Aquaculture 287 (2009) 191–202 Contents lists available at ScienceDirect Aquaculture j o u r n a l h o m e p a g e : w w w e l s ev i e r c o m / l o c a t e / a q u a - o n l i n e Gonad development during sexual differentiation in hatchery-produced orange-spotted grouper (Epinephelus coioides) and humpback grouper (Cromileptes altivelis) (Pisces: Serranidae, Epinephelinae) Min Liu ⁎, Yvonne Sadovy de Mitcheson The Swire Institute of Marine Science, The Division of Ecology & Biodiversity, The School of Biological Sciences, The University of Hong Kong, Pokfulam Road, Hong Kong SAR a r t i c l e i n f o Article history: Received 24 May 2008 Received in revised form 11 October 2008 Accepted 15 October 2008 Keywords: Grouper mariculture Primary male Protogynous hermaphroditism Sex-change Sexual differentiation a b s t r a c t Juvenile sexual differentiation of two commercially important epinephelines, orange-spotted grouper (Epinephelus coioides) and humpback grouper (Cromileptes altivelis) (Serranidae, Epinephelinae), produced in hatcheries, was studied until first sexual maturation using gonadal histology Diandric, protogynous hermaphroditism is confirmed for E coioides with evidence of primary male differentiation directly from the juvenile phase as well as of secondary male development through the sex-change of functional females All juveniles developed first an ovarian-phase then entered a bisexual-phase gonad Sexual differentiation occurred in bisexual gonads at around 105 weeks after hatching (wah), with the growth of oocytes beyond the primary-growth stage for females, and the appearance of sperm sinuses and proliferation of spermatogenic cysts in primary males The minimum size and age of first sexual maturation for females were 355 mm standard length (SL) and 113 wah No mature males were found by 141 wah, suggesting that first maturation in males takes longer than in females, at least under mariculture conditions For C altivelis, all juveniles developed an ovarian-phase gonad Minimum body size and age of first sexual maturation for females were 155 mm SL and 83 wah In contrast to E coioides, there was no sign of spermatogenic cysts in juvenile gonads and any indication of primary male differentiation in C altivelis This study reveals the variation in early gonad development in the epinephelines and highlights the importance of studying juvenile sexual differentiation for fully understanding sexual pattern in this fish subfamily © 2008 Elsevier B.V All rights reserved Introduction Sexual patterns in the subfamily Epinephelinae (Family: Serranidae) are diverse and include gonochorism, protogynous hermaphroditism and bi-directional sex-change (Sadovy de Mitcheson and Liu, 2008) Protogynous hermaphroditism has two forms, monandry and diandry, based on the number of male developmental pathways involved (Reinboth, 1967) In monandry all males (i.e secondary males) are derived exclusively from functional females through sexchange In diandry there are two male developmental pathways; primary males that develop directly from the juvenile phase through sexual differentiation and function as males at first sexual maturation, and secondary males, as in monandry Diagnosis of sexual pattern is particularly challenging in the epinephelines because differences in testicular morphology between primary and secondary male developmental pathways are not as readily apparent as they are in other fish families such as Labridae and Scaridae (Sadovy de Mitcheson and Liu, 2008) All testes, including ⁎ Corresponding author Tel.: +852 2809 2179; fax: +852 2809 2197 E-mail address: lium@graduate.hku.hk (M Liu) 0044-8486/$ – see front matter © 2008 Elsevier B.V All rights reserved doi:10.1016/j.aquaculture.2008.10.027 those of gonochoristic (e.g Epinephelus striatus and Mycteroperca rosacea), protogynous (e.g Cephalopholis, Epinephelus and Plectropomus species) and bi-directional sex-changing (e.g C boenak and E akaara) epinephelines studied to date, exhibit an ovarian structure, with a lumen and sperm sinuses within the gonadal