HYEJIN KIM, 1 BYEONG HAK KIM, 2 MAENG HYUN SON, 2 MI AE JEON, 1 YEON GYU LEE 3 AND JUNG SICK LEE 1 *

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1 Journal of Shellfish Research, Vol. 35, No. 3, , GONADAL DEVELOPMENT AND REPRODUCTIVE CYCLE OF CULTURED ABALONE, HALIOTIS DISCUS HANNAI (GASTROPODA: HALIOTIDAE) IN KOREA: IMPLICATIONS FOR SEED PRODUCTION HYEJIN KIM, 1 BYEONG HAK KIM, 2 MAENG HYUN SON, 2 MI AE JEON, 1 YEON GYU LEE 3 AND JUNG SICK LEE 1 * 1 Department of Aqualife Medicine, Chonnam National University, Yeosu 59626, Republic of Korea; 2 South Sea Fisheries Research Institute, National Fisheries Research and Development Institute, Yeosu 59780, Republic of Korea; 3 Faculty of Marine Technology, Chonnam National University, Yeosu 59626, Republic of Korea ABSTRACT The objective of this study was to examine gonad development and reproductive cycle of the abalone Haliotis discus hannai at three main abalone aquaculture farms (Uljin, Wando, and Jeju Island) in Korea to obtain information on artificial seed production. The sex ratio (female:male) was approximately 1:1 without significant difference among the three regions or between females and males. Average gonad index was the highest during July in Uljin and Jeju Island and during August in Wando. There was no definitive seasonal change in condition index and meat weight index throughout the year in the three regions. The gonad of H. discus hannai displayed seasonal change histologically, thereby enabling us to distinguish the following stages: inactive (November December), early active (January March), late active (March April), ripe (May July), spent (August October), and degenerative (October November). The main spawning period of H. discus hannai was determined to be during September October in Uljin and Wando, and August September in Jeju Island. KEY WORDS: abalone, Haliotis discus hannai, reproductive cycle, Korea INTRODUCTION Members of the family Haliotidae exist in most tropical and temperate oceans, particularly in the shallow subtidal zone. A total of 55 species with 10 additional subspecies have been reported (Lindberg 1992, Geiger 1998). Two species and two subspecies (Haliotis madaka, Haliotis gigantea, Haliotis discus discus, Haliotis discus hannai) ofgenushaliotis and two subspecies (Sulculus diversicolor diversicolor, Sulculus diversicolor supertexta) ofgenussulculus have been reported in Korea (Lee & Min 2002). In Korea, Haliotis discus hannai have been produced by aquaculture at 8,977 tons in Among these, the regions of Uljin, Wando, and Jeju Island produced a total of 7,550 tons, which accounts for 85% of the total production of abalone in Korea (KOSIS 2014). Studies on the reproductive ecology of marine molluscs including gastropods can provide essential information on basic ecological data to protect wild populations, increase biological resources, and develop aquaculture technology (Gosling 2004, Bigatti et al. 2008). Studies on the reproduction of abalones in Korea have been focused on gametogenesis and reproductive cycle of abalones (Lee 1974, Kim et al. 2014). In these researches, the main spawning period of abalone in Korea has been reported to be in around October. Nevertheless, seed production in abalone aquaculture of Korea is executed during April May by maturing abalone earlier through artificially increasing the water temperature over a period (approximately 1 mo) to enable the abalone to grow sufficiently during the period of high water temperature in summer by producing the young abalone earlier. Although *Corresponding author. ljs@jnu.ac.kr DOI: / such practice has problems, such as egg quality including yolk accumulation since the ripe and fertilized eggs produced through such artificial process do not have sufficient maturation period compared with those obtained naturally. Therefore, the objective of this study was to provide information on the sexual maturation and reproductive cycle of abalone as wellastheperiodfrommaturationtospawninginartificial seed production of Haliotis discus hannai at three main regions of abalone aquaculture in Korea. MATERIALS AND METHODS Sampling Abalone Haliotis discus hannai were collected from Uljin (on the eastern sea of Korea), Wando, and Jeju Island (on southern sea of Korea) (Fig. 1). A total of 788 H. discus hannai were collected monthly from September 2012 to August 2013 with shell length of (±11.60) mm. Environmental Conditions Data from the Korea Hydrographic and Oceanographic Administration were used to obtain water temperature profiles of the three areas (KHOA 2013). Water temperatures were measured at every hour during the period of survey, and the monthly average and SD are indicated in Figure 4. Sex Ratio, Condition Index, and Meat Weight Index Sex ratio was calculated by female:male ratio. Condition index (CI) and meat weight index (MWI) were calculated using the following equations according to the methodology of Jeon et al. (2012): CI ¼ [body weight (g)/shell length (mm) 3 ] 3 1,000; MWI ¼ [body weight (g)/total weight (g)]

2 654 KIM ET AL. Histology Specimens were dissected, and the gonads were prepared after measuring the morphometric characteristics of shell length, total weight, and body weight. Specimens were prepared for a light microscopy according to the methodology of Drury and Wallington (1980). The visceral mass including the gonad was fixed in BouinÕs solution for 24 h, rinsed with running water for h, and dehydrated through a graded ethyl alcohol series (70% 100%). The preparations were then embedded in paraplast (Leica, Germany). Embedded tissues were sectioned at thickness of 4 6 mm using a microtome (RM2235, Leica, Germany). Samples were stained with MayerÕs hematoxylin 0.5% eosin. Gonad Index Gonad index (GI) was partially corrected using the method of Eversole (1997). Gonad development was categorized into the following six stages: inactive stage (In), early active stage (Ea),lateactivestage(La),ripestage(R),spent(Sp),and degenerative stage (D) (Figs. 2 and 3). Samples were quantified by multiplying each individual with a constant (In ¼ 1, Ea ¼ 2, La ¼ 3, R ¼ 4, Sp ¼ 3, and D ¼ 2) for each gonad development stage. The GI was calculated using the following equation. GI ¼ ðin 3 1Þ + ðea 3 2Þ + ðla 3 3Þ + ðr 3 4Þ + ðsp 3 3Þ + ðd 3 2Þ Total number Statistical Analysis The sex ratio data were analyzed using SPSS 21.0 software (SPSS Inc.). The observed female:male ratio for each shell length group was compared with the expected ratio of 1:1 using a c 2 test. Statistical significance was considered when P value was <0.05. RESULTS Water Temperature The average water temperature during the collection period (September 2012 to August 2013) was 15.0 C in Uljin, 14.6 C in Wando, and 17.1 C in Jeju Island. The three areas reported the highest water temperatures in August or September, and the lowest temperatures in January or February (Fig. 4). Sex Ratio The sex ratio (female:male) of Haliotis discus hannai was approximately 1:1 in the three areas. There was no statistical significance in the difference of sex ratios among the three areas (Table 1). Gonadal Development Stage Monthly changes in gonadal development stage displayed differences among the three regions (Uljin, Wando, and Jeju Island). The gonad of Haliotis discus hannai displayed histological seasonal changes in the inactive stage (November December), early active stage (January March), late active stage (March April), ripe stage (May July), spent stage (August October), and degenerative stage (October November; Fig. 4). Gonad Index Gonad index of Haliotis discus hannai displayed differences in the three regions. The average GI was 2.7 in Uljin, 2.2 in Wando, and 2.6 in Jeju Island. In Uljin, the lowest GI (1.3) occurred in December (WT 10.6 C) 2012 while the highest GI (3.6) occurred in July (WT 17.4 C) In Wando, the lowest GI (1.2) was in December (WT 10.1 C) 2012 while the highest GI (3.5) was in August (WT 22.7 C) For Jeju Island, the lowest GI (1.8) occurred in December (WT 12.9 C) 2012 while the highest GI (3.6) occurred in July (WT 22.6 C) 2013 (Fig. 5). Main Spawning Period On the basis of the monthly changes in GI and the stages of gonad development, the main spawning period of Haliotis discus hannai was determined to be during September October in Uljin and Wando, and August September in Jeju Island. Condition Index The order of average CI of Haliotis discus hannai for the three regions was: Uljin (0.086) > Wando (0.076) > Jeju Island (0.067). Although CI displayed increases in May for Uljin, and CI of Wando and Jeju Island did not display definitive seasonal changes (Fig. 6). Meat Weight Index The average MWI was 68% in Uljin and 66% in both Wando and Jeju Island. Although MWI displayed decreases in January and November on Uljin and Jeju Island, respectively, and CI did not display definitive seasonal changes at three regions (Fig. 7). DISCUSSION Figure 1. Sampling areas of the abalone Haliotis discus hannai. The gonad of Haliotis discus hannai is composed of numerous gametogenic follicles. Germ cells in each of these follicles are developed asynchronously with definitive seasonal changes. These structural characteristics have been confirmed in Haliotis iris and Haliotis australis (Wilson & Schiel 1995), Haliotis asinina (Capinpin et al. 1998, Sobhon et al. 1999),

3 GONADAL DEVELOPMENT AND REPRODUCTIVE CYCLE OF H. DISCUS HANNAI 655 Figure 2. Photomicrographs showing the ovarian developmental stage of the abalone Haliotis discus hannai. (A) Inactive stage, (B) early active stage, (C) late active stage, (D) ripe stage, (E): spent stage, and (F) degenerative stage. El, epithelial layer; Es, egg stalk; N, nucleus; Oc, oocyte; Ogf, oogenic follicle; Om, outer membrane. Haliotis varia (Najmudeen & Victor 2004), Batillus cornutus (Jung et al. 2007a, 2007b), Haliotis midae (Visser-Roux 2011), and H. discus discus (Kim et al. 2014). For molluscs, genetic and environmental factors such as water temperature and the availability of food can affect sex ratio and sex determination (Yusa 2007, Ch avez-villalba et al. 2011). The sex ratio (female:male) of Haliotidae has been reported to be approximately 1:1 in Haliotis cracheroidii (Webber & Giese 1969), Haliotis rufescens (Young 1970), Haliotis gigantea, Haliotis sieboldii, Haliotis discus, Haliotis discus hannai (Lee 1974), Haliotis australis (Wilson & Schiel 1995), Haliotis asinina (Capinpin et al. 1998), and Haliotis varia (Najmudeen & Victor 2004). On the other hand, for Haliotis iris collected in the vicinity of Dunedin in the southern island of New Zealand, different sex ratios (1:1 and 1.7:1) have been reported for two different locations (Wilson & Schiel 1995). In this study, the sex ratio of Haliotis discus hannai was approximately 1:1 in the three areas. The sex ratio of Haliotis discus discus collected on Jeju Island has been reported to be 1:0.8 (Kim et al. 2014). Data such as monthly frequencies of gonad development stages, GI, and CI, can provide important information to distinguish the reproductive cycles and determine the range of spawning period of molluscs including gastropods (Boolootian et al. 1962, Marsden 1999, Sobhon et al. 1999, Park et al. 2003, Najmudeen & Victor 2004, Liu et al. 2008, Limpanont et al. 2011, Jeon et al. 2012). The reproductive cycle and sexual maturation of aquatic animals can be affected by exogenous and endogenous factors. Water temperature is an important exogenous factor that determines the sexual maturity of many species of molluscs (Mackie 1984, Eversole 2001), including Haliotis varia (Najmudeen & Victor 2004), Spisula sachalinensis (Lee et al. 1997), Gomphina veneriformis (Park et al. 2003), Fulvia mutica (Liu et al. 2008), Pecten sulcicostatus (Arendse et al. 2008), and Heteromacoma irus (Limpanont et al. 2011). Gonadal activation of these Figure 3. Photomicrographs showing the testicular developmental stage of the abalone Haliotis discus hannai. (A) Inactive stage, (B) early active stage, (C) late active stage, (D) ripe stage, (E) spent stage, and (F) degenerative stage. Sg, spermatogonia; Sgf, spermatogenic follicle; Sp, sperm; St, spermatids; Uds, undischarged sperm.

4 656 KIM ET AL. TABLE 1. Sex ratio of the abalone Haliotis discus hannai. Uljin Wando Jeju Island Shell length (mm) Sex ratio (F:M) x 2 P value Sex ratio (F:M) x 2 P value Sex ratio (F:M) x 2 P value : : : : : : : : : : : : : : : : Average 1: : : species begins in the spring when water temperature begins to increase. They mature in the summer when water temperature is high. Spawns mainly occur in early fall when the water temperature begins to decrease. In general, the reproductive period of invertebrates that thrive in low latitude regions is long compared with that of species in high latitude regions. These characteristics are due to the effects of different light or water temperatures, in accordance with the effect of latitude on reproductive characteristics of the species (Fretter 1984). The gonad of Haliotis discus hannai displayed histological seasonal changes in the inactive stage (November December, WT C), early active stage (January March, WT C), late active stage (March April, WT C), ripe stage (May July, WT C), spent stage (August October, WT C), and degenerative stage (October November, WT C). The highestgi wasobservedin thesummer and the lowest GI was found in the winter for both females and males. Gonadal tissues displayed similar seasonal changes in appearances. On the basis of the results of this study, it was suggested that the gonadal activation of H. discus hannai commences in the spring as water temperature begins to increase. After undergoing maturation and ripe stages during Figure 4. Monthly variation of water temperature in the sampling area (KHOA 2013) and gonad developmental stage of the abalone, Haliotis discus hannai. In: Inactive stage; Ea: Early active stage; La: Late active stage; R: Ripe stage; Sp: Spent stage; D: Degenerative stage; s: Water temperature. Vertical bars indicate standard deviation.

5 GONADAL DEVELOPMENT AND REPRODUCTIVE CYCLE OF H. DISCUS HANNAI 657 Figure 5. Monthly variation of GI in the abalone Haliotis discus hannai. Vertical bars indicate SD. the summer, spawning begins in early fall when the water temperature starts to decrease. Accordingly, the sexual maturation and reproductive cycles of H. discus hannai are affected by the water temperature. According to the results of monthly frequencies of gonad development stages of Haliotis discus hannai, it takes an average period of 2 3 mo from ripe stage to spent stage in the three regions studied. Therefore, artificial seed production of H. discus hannai in Korea needs to be improved. In other words, the time of early maturation must be brought forward through increasing water temperature. Artificial seed production needs to undergo a minimum of 2 3 mo of ripe stage after maturation. As the results of this study, early and late active vitellogenic oocytes (Lavo) were observed mostly in the ovary of Haliotis discus hannai in April while ripe oocytes (Ro) were observed mostly in the ovary in August. The size of Ro ( mm) of H. discus hannai is larger than the Lavo ( mm). Moreover, Ro displays stronger eosinophilic stainability than Lavo in the hematoxylin 0.5% eosin stain, along with approximately 30% greater thickness of outer jelly membrane (Ju & Lee 2016). Such results signify that oocytes spawned in the spring has lower degree of yolk accumulation, as well as the ability to adapt to the physical environmental factors in the water in comparison with the oocytes spawned in autumn. The oocytes of invertebrates spawned in the water are equipped with variable structures in the egg membrane to protect the eggs from environmental elements (Bolton et al. 2000). Therefore, it is necessary to pursue additional researches on the biochemical composition of oocyte and survival rates of juveniles to make biological evaluation of the oocytes spawned in spring and those spawned in autumn of H. discus hannai. Figure 6. Monthly variation of CI in the abalone Haliotis discus hannai. Vertical bars indicate SD. Molluscs can be divided into two categories based on the number of spawning: (1) species that spawn once a year, such as Gomphina veneriformis (Park et al. 2003), Hexaplex trunculus (Elhasni et al. 2010), Barnea davidi (Jeon et al. 2012), and Figure 7. Monthly variation of MWI in the abalone Haliotis discus hannai. Vertical bars indicate SD.

6 658 KIM ET AL. Haliotis discus discus (Kim et al. 2014) and (2) species that spawn more than twice a year, such as Haliotis asinina (Sobhon et al. 1999) in Thailand. Molluscs can also be divided based on their spawning season: (1) year-around breed species found mostly in the tropical region, (2) winter breeders that spawn from late fall to early spring the next year, and (3) summer breeders that spawn from late spring to early fall (Boolootian et al. 1962, Capinpin et al. 1998). The main spawning period of Haliotis discus hannai in Korea was found to be September October in Uljin and Wando and August September in Jeju Island. These results are similar to those reported by Lee (1974), in which August October is the main spawning period of H. discus hannai as a summer breeder collected in the coastal waters of Busan in the South Sea of Korea. Regional differences in the main spawning period in this study could be due to fluctuation in water temperature in accordance with the size of water mass arising from the differences in geographical features. Jeju Island is in the vicinity of open seas but Wando is in a bay as illustrated in Figure 1. Germ cell development and spawning of bivalves are important factors that induce the changes in CI, which is correlated with the reproductive cycle. The correlation between CI and the reproductive cycle has been seen quite clearly in Hexaplex trunculus (Gaspar 2008), Spisula sachalinensis (Lee et al. 1997), Tegillarca granosa (Lee 1997), Gomphina veneriformis (Park et al. 2003), and Mercenaria mercenaria (Marroquin-Mora & Rice 2008). Though, it is difficult to find researches reporting such correlation in abalones. In this study, the CI and MWI of Haliotis discus hannai did not display definitive changes throughout the year. 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