Early Larval Development and Annual Gametogenesis of the Brooding Oyster Ostrea circumpicta (Pilsbry, 1904) in the Shallow Subtidal Benthic Ecosystem in Jeju Island, Off the South Coast of Korea

© 2019 Academia Sinica, Taiwan Open Access Early Larval Development and Annual Gametogenesis of the Brooding Oyster Ostrea circumpicta (Pilsbry, 1904) in the Shallow Subtidal Benthic Ecosystem in Jeju Island, Off the South Coast of Korea Na-Lae Lim1, Hye-Mi Lee1, Hee-Do Jeung1,2, Ronald G. Noseworthy1, Sukgeun Jung1, and KwangSik Choi1,3,* 1School of Marine Biomedical Science (BK21 PLUS), Jeju National University, 102 Jejudaehakno, Jeju 63243, Republic of Korea. E-mail: [email protected] (Lim), [email protected] (Lee), [email protected] (Jeung), [email protected] (Noseworthy), [email protected] (Jung) 2Tidal Flat Research Institute, National Institute of Fisheries Science (NIFS), Kunsan 54014, Republic of Korea 3Marine Science Institute, Jeju National University, Jeju Self-Governing Province 63333, Republic of Korea. *Correspondence: Tel: +82-64-754-3422. Fax: +82-64-756-3493. E-mail: [email protected] Received 23 March 2019 / Accepted 29 June 2019 / Published 19 September 2019 Communicated by Benny K.K. Chan The subtropical brooding oyster Ostrea (= Striostrea) circumpicta (Pilsbry, 1904) occurs at high density in the shallow, subtidal, rocky bottom in Jeju Island, off the south coast of Korea, where the sea surface temperature and salinity varies annually from 13 to 25°C and 30 to 33 ppt, respectively. In this study, the annual gametogenesis and early larval development of O. circumpicta was examined, using histology and scanning electron microscopy (SEM). Histology indicated that the females and males initiated gonial mitosis in September, shortly after sexual resting in August. In December, ripe eggs first appeared in the follicles, and most of the females exhibited fully mature oocytes in May, as the water temperature reached 17 to 18°C. Spawning females were dominant in June and July, when the trochophore and straithinged veliger larvae were also identified in the branchial chambers, their size ranging from 111 to 130 µm and 135–205 µm in diameter, respectively. The veliger larvae in the brooding chamber exhibited a welldeveloped velum and digestive tract, suggesting that the larvae are engaged in feeding in the branchial chamber. Unlike other marine bivalves in temperate coastal ecosystems, O. circumpicta has a long period of gonad maturation and a short resting phase. It has been believed that such a long period of reproductive maturation is associated with a low level of food in the environment and the comparatively large size of the oocytes, which may require a relatively longer time to accumulate necessary nutrients to produce large eggs in a food-poor environment. Key words: Ostrea circumpicta, Larval development, Gametogenesis, Microscopy, Jeju Island Korea. Citation: Lim NL, Lee HM, Jeung HD, Noseworthy RG, Jung S, Choi KS. 2019. Early larval development and annual gametogenesis of the brooding oyster Ostrea circumpicta (Pilsbry, 1904) in the shallow subtidal benthic ecosystem in Jeju Island, off the south coast of Korea. Zool Stud 58:29. doi:10.6620/ZS.2019.58-29. BACKGROUND The subtropical larviparous oyster Ostrea (= Striostrea) circumpicta (Pilsbry, 1904) is widely distributed in the tropical to subtropical northwest Pacific Ocean, from Jeju Island to Taiwan, Hong Kong, southern Japan, and southern China (Hirase 1930; Bernard et al. 1993; Kwon et al. 1993; Okutani Zoological Studies 58: 29 (2019) doi:10.6620/ZS.2019.58-29 1 © 2019 Academia Sinica, Taiwan 2000; Min et al. 2004). In the shallow, subtidal, rocky substrate in Jeju Island, O. circumpicta occurs at a high density, and is often encrusted with various sessile flora and fauna including soft corals, sponges, and small algae, which play a crucial role in the ecosystem by providing substrata for other benthic organisms (Je et al. 2002; Kang et al. 2004a). Despite its abundance and ecological importance, few studies have been carried out on the life history of this larviparous oyster. Kang et al. (2004a) observed, for the first time, microscopic features of the larvae and gonads of O. circumpicta collected from Munseom Island, off the south coast of Jeju Island. They reported that females collected in early summer brood the early veliger larvae (115–135 µm) in their infra-branchial chamber. Although they reported the spawning and appearance of larvae, complete annual gametogenesis of O. circumpicta has yet to be investigated. Located off the south coast of Korea, Jeju Island (33.10° to 33.50°N, 126.10° to 127.0°E) is a typical volcanic island with a well-developed rocky shoreline. Coastal Jeju Island is a complex region, where three major water masses from the north and northwest are mixed. The warm Tsushima Current branches from the northeastward flowing Kuroshio Current and travels through the southern coastal area of the island, giving this area somewhat warmer water temperatures. The prevailing winds from the Pacific Ocean are southeasterly in summer, and also help to raise the water temperature. Due to the blending of warm and cool currents around the island, Jeju has a high diversity of marine flora and fauna (Oh et al. 1994; Lee 1999; Kang et al. 2005; Lee et al. 2019). Munseom Island, an uninhabited island located off the southern coast of Jeju (33°13'25"N, 126°33'58"E), is well known for its high species diversity and richness of marine flora and fauna (Je et al. 2002, Fig. 1). Munseom is characterized by rocky, volcanic intertidal and subtidal zones that are subjected to strong wave action. The rocky substrata are covered with a rich assortment of sessile fauna such as oysters, barnacles, and numerous species of hydrozoans. Surface water temperature and salinity in this area vary annually from 14 to 22°C and from 32.2–34.4 psu, respectively (Choa and Lee 2000; Lee et al. 2000). In the shallow subtidal of southern Jeju Island, numerous species of mollusks have been identified; some species occur only in Jeju Island (Je et al. 2002; Noseworthy et al. 2007 2016; Limpanont et al. 2010). According to Min et al. (2004), there are 17 oyster species in Korean waters; O circumpicta occurs only in Jeju Island, mainly on the south coast. The present study investigated seasonal changes Fig. 1. Map showing the sampling location, Munseom Island, off the south coast of Jeju Island. N page 2 of 12Zoological Studies 58: 29 (2019) © 2019 Academia Sinica, Taiwan in the gonad and early larval development of O. circumpicta from the southern coast of Jeju Island using histology and scanning electron microscopy (SEM). This is the first study to report on the annual reproductive cycle and larval development of this brooding oyster. MATERIALS AND METHODS From September, 2003 to September, 2004, adult oysters with shell lengths ranging from 50–80 mm were collected monthly using SCUBA, from Munseom Island at depths of 5–7 m (Fig. 2). Seasonal changes in the surface seawater temperature and salinity of the sampling site during sampling were obtained from the National Oceanographic Research Institute of Korea (NORI). The monthly mean chlorophyll a level in each sampling period was collected from the NASA EARTHDATA database (https://earthdata.nasa.gov/, USA). The chlorophyll a varied from 0.3 (July 2004) to 1.7 µg/L (October 2003), while the surface seawater temperature varied from 14 to 25°C, and salinity ranged from 24.2 to 32.1 psu during the sampling period (Fig. 3). To examine gonad development, a longitudinal section was cut from the middle of the body, including the gills and gonad. The tissue sections were fixed in Davidson’s fixative for 48 hrs and dehydrated in ethanol. The dehydrated tissues were embedded in paraffin, sectioned at 6 μm, stained with Harris’ hematoxylin, and counter stained with eosin Y. The histological preparations were examined under a light compound microscope to evaluate the gonad maturation level. Based on the microscopic appearance of the gonad, the reproductive stages of each specimen were assigned to one of six categories according to Siddiqui and Ahmed (2002): 1) resting, 2) early developing, 3) late developing, 4) ripe, 5) partially spawned, and 6) spent. Egg diameter in randomly-selected microscopic fields was also measured