wall (Shapiro et al., 1993; Siau, 1994; Sadovy and Colin, 1995; Okumura, 2001; Fennessy and Sadovy, 2002; Adams, 2003; Liu and Sadovy, 2004a,b; Erisman et al., 2008; Sadovy de Mitcheson and Liu, 2008) This differs from the gonads of primary males in some species of the labrids and scarids that exhibit structurally gonochoristic characteristics including an epithelial lined central sperm duct located dorsally and have no lumen (Choat and Robertson, 1975; Robertson and Warner, 1978; Warner and Robertson, 1978; Dipper and Pullin, 1979) In such species, the gonads of secondary males differ clearly from primary males and retain the lumen from the functional female phase and develop sperm sinuses within the gonadal wall during sex-change Therefore, understanding the sexual pattern in the epinephelines requires considerable care and examination of a wide range of body sizes and ages, including both juvenile and adult phases Despite the importance of studying juvenile sexual differentiation for fully understanding sexual patterns in the epinephelines, few studies involve examination of juveniles (Tanaka et al., 1990; Adams, 192 M Liu, Y Sadovy de Mitcheson / Aquaculture 287 (2009) 191–202 2003; Liu and Sadovy, 2004a,b) In part, this is because of the difficulty in finding larvae and small juveniles of many grouper species in the wild Therefore, the epinephelines available from hatcheries for mariculture operations provide appropriate, and otherwise hard to capture, materials for studying juvenile sexual differentiation The live reef food fish (LRFF) are considered luxury seafood in Asia because of their superior taste or texture Despite the wide species diversity in the LRFF trade the epinephelines are dominate in the trade in terms of both volume and value (Sadovy et al., 2003) Some epinephelines including the orange-spotted grouper (E coioides, as ‘E suillus’ or ‘E tauvina’ in earlier publications) and humpback grouper (Cromileptes altivelis) are overfished and threatened (Heemstra and Randall, 1993; Sadovy et al., 2003;) Mariculture of epinephelines has been developed in Asia since the 1980s; E coioides and C altivelis are examples of the few epinepheline species available from hatcheries, which provided appropriate materials for this study (Liao et al., 2001; Hong and Zhang, 2003; Rimmer et al., 2004) E coioides is an important mariculture species in Asia including southern mainland China, Hong Kong and Taiwan, and mariculture of C altivelis is at an experimental scale in Hong Kong (Liu and Sadovy de Mitcheson, 2008) E coioides is confirmed as protogynous with occasional occurrence of small males although the pathways of male development are unknown (Tan and Tan, 1974; Chen et al., 1977; Heemstra and Randall, 1993; Quinitio et al., 1997) The smallest body size and age of female sexual maturation in E coioides are reported to be 450 mm in standard length (SL) and two years (Tan and Tan, 1974; Chen et al., 1977) C altivelis is proposed to be a protogynous hermaphrodite based on body size- and age-biased sex ratios, with females smaller and younger than males in Australian waters (Davies et al., 2006) The smallest body size and age of female sexual maturation in C altivelis are reported to be 350 mm in total length (TL) and two years (Davies et al., 2006) However, there are no detailed studies on gonad development during juvenile sexual differentiation for either species The objectives of this study were to describe gonad development of E coioides larvae and juveniles during sexual differentiation until first sexual maturation to better elucidate its sexual pattern, and of C altivelis juveniles during sexual differentiation until first sexual maturation to provide information on early sexual development Materials and methods between SL and TL were described from data pooled across all sources; same for C altivelis (see Section 2.1.2) 2.1.2 C altivelis A total of 273 specimens were collected between November 2006 and February 2008 with sampling conducted every 2-10 weeks but mainly every 2-7 weeks Body sizes of all