Gonadal Development and Reproductive Cycle of Cultured Abalone, Haliotis Discus Hannai (Gastropoda: Haliotidae) in Korea: Implications for Seed Production

Journal of Shellﬁsh Research, Vol. 35, No. 3, 653–659, 2016.

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 LEE3 AND JUNG SICK LEE1* 1Department of Aqualife Medicine, Chonnam National University, Yeosu 59626, Republic of Korea; 2South Sea Fisheries Research Institute, National Fisheries Research and Development Institute, Yeosu 59780, Republic of Korea; 3Faculty 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 such practice has problems, such as egg quality including yolk accumulation since the ripe and fertilized eggs produced Members of the family Haliotidae exist in most tropical through such artificial process do not have sufficient matura- and temperate oceans, particularly in the shallow subtidal tion period compared with those obtained naturally. There- zone. A total of 55 species with 10 additional subspecies have fore, the objective of this study was to provide information on been reported (Lindberg 1992, Geiger 1998). Two species and the sexual maturation and reproductive cycle of abalone as two subspecies (Haliotis madaka, Haliotis gigantea, Haliotis wellastheperiodfrommaturationtospawninginartificial discus discus, Haliotis discus hannai)ofgenusHaliotis and seed production of Haliotis discus hannai at three main regions two subspecies (Sulculus diversicolor diversicolor, Sulculus of abalone aquaculture in Korea. diversicolor supertexta)ofgenusSulculus have been reported in Korea (Lee & Min 2002). MATERIALS AND METHODS In Korea, Haliotis discus hannai have been produced by aquaculture at 8,977 tons in 2014. Among these, the regions of Sampling Uljin, Wando, and Jeju Island produced a total of 7,550 tons, which accounts for 85% of the total production of abalone in Abalone Haliotis discus hannai were collected from Uljin (on Korea (KOSIS 2014). the eastern sea of Korea), Wando, and Jeju Island (on southern Studies on the reproductive ecology of marine molluscs sea of Korea) (Fig. 1). A total of 788 H. discus hannai were including gastropods can provide essential information on basic collected monthly from September 2012 to August 2013 with ecological data to protect wild populations, increase biological shell length of 70.68 (±11.60) mm. resources, and develop aquaculture technology (Gosling 2004, Bigatti et al. 2008). Studies on the reproduction of abalones in Environmental Conditions Korea have been focused on gametogenesis and reproductive cycle of abalones (Lee 1974, Kim et al. 2014). In these researches, Data from the Korea Hydrographic and Oceanographic the main spawning period of abalone in Korea has been Administration were used to obtain water temperature profiles reported to be in around October. of the three areas (KHOA 2013). Water temperatures were Nevertheless, seed production in abalone aquaculture of measured at every hour during the period of survey, and the Korea is executed during April–May by maturing abalone monthly average and SD are indicated in Figure 4. earlier through artificially increasing the water temperature over a period (approximately 1 mo) to enable the abalone to Sex Ratio, Condition Index, and Meat Weight Index grow sufficiently during the period of high water temperature Sex ratio was calculated by female:male ratio. Condition in summer by producing the young abalone earlier. Although index (CI) and meat weight index (MWI) were calculated using the following equations according to the methodology of Jeon 3 *Corresponding author. E-mail: [email protected] et al. (2012): CI ¼ [body weight (g)/shell length (mm) ] 3 1,000; DOI: 10.2983/035.035.0311 MWI ¼ [body weight (g)/total weight (g)] 3 100.

653 654 KIM ET AL.

Histology in Wando, and 17.1°C in Jeju Island. The three areas reported the highest water temperatures in August or September, and the Specimens were dissected, and the gonads were prepared lowest temperatures in January or February (Fig. 4). after measuring the morphometric characteristics of shell length, total weight, and body weight. Specimens were prepared Sex Ratio for a light microscopy according to the methodology of Drury and Wallington (1980). The visceral mass including the gonad The sex ratio (female:male) of Haliotis discus hannai was was fixed in BouinÕs solution for 24 h, rinsed with running water approximately 1:1 in the three areas. There was no statistical for 36–48 h, and dehydrated through a graded ethyl alcohol significance in the difference of sex ratios among the three areas series (70%–100%). The preparations were then embedded in (Table 1). paraplast (Leica, Germany). Embedded tissues were sectioned at thickness of 4–6 mm using a microtome (RM2235, Leica, Gonadal Development Stage Germany). Samples were stained with MayerÕs hematoxylin– 0.5% eosin. Monthly changes in gonadal development stage displayed differences among the three regions (Uljin, Wando, and Jeju Gonad Index Island). The gonad of Haliotis discus hannai displayed histological seasonal changes in the inactive stage (November– Gonad index (GI) was partially corrected using the method December), early active stage (January–March), late active of Eversole (1997). Gonad development was categorized into stage (March–April), ripe stage (May–July), spent stage (August– the following six stages: inactive stage (In), early active stage October), and degenerative stage (October–November; Fig. 4). (Ea),lateactivestage(La),ripestage(R),spent(Sp),and degenerative stage (D) (Figs. 2 and 3). Samples were quantified Gonad Index 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 Gonad index of Haliotis discus hannai displayed differences in stage. The GI was calculated using the following equation. 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 ðÞIn 3 1 + ðÞEa 3 2 + ðÞLa 3 3 + ðÞR 3 4 + ðÞSp 3 3 + ðÞD 3 2 ° GI ¼ December (WT 10.6 C) 2012 while the highest GI (3.6) occurred Total number in July (WT 17.4°C) 2013. 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) 2013. For Jeju Island, the lowest GI (1.8) Statistical Analysis occurred in December (WT 12.9°C) 2012 while the highest The sex ratio data were analyzed using SPSS 21.0 software GI (3.6) occurred in July (WT 22.6°C) 2013 (Fig. 5). (SPSS Inc.). The observed female:male ratio for each shell length group was compared with the expected ratio of 1:1 using Main Spawning Period a c2 test. Statistical significance was considered when P value On the basis of the monthly changes in GI and the stages of was <0.05. gonad development, the main spawning period of Haliotis discus hannai was determined to be during September–October RESULTS in Uljin and Wando, and August–September in Jeju Island.

Water Temperature Condition Index

The average water temperature during the collection period The order of average CI of Haliotis discus hannai for the (September 2012 to August 2013) was 15.0°C in Uljin, 14.6°C 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

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), Figure 1. Sampling areas of the abalone Haliotis discus hannai. Haliotis asinina (Capinpin et al. 1998, Sobhon et al. 1999), 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 The sex ratio of Haliotis discus discus collected on Jeju Island (Jung et al. 2007a, 2007b), Haliotis midae (Visser-Roux 2011), has been reported to be 1:0.8 (Kim et al. 2014). and H. discus discus (Kim et al. 2014). Data such as monthly frequencies of gonad development For molluscs, genetic and environmental factors such as stages, GI, and CI, can provide important information to water temperature and the availability of food can affect sex distinguish the reproductive cycles and determine the range of ratio and sex determination (Yusa 2007, Chavez-Villalba et al. spawning period of molluscs including gastropods (Boolootian 2011). The sex ratio (female:male) of Haliotidae has been et al. 1962, Marsden 1999, Sobhon et al. 1999, Park et al. 2003, reported to be approximately 1:1 in Haliotis cracheroidii Najmudeen & Victor 2004, Liu et al. 2008, Limpanont et al. (Webber & Giese 1969), Haliotis rufescens (Young 1970), 2011, Jeon et al. 2012). Haliotis gigantea, Haliotis sieboldii, Haliotis discus, Haliotis The reproductive cycle and sexual maturation of aquatic discus hannai (Lee 1974), Haliotis australis (Wilson & Schiel animals can be affected by exogenous and endogenous factors. 1995), Haliotis asinina (Capinpin et al. 1998), and Haliotis varia Water temperature is an important exogenous factor that de- (Najmudeen & Victor 2004). termines the sexual maturity of many species of molluscs (Mackie On the other hand, for Haliotis iris collected in the vicinity of 1984, Eversole 2001), including Haliotis varia (Najmudeen & Dunedin in the southern island of New Zealand, different sex Victor 2004), Spisula sachalinensis (Lee et al. 1997), Gomphina ratios (1:1 and 1.7:1) have been reported for two different veneriformis (Park et al. 2003), Fulvia mutica (Liu et al. 2008), locations (Wilson & Schiel 1995). In this study, the sex ratio of Pecten sulcicostatus (Arendse et al. 2008), and Heteromacoma Haliotis discus hannai was approximately 1:1 in the three areas. 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. 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) x2 P value Sex ratio (F:M) x2 P value Sex ratio (F:M) x2 P value 40.01–50.00 1:0.33 1.00 0.317 1:3.25 4.77 0.029 – – – 50.01–60.00 1:0.60 0.50 0.480 1:1.00 0.00 1.000 1:1.18 0.17 0.683 60.01–70.00 1:0.69 2.98 0.084 1:0.71 3.53 0.060 1:0.76 1.49 0.222 70.01–80.00 1:1.31 1.60 0.206 1:0.97 0.01 0.907 1:1.36 0.76 0.384 80.01–90.00 1:1.00 0.00 1.000 1:0.90 0.05 0.819 1:0.76 1.25 0.264 90.01–110.00 1:0.67 0.20 0.655 – – – 1:0.73 0.95 0.330 Average 1:0.93 0.33 0.564 1:0.91 0.95 0.329 1:0.86 1.06 0.304 species begins in the spring when water temperature begins to WT 16.1–10.0°C), early active stage (January–March, WT 7.6– increase. They mature in the summer when water temperature is 12.8°C), late active stage (March–April, WT 9.4–14.5°C), ripe high. Spawns mainly occur in early fall when the water temper- stage (May–July, WT 14.4–22.6°C), spent stage (August–October, ature begins to decrease. In general, the reproductive period of WT 26.2–18.9°C), and degenerative stage (October–November, invertebrates that thrive in low latitude regions is long compared WT 20.3–13.8°C). The highest GI was observed in the summer with that of species in high latitude regions. These characteristics and the lowest GI was found in the winter for both females and are due to the effects of different light or water temperatures, in males. Gonadal tissues displayed similar seasonal changes in accordance with the effect of latitude on reproductive character- appearances. On the basis of the results of this study, it was istics of the species (Fretter 1984). suggested that the gonadal activation of H. discus hannai The gonad of Haliotis discus hannai displayed histological commences in the spring as water temperature begins to seasonal changes in the inactive stage (November–December, 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. GONADAL DEVELOPMENT AND REPRODUCTIVE CYCLE OF H. DISCUS HANNAI 657

Figure 6. Monthly variation of CI in the abalone Haliotis discus hannai. Figure 5. Monthly variation of GI in the abalone Haliotis discus hannai. Vertical bars indicate SD. Vertical bars indicate SD. Molluscs can be divided into two categories based on the the summer, spawning begins in early fall when the water number of spawning: (1) species that spawn once a year, such as temperature starts to decrease. Accordingly, the sexual matu- Gomphina veneriformis (Park et al. 2003), Hexaplex trunculus ration and reproductive cycles of H. discus hannai are affected (Elhasni et al. 2010), Barnea davidi (Jeon et al. 2012), and by the water temperature. According to the results of monthly frequencies of gonad development stages of Haliotis discus hannai,ittakesan 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 (282.6 3 230.8 mm) of H. discus hannai is larger than the Lavo (202.3 3 73.3 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 Figure 7. Monthly variation of MWI in the abalone Haliotis discus spawned in autumn of H. discus hannai. hannai. Vertical bars indicate SD. 658 KIM ET AL.

Haliotis discus discus (Kim et al. 2014) and (2) species that Germ cell development and spawning of bivalves are spawn more than twice a year, such as Haliotis asinina (Sobhon important factors that induce the changes in CI, which is et al. 1999) in Thailand. Molluscs can also be divided based correlated with the reproductive cycle. The correlation between on their spawning season: (1) year-around breed species found CI and the reproductive cycle has been seen quite clearly in mostly in the tropical region, (2) winter breeders that spawn Hexaplex trunculus (Gaspar 2008), Spisula sachalinensis from late fall to early spring the next year, and (3) summer (Lee et al. 1997), Tegillarca granosa (Lee 1997), Gomphina breeders that spawn from late spring to early fall (Boolootian veneriformis (Park et al. 2003), and Mercenaria mercenaria et al. 1962, Capinpin et al. 1998). (Marroquin-Mora & Rice 2008). Though, it is difficult to find The main spawning period of Haliotis discus hannai in Korea researches reporting such correlation in abalones. In this study, was found to be September–October in Uljin and Wando and the CI and MWI of Haliotis discus hannai did not display August–September in Jeju Island. These results are similar to definitive changes throughout the year. This could be due to those reported by Lee (1974), in which August–October is the relatively low quantity of germ cells released by H. discus hannai main spawning period of H. discus hannai as a summer breeder compared with bivalves. collected in the coastal waters of Busan in the South Sea of Korea. Regional differences in the main spawning period in this ACKNOWLEDGMENT study could be due to fluctuation in water temperature in accordance with the size of water mass arising from the This work was supported by grants from the National differences in geographical features. Jeju Island is in the vicinity Fisheries Research and Development Institute, South Sea of open seas but Wando is in a bay as illustrated in Figure 1. Fisheries Research Institute (RP-15-AQ-51).

LITERATURE CITED Arendse, D. C. Z., S. Andersen, N. J. Blake & G. C. Pitcher. 2008. The Jeon, M. A., M. W. Park, K. Ku, S. M. Ju, P. Ko, B. H. Kim, J. I. reproductive cycle of the scallop Pecten sulcicostatus from the Myeong & J. S. Lee. 2012. Gonadal development and reproductive southern Benguela upwelling system. J. Shellfish Res. 27:281–287. cycle of the boring bivalve, Barnea davidi (Deshayes, 1874) in Bigatti, G., E. M. Marzinelli & P. E. Penchaszadeh. 2008. Seasonal Hampyeong Bay off the west coast of Korea (Bivalvia: Pholadidae). reproduction and sexual maturity in Odontocymbiola magellanica J. Shellfish Res. 31:951–958. (Neogastropoda, Volutidae). Invertebr. Biol. 127:314–326. Ju, S. M. & J. S. Lee. 2016. Changes of morphology and morphometric Bolton, T. F., F. I. M. Thomas & C. L. Leonard. 2000. Maternal energy characteristics of the oocyte during oogenesis of the abalone, investment in eggs and jelly coats surrounding eggs of the echinoid Haliotis discus hannai. Korean J. Malacol. 32:1–7. Arbacia punctulata. Biol. Bull. 199:1–5. Jung, G. K., J. J. Park, J. W. Lee & J. S. Lee. 2007a. Spermatogenesis of Boolootian, R. A., A. Farmanfarmaian & A. C. Giese. 1962. On the the spiny top shell, Batillus cornutus (Lightfoot, 1786) (Gastropoda: reproductive cycle and breeding habits of two western species of Turbinidae). Dev. Reprod. 11:97–104. Haliotis. Biol. Bull. 122:183–193. Jung, G. K., J. J. Park, S. M. Ju, Y. G. Jin & J. S. Lee. 2007b. Ovarian Capinpin, E. C., Jr., V. C. Encena, II & N. C. Bayona. 1998. Studies on structure and oogenesis of the spiny top shell, Batillus cornutus the reproductive biology of the DonkeyÕs ear abalone, Haliotis (Lightfoot, 1786) (Gastropoda: Turbinidae). Korean J. Malacol. asinina Linne. Aquaculture 166:141–150. 23:209–216. Chavez-Villalba, J., C. Soyez, A. Huvet, Y. Gueguen, C. Lo & G. Le KHOA. 2013. Real time coastal data; Uljin, Wando and Jeju Island. Moullac. 2011. Determination of gender in the pearl oyster Pinctada Accessed February 2013. Available at: http://sms.khoa.go.kr/koofs/ margaritifera. J. Shellfish Res. 30:231–240. kor/observation/obs_past_search.asp. Drury, R. A. B. & E. A. Wallington. 1980. CarletonÕs histological Kim, J. W., B. W. Lee, J. C. Kang, E. Y. Min, S. H. Won, H. G. Lim, technique. Oxford, United Kingdom: Oxford University Press. 520 S. W. Kang, M. A. Jeon & J. S. Lee. 2014. Reproductive cycle of the pp. abalone, Haliotis discus discus collected from Jeju Island of Korea. Elhasni, K., M. Ghorbel, P. Vasconcelos & O. Jarboui. 2010. Re- Korean J. Malacol. 31:21–26. productive cycle and size at first sexual maturity of Hexaplex KOSIS 2014. Fishery, species and fishing method statistics. Accessed trunculus (Gastropoda: Muricidae) in the Gulf of Gabes (southern March 2015. Available at: kosis.kr. Tunisia). Invertebr. Reprod. Dev. 54:213–225. Lee, T. Y. 1974. Gametogenesis and reproductive cycle of abalones. Eversole, A. G. 1997. Gametogenesis of Mercenaria mercenaria, M. Publ. Mar. Lab. Busan Fish. Coll. 7:21–50. campechiensis and their hybrids. Nautilus 110:107–110. Lee, J. H. 1997. Studies on the gonadal development and gametogenesis Eversole, A. G. 2001. Reproduction in Mercenaria mercenaria. In: of the granulated ark, Tegillarca granosa (Linne). Korean J. Kraeuter, J. N. & M. Castagna, editors. Biology of the hard clam. Malacol. 13:55–64. New York, NY: Elsevier. pp. 221–260. Lee, J. Y., Y. J. Chang & C. S. Lee. 1997. Reproductive cycle of surf Fretter, T. 1984. Prosobranchs. In: Tompa A. S., N. H. Verdonk & clam, Spisula sachalinensis. J. Kor. Fish. Soc. 30:132–138. J. A. M. van den Biggelaar, editors. The mollusca: reproduction, Lee, J. S. & D. K. Min. 2002. A catalogue of molluscan fauna in Korea. vol. 7. New York, NY: Academic Press. pp. 1–45. Korean J. Malacol. 18:93–217. Gaspar, M. B. 2008. Comparison of indices for the assessment of Limpanont, A., H. S. Yang, K. I. Park & K. S. Choi. 2011. First report reproductive activity in Hexaplex trunculus (Gastropoda: Murici- on the annual gametogenesis of Heteromacoma irus (Hanley, 1845) dae). Mar. Biol. Res. 4:392–399. in a rocky intertidal area, northern Jeju Island, Korea. J. Shellfish Geiger, D. L. 1998. Recent genera and species of the family Haliotidae. Res. 30:39–46. Rafinesque, 1815 (Gastropoda: Vestigastropoda). Nautilus Lindberg, D. R. 1992. Evolution, distribution, and systematics of 111:85–116. Haliotidae. In: Shepherd S. A., M. J. Tegner & S. A. Guzman del Gosling, E. 2004. Bivalve molluscs: biology, ecology and culture. Proo, editors. Abalone of the world: biology, fisheries, and culture. Oxford, United Kingdom: Blackwell Science. 443 pp. Oxford, United Kingdom: Blackwell. pp. 3–18. GONADAL DEVELOPMENT AND REPRODUCTIVE CYCLE OF H. DISCUS HANNAI 659

Liu, W., Q. Li, Y. Yuan & S. Zhang. 2008. Seasonal variations in Sobhon, P., S. Apisawetakan, M. Chanpoo, C. Wanichanon, V. reproductive activity and biochemical composition of the cockle Linthong, A. Thongkukiatkul, P. Jarayabhand, M. Kruatrachue, Fulvia mutica (Reeve) from the eastern coast of China. J. Shellfish S. E. Upatham & T. Poomthong. 1999. Classification of germ cells, Res. 27:405–411. reproductive cycle and maturation of gonads in Haliotis asinina Mackie, G. L. 1984. Bivalves. In: Tompa A. S., N. H. Verdonk & Linnaeus. Sci. Asia 25:3–21. J. A. M. van den Biggelaar, editors. The mollusca: reproduction, Visser-Roux, A. 2011. Reproduction of the South African abalone, vol. 7. New York, NY: Academic Press. pp. 351–418. Haliotis midae. PhD diss., University of Stellenbosch, Matieland, Marroquin-Mora, D. C. & M. A. Rice. 2008. Gonadal cycle of northern South Africa. 88 pp. quahogs, Mercenaria mercenaria (Linne, 1758), from fished and Webber, H. H. & A. C. Giese. 1969. Reproductive cycle and gameto- non-fished subpopulations in Narragansett Bay. J. Shellfish Res. genesis in the black abalone, Haliotis cracheroidii (Gastropoda: 27:643–652. Prosobranchiata). Mar. Biol. 4:152–159. Marsden, I. D. 1999. Reproductive cycle of the surf beach clam, Paphies Wilson, N. H. F. & D. R. Schiel. 1995. Reproduction in two species of donacina (Spengler, 1973) from New Zealand. J. Shellfish Res. 18:539–546. abalone (Haliotis iris and H. australis) in southern New Zealand. Najmudeen, T. M. & A. C. C. Victor. 2004. Reproductive biology of the Mar. Freshwater Res. 46:629–637. tropical abalone Haliotis varia from Gulf of Mannar. J. Mar. Biol. Young, J. S. 1970. The reproductive cycle, gonadal histology and Assoc. India 46:154–161. gametogenesis of the red abalone Haliotis rufescens (Swaison). Calif. Park, J. J., J. Y. Lee, J. S. Lee & Y. J. Chang. 2003. Gonadal Fish Game 56:298–309. development and gametogenic cycle of the equilateral venus, Yusa, Y. 2007. Causes of variation in sex ratio and modes of sex Gomphina veneriformis (Bivalvia: Veneridae). J. Kor. Fish. Soc. determination in the mollusca—an overview. Am. Malacol. Bull. 36:352–357. 23:89–98.