from the histological preparation, using image analyzing software. During May and August, the presence of larvae in the branchial chamber was examined directly under a dissecting light microscope. As the shells were opened, the gills which may contain the larvae were gently washed using filtered seawater; the filtrates were harvested and examined under a microscope, and the size, i.e., distance of the longest axis, was measured using image analyzing software. For SEM observation, the larvae collected from the infra-branchial chamber were fixed in 2% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.3 at 4°C), then washed for 30 mins in the same buffer. The fixed specimens were finally dehydrated in an ethanol series, freeze-dried, and coated with platinum. The platinum-coated larvae were examined under a JEM 1200EX-II SEM (JEOL, Japan). Shell development during the larval period was categorized according to the criteria described by Marin and Luquet (2004). RESULTS Microscopic features of the gonad and annual gametogenesis Histology revealed that 40% of the female oysters collected in September, 2003 were in the late developing stage. During early and late developing stages, oogonia and oocytes propagated along the germinal epithelia (Fig. 4a, b). Female oysters in the late developing stage Fig. 2. Animal and shell of O. circumpicta. , Gonad, , Gills. page 3 of 12Zoological Studies 58: 29 (2019) © 2019 Academia Sinica, Taiwan were characterized by greatly expanded and coalesced follicles, and the cytoplasm became less basophilic than in the early developing stage (Fig. 4b). Spermatogonia and spermatocytes appeared in the follicles during the late developing stage (Fig. 5a), in December. During the late developing phase, the spermatocytes developed into spermatids, and several groups of spermatids were distributed in the center of the follicle; groups of spermatocytes were arranged along the spermatogenic follicles (Fig. 5b). In February, as the surface seawater temperature increased from 14 to 17°C; the females became sexually mature, exhibiting fully mature oocytes containing large nuclei with nucleoli; and the follicles were greatly expanded (Fig. 4c). During this month, the ripe males also appeared, demonstrating expanded follicles containing a large number of concentrated spermatids and spermatozoa (Fig. 5c). In May, most males were actively engaged in spawning, exhibiting scattered masses of spermatozoa and vacated testis (Fig. 5d). Spawning males were observed until July; spent testis was recognized from some individuals (Fig. 5e). Spawning females first appeared in May, exhibiting free mature oocytes in the lumen of the follicles, while the follicles became partially empty (Fig. 4d). The spawning females could be observed until July, as the SST increased from 17°C to 20°C. In July and August, most oysters were in the spent stage exhibiting a few relict oocytes in the follicles (Fig. 4e). During this stage, the follicles were mostly vacated, and the follicle area become reduced to a great extent. In males, most follicles appeared empty, while some residual spermatozoa and spermatids become degenerated (Fig. 5e). Sexually undifferentiated oysters appeared in August and September, and no gametogenic cells could be identified from the thin follicle wall (Figs. 4f, 5f). Seasonal changes in frequency distribution in the different reproductive stages are plotted in figure 6. In females, ripe oysters could be seen from December to July, while spawning females could be seen during June and July. The high percentage of spawning/spent oysters in June and July indicated that O. circumpicta spawned during this period. Contrary to the females, spawning males were first observed in May, and spawning in males continued until July, suggesting that the major spawning period of this species in Jeju Island is June and July. Figure 7 plots monthly variation in the oocyte size. In the early developing stage, young oocytes (60 μm in diameter) were dominant during September and October. Fully mature eggs began to appear in March, and were dominant in May, measuring 104 µm in diameter. The proportion of mature eggs decreased in June, possibly due to infernal fertilization. Histology revealed that O. circumpicta in Jeju Island carries the oocytes in the gonad all year round, representing a characteristic of tropical/subtropical oysters. Larval development Trochophore and veliger larvae first appeared as early as May. In June, 50% of the females reared the larvae in their gill chambers. The proportion of the females exhibiting D-shaped veliger and/or trochophore larvae declined in July to 29%, and none of the females examined in August exhibited the larvae in the gill chambers (Table 1). The data suggest that spawning and subsequent larval rearing in O. circumpicta was finished by July in southern Jeju Island. Histology also confirmed the presence of the Fig. 3. Monthly variations in the surface water temperature, salinity and chlorophyll a at the sampling location from September 2003 to September 2004. The sea surface temperature and salinity data were obtained from Korea Hydrographic and Oceanographic Agency, and the chlorophyll a were referred from NASA EARTHDATA (https:// earthdata.nasa.gov/, USA) page 4 of 12Zoological Studies 58: 29 (2019) © 2019 Academia Sinica, Taiwan trochophore and veliger larvae in the infra-branchial chamber in the mantle cavity in June and July (Fig. 8a, b); the former consisted of a rudimentary shell and foregut (Fig. 8c), and the latter showed well-developed organs including locomotory cilia, digestive gland, esophagus, rudimentary gill, intestine, mantle, retractor muscle, rudimentary shell, stomach, velum, and visceral cavity (Fig. 8d). Under SEM observation, sizes of the trochophores ranged from 111 µm to 130 µm (Fig. 9a), and the veligers ranged from 135 µm to 205 µm in shell length (Fig. 9b, 9c). Characteristics of the veliger larvae observed from SEM included relatively small size, straight hinge, and absence of the rim of prodissoconch II; it was concluded that the shell stage of the veliger larvae observed in the present study belonged to prodissoconch I (Fig. 9d). Fig. 4. Photomicrographs of reproductive stages of female O. circumpicta. (A) early developing, (B) late developing, (C) ripe, (D) partially spawned, (E) spent/absorbing, (F) resting. (A) (C) (E) (B) (D) (F) page 5 of 12Zoological Studies 58: 29 (2019) © 2019 Academia Sinica, Taiwan DISCUSSION According to the World Register of Marine Species (WoRMS 2019), the genus Ostrea (Linnaeus, 1758) includes 17 accepted living species, while approximately 400 synonymized and unaccepted species names have been assigned to the genus worldwide. In the north-west Pacific region, currently four species of brooding oysters have been identified, including O. circumpicta, O. denselamellosa, O. stentina, and O. futamiensis (Bernard et al. 1993; Kwon et al. 1993; Okutani 2000; Min et al. 2004; Hamaguchi et al. 2017). In Korean waters, where 17 living oyster species have been recognized, O. circumpicta is limited in distribution to Jeju Island, where the sea surface water temperature ranges from 13 to 26°C annually (Min et al. 2004; Kang et al. 2004b; Hong et al. 2013). Recently, Song et al. (2017) reported the presence of Fig. 5. Photomicrographs of reproductive stages of male O. circumpicta. (A) early developing, (B) late developing, (C) ripe, (D) partially spawned, (E) spent/absorbing, (F) resting. (A) (C) (E) (B) (D) (F) page 6 of 12Zoological Studies 58: 29 (2019) © 2019 Academia Sinica, Taiwan O. circumpicta subtidally in Dokdo Island in the East/ Japan Sea (37°14'26.8"N, 13151'54.6"E), a typical offshore island where the Tsushima current supplies warmer water from the south. In the northwest Pacific region, O. circumpicta has also been reported from the sub-tropical East China Sea, at Kyushu and the Seto Inland Sea in Japan (Hirase 1930; Bernard et al. 1993; Okutani 2000). Like other species in the genus Ostrea, O. circumpicta harbors its larvae in the branchial chamber; however, studies on its annual reproductive cycle are limited (Kang et al. 2004a). This study is the first to examine the annual reproductive cycle of an O. circumpicta population on the shallow, subtidal, rocky substrate in Jeju Island. It is noticeable that