specimens ranged between 42 and 211 mm SL, ages between 10 and 109 wah, and life stages ranging from small juveniles to first sexual maturation Three batches of juveniles from different age groups were sampled; all juveniles were hatcheryproduced in Gondol, Indonesia (8°582′ S, 116°272′ E), and imported to Hong Kong (22°209′ N, 114°256′ E) at 10 wah for mariculture grow-out; earlier life stage samples between and wah were not available Fish were reared in indoor recirculation tanks with artificial sea water from 10 to 30 wah under controlled water temperatures of 24.7-28.9 °C After 30 wah, fish were moved to outdoor tanks with a continuous seawater supply pumped directly from the sea and passed through a biofilter with natural water temperatures of 12.4-31.4 °C Fish were fed on commercial pellet diets at different particle sizes (T.P.C., Taiwan) 2.2 Gonadal histology 2.2.1 Gonadal fixation Two fixatives were used; Dietrich's (900 ml distilled water, 450 ml 95% ethanol, 150 ml 40% formaldehyde and 30 ml acetic acid) for paraffin embedding, and Karnovsky's (100 ml 8% paraformaldehyde, 17 ml 25% glutaraldehyde and 93 ml 0.2 M phosphate buffer at pH 7.2) for resin (i.e plastic or glycolmethacrylate) embedding Gonads lie in the dorsal part of the body cavity and consist of a pair of elongate lobes separated anteriorly but united posteriorly, as in other epinephelines (Smith, 1965; Moe, 1969; Liu and Sadovy, 2004a) Gonads were removed and cut into several pieces of about 10 mm long each, randomly fixed in either Dietrich's or Karnovsky's fixatives, and stored at room temperature or °C, respectively For larvae and small juveniles from which gonads could not be removed, the entire body cavity was fixed 2.2.2 Histological procedures Body cavities containing gonads were decalcified in Fisher's Cal-Ex solution (Fisher Scientific, USA) overnight then washed with running tap water for 24 h before dehydration in preparation for histology 2.1 Fish collection 2.1.1 E coioides A total of 777 specimens were collected for gonadal histology based on samples taken every 2-5 weeks between October 2004 and December 2006 Body sizes of specimens ranged between 2.6 and 425 mm SL, ages between and 141 weeks after hatching (wah) and life stages ranging from larvae to first sexual maturation Specimens were collected from two sources Two hundred and seventy two specimens, aged between and 26 wah, were collected between April and September 2005 from a marine fish hatchery in Guangdong, southern mainland China (22°425′ N, 114°325′ E) Fish were reared under controlled water temperatures of 22.7-27.8 °C, and fed on oyster trochophores, rotifers, nauplii and adult Artemia, cladocerans and mixed fish feed caught by trawlers The remaining 505 specimens aged between 30 and 141 wah were collected between October 2004 and December 2006 from grouper culture cages in northeastern Hong Kong waters (22°458′ N, 114°353′ E), having been imported for mariculture operations at 20 wah from a hatchery in Gaoxiong (Kaohsiung), Taiwan (22°572′ N, 120°307′ E) In floating cages, fish were fed on mixed fish feed caught by trawlers, and natural water temperatures year-around ranged between 13.5 and 32.4 °C After each collection, fish were anaesthetized preserved on ice and transported to the laboratory, and SL, TL, body weight (BW) and gonad weight (GW) were measured Relationships between SL and BW and Table Gonadal categories and sexes of Epinephelus coioides and Cromileptes altivelis according to gonadal morphology present (+) or absent (−) Gonadal phase Sex Ud Ov-1 Ov-2 Ov-3 Bi-1 Bi-2 Bi-3 Ovary Testis UJ UJ UJ DF UJ DF DM MF MM Gonadal morphology GP OL SS G O1 O2 O3 SC + − − − − − − − − − + + + + + + + + − − − − − − +b − +d + + + + + + + + + − − + + + + + + + − − − + − + − + − − − − − − − − +e − − − − − +a +a +c +a/− +c For gonadal phase: Ud, undifferentiated-phase; Ov-1, ovarian-phase-1; Ov-2, ovarianphase-2; Bi-1, bisexual-phase-1; Bi-2, bisexual-phase-2; Bi-3, bisexual-phase-3 For sex: UJ, undifferentiated juvenile; DF, differentiating female; DM, differentