digestive diverticular (Fig. 5B). The resting stage was observed in all months except July, August, and September (Fig. 6).

Seasonal Gonadal Development

Clams used in this study ranged from 6.10-31.80 mm in SL. Clams with SL over 15.7 mm exhibited mature gonads, indicating that this size may be a biological minimum for reproduction. Figure 6 shows the frequency of each gonadal development stage in each sampling period. Sexually mature female clams appeared in July 1999 (26.3%) and the proportion of mature individuals increased until August (45.5%). Spawning clams were observed from August (50%) to November (11.0%) and spawning peaked in September (66.7%). In November, 92% of the clams were in the spent stage. Most of the female clams were in the early active stage in April 2000 (70%) and clams in the late active stage were first observed in May (29.4%). Male clams showed a similar annual gametogenic pattern to females, although gonadal maturation in males tended to be slower (Fig. 6). Our data indicate that C. chinensis along the east coast of Jeju become sexually mature in the middle of summer and spawn in late summer to early fall.

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Seasonal Changes in CI, OD, and FI

Figure 7 plots annual variation in CI, which ranged from 0.09 (December) to 0.18 (July). A gradual increase of CI from April (0.10) to July (0.18) coincided with gonadal maturation, from the early active to late active stage. CI dropped dramatically from August (0.16) to September (0.11), when most of clams released their gonadal mass through spawning.

Figure 8 shows annual variation in oocyte size. Oocyte diameter varied from 5.1-31.2 [micro]m, with a mean of 14.5 [micro]m. Eggs with a diameter of 1.5-10.4 [micro]m were dominant in April, whereas oocytes 5 15 [micro]m in diameter were dominant (92.6%) in May. Eggs were significantly larger in May and June than in April (P < 0.001). In July, oocyte growth reached an annual peak. The frequency of oocytes 21-30 [micro]m in diameter suddenly decreased in August. In October, relict and phagocytized eggs 20-23 [micro]m in diameter were dominant.

Figure 9 shows seasonal variation in FI and the percentage area of egg within the follicle, which ranged from 14.60 (October) to 63.47% (July). The maximum FI was observed in July, when clams were mostly in the mature stage. From April to July. FI gradually increased as eggs or sperm in follicles increased in size and prepared for spawning. FI then decreased from July to August and September, suggesting that the clams spawned during this period.

Annual Gametogenesis of C. chinensis

We report for the first time annual gametogenesis in the Chinese anapella clam. As shown in Figure 6, gametogenesis begins as early as January, when water temperature is 14-16[degrees]C. The early active stage lasts from January to July, although this stage is most frequently observed in April and May. A mature female gonad was first observed in July, when the water temperature was 19[degrees]C. In August, 50% of female clams were partially spawned and spawning activity continued until the end of November. Spawning males were also observed from August to October, although male spawning peaked in September, when the water temperature reached 23[degrees]C. Sexually indifferent or resting clams were dominant from January to April in females and from November to April in males. Our data indicate that the gametogenic development of Chinese anapella clams is cyclic and it can be summarized as a resting stage from January to April, an early active stage from February to May, a late active phase from July to August, maturity or spawning readiness in July and August, spawning or the partially spent stage from August to October, and finally the spent stage from September to December.

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Seasonal changes in gametogenesis were also monitored by measuring the area of egg within follicles (i.e., FI) and egg diameter from histological sections. As shown in Figure 9, FI was measured from April to October, with a peak in July. FI increased quickly from June to July, suggesting that gonadal maturation is a rather fast process. From July to August, FI dropped quickly and decreased until October. Variation in FI indicates that the majority of Chinese anapella clams in the study site spawn during July and September, with a peak between July and August. Monthly monitoring of CI (Fig. 7) also indicates that the anapella clam has a single spawning event, because CI increased rapidly from April to July, then dropped from July to September. Several papers have reported that CI peaks when clams are ready for spawning and drops dramatically after spawning caused by weight loss through gonadal discharge (Deslous-Paoli & Heral 1988, Morvan & Ansell 1988, Pouvreau et al. 2000).

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Seasonal variation in CI is often associated with seasonal fluctuations in food availability. Lee et al. (1999) and Choa and Lee (2000) reported seasonal phytoplankton blooms in coastal Jeju, a spring bloom from March to May, and a fall bloom in September. The spring bloom likely provides energy for the clams to mature eggs or sperm for spawning in July to August, whereas the fall bloom increases the survivorship of their offspring (Shafee 1989, Hadfield & Anderson 1998). However, the scale of the phytoplankton bloom along coastal Jeju is much smaller than the phytoplankton blooms along the west and south coasts of Korea (Kang et al. 2000, Hyun et al. 2001, Park et al. 2006).

The single spawning peak of the Chinese anapella clam observed in this study may also be associated with intensity of the phytoplankton bloom. In contrast to this study, several others have reported multiple spawning of marine bivalves along the west and south coasts of Korea, where phytoplankton biomass or primary production is much higher than in Jeju (Park et al. 1999, Kang et al. 2003, Park et al. 2006). Park et al. (2006) reported on the annual reproductive cycle of the Manila clam Ruditapes philippinarum in Gomso Bay, along the western coast of Korea. The Manila clam may spawn three times annually, from late May to September, although the number of eggs released in the three spawning periods differs substantially. Pacific oysters along the southern coast of Korea also spawn at least twice during their spawning period, from May to September, and these multiple spawnings are strongly associ ated with the amount of food in the water column (Park et al. 1999, Kang et al. 2000, Kang et al. 2003). The relatively longer period in the spent or resting stages of the anapella clam (September to May) observed in this study could be explained by low food availability.

Seasonal changes in water temperature initiate the annual gametogenesis cycle of clams in the family Mesodesmatidae (Redfearn 1982, Hooker & Creese 1995a, Hooker & Creese 1995b). Initiation of gametogenesis in the Manila clam in the study area takes place in March, when the water temperature is over 14[degrees]C (Kang et al. 2004, Ngo & Choi 2004). However, gonadal development of clams in the upper intertidal zone in this study may be associated with not just seawater temperature, but also sediment temperature, because the exposure of clams to the atmosphere during low tide is much longer than the time they are submerged. Jones (1981) suggested that the rate of gonadal maturation and initiation in the ocean quahog (Arctica islandica) and surf clam (Spisula solidissima) were in part governed by sediment temperature. As shown in Figure 2, seasonal fluctuations in sediment temperature are much larger than the fluctuations in seawater temperature. In particular, the rate of sediment temperature increase from April to June is dramatic, from 11-31[degrees]C. During this period, the anapella clam quickly matures its gonads and becomes ready for spawning by the end of June. In contrast, water temperature during this period increases comparatively little, from 13.9[degrees]C in April to 17.8[degrees]C in June.

Table 1 summarizes the body and egg sizes of clams in the order Veneroida. Note that egg diameter exceeds 50 [micro]m, regardless of body size. Macoma balthica, a small clam with a shell length of only 14-17 mm produces relatively large eggs, with a mean diameter of 105 [micro]m (Honkoop & van der Meer 1998). Mactra venerformis (Chung & Ryou 2000) and Mya arenaria (Brousseau 1987) are also considered smaller clams with shell lengths of 21-45 and 21-90 mm. respectively. Despite their small body size, they produce eggs with a mean diameter of 60-70 and 65-70 [micro]m, respectively (Fig. 2). In contrast, the Chinese anapella clam produces relatively small eggs, 21-30 [micro]m in diameter. The small egg size observed here is one of the smallest eggs reported from bivalve molluscs (see Table 1). The small size of C. chinensis eggs could be the result of physiological adaptations to poor food conditions in the higher intertidal area, although we have no data on available food in the sampling area. This clam may favor producing small but numerous planktotrophic larvae instead of a few large brooding eggs, as is seen in many small marine bivalves.

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ACKNOWLEDGMENTS

The authors thank the staff of the Shellfish Aquaculture and Research Laboratory at Cheju National University for their help with data collection and analysis. This study was funded by grants from the Korea Ocean Research and Development Institute (PE 97801 and PE 97802).

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DO-HYUNG KANG, (2) CHANG-YONG HYUN, (1) YANIN LIMPANONT (1) AND KWANG-SIK CHOI (1) *

(1) School of Applied Marine Science, College of Ocean Sciences, Cheju National University, 66 Jejudaehakno, Jeju 690-756, Republic of Korea; (2) Korea Ocean Research and Development Institute (KORDI), Ansan Korea

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

TABLE 1.
Summary of egg diameter (ED), shell length (SL), and gametogenic and
spawning periods in various bivalves of the order Veneroida.

                                                    Gametogenic
Species                   ED ([micro]m)   SL (mm)     Period

Corbicular japonica           70-90        20-35     Feb.-Jul.
Mercenaria mercenaria         51-55        69-75     Jan.-Apr.
Spisula sachalinensis         50-60        49-80     Feb.-Jun.
Macoma halthica              105           14-17     Oct.-Mar.
Cerastoderma echcle           77           28-33     Mar.-May
Meretrix lusoria              70          Absent     Jan.-Jul.
Ruditapes philippinarum       55-65        17-55     Mar.-Jun.
Mactra veneriformis           60-70        21-45     Feb.-Jun.
Mya arenaria                  65-70        21-90     Jan.-Jun.
Coecella chinensis            21-30        6-32      Mar.-Aug.

Species                   Spawning Peak      Habitats

Corbicular japonica         Jul.-Sep.     Subtidal
Mercenaria mercenaria       Apr.-Jun.     Intertidal
Spisula sachalinensis       Jun.-Jul.     Subtidal
Macoma halthica             Mar.-Apr.     Intertidal
Cerastoderma echcle         May-Jun.      Intertidal
Meretrix lusoria           Jul.-Aug.      Intertidal
Ruditapes philippinarum    Jul.-Aug.      Intertidal
Mactra veneriformis        Jul.-Aug.      Intertidal
Mya arenaria               Jun.-Jul.      Intertidal
Coecella chinensis         Aug.-Sep.      High intertidal

Species                                Authors

Corbicular japonica       Chung et al. (2004)
Mercenaria mercenaria     Heffernan et al. (1989)
Spisula sachalinensis     Lee et al. (1997)
Macoma halthica           Honkoop and van derMeer (1998)
Cerastoderma echcle       Honkoop and van der Meer (1998)
Meretrix lusoria          Lee (1997)
Ruditapes philippinarum   Kang et al. (2004)
Mactra veneriformis       Chung and Ryou (2000)
Mya arenaria              Brousseau (1987)
Coecella chinensis        This study