O. circumpicta exhibits a short period of reproductively resting stage in August and a relatively longer period of gonad maturation. Shortly after resting in summer, the oysters initiated gonial mitosis in September and they first spawned in May, indicating that gonad growth takes approximately 8 months. It is also noticeable that the spawning activity of the females is limited to a short period, from late May to July. During this period, the females also exhibited early developing larvae such as trochophores and veligers in the mantle cavity. Histology also indicated that O. circumpicta carries the early developing larvae during June and July, suggesting that the spawning period coincides with the early larval brooding period. Accordingly, the annual gametogenesis of O. circumpicta can be summarized as a short period of resting and early developing, i.e., gonial mitosis, in summer to early fall, maturing of the gametes in late fall to late spring, and spawning with the larvae developed and released in summer (Fig. 10). Several studies have reported that bivalves distributed in warmer water have a short period of resting or no inactive/resting phase, due to the warmer water temperature which enables the oyster to actively remain in gametogenesis. In the tropical lagoons in French Polynesia, black-lip pearl oysters do not have a resting stage during their annual reproductive cycle, where the SST ranges 22–29°C annually (Pouvreau et al. 2000; Le Moullac et al. 2012). No sexual rest was also reported from the southern Gulf of Hammamet on the eastern coast of Tunisia, as the dwarf oyster Fig. 6. Frequency distribution of gametogenic stages of O. circumpicta. page 7 of 12Zoological Studies 58: 29 (2019) © 2019 Academia Sinica, Taiwan Ostreola (= Ostrea) stentina exhibited gametes all year round (Salah et al. 2012). In a lagoon in the southern Gulf of California where the SST varies from 19–31°C seasonally, Crassostrea corteziensis rested for a month during its annual reproductive cycle (RodrÍguezJaramillo et al. 2008). Unlike other tropical and/or subtropical oysters, the tropical rock oyster Striostrea prismatica, on the southern coast of Ecuador, has a distinct sexual resting period from June to August (Loor and Sonnenholzner 2016). Temperature is one of the key environmental factors that govern the annual gametogenesis of marine bivalves, especially in temperate waters, where the SST fluctuates seasonally with a wide range. On the south coast of Korea, where the SST ranges 9 to 26°C annually, the Pacific oyster, Crassostrea gigas, exhibits Fig. 7. Monthly mean changes in egg size of O. circumpicta. Table 1. Percentage of the female oysters brooding the trochophore and/or veliger larvae in the infra-branchial chamber Period Percentage May 0 2004 June 50 July 29 August 0 Fig. 8. Light microscopic images of O. circumpicta larvae in infrabranchial chamber. (A) trochophores; (B) veligers; (C) single trochophore (R, rudimentary shell; Fg, foregut); (D) Frontal section of larval of S. circumpicta stained with Hemotoxylin and Eosin. (Lcl, locomotory cilia; Dg, digestive gland; Es, esophagus; Gr, rudimentary gill; In, intestine; Mn, Mantle; Rm, retractor muscle; Sr, rudimentary shell; St, stomach; Vel, velum; Vc, visceral cavity) (A) (C) (B) (D) page 8 of 12Zoological Studies 58: 29 (2019) © 2019 Academia Sinica, Taiwan a series of discrete gonad development phases in an annual reproductive cycle, which are closely linked to changes in water temperature (Kang et al. 2000; Ngo et al. 2002 2006; Mondol et al. 2016). In Gamakman Bay, on the south coast, C. gigas attached to subtidal long-lines spawned in June and July, when the SST ranged from 22–25°C. After spawning is completed, this species has a 4 months of resting stage, from Fig. 9. Scanning electron microscopic images of O. circumpicta larvae. (A) trochophore, (B) hinge; (C) velum; (D) veliger, lateral view. TP: trochophore, IC: infrabranchial chamber, HL: straight hinge line; CL, cilium; VL, velum; P1, prodissoconch I. Fig. 10. Comparison of gametogenic stages of seven bivalve species from Jeju Island. (A) (C) (B) (D) page 9 of 12Zoological Studies 58: 29 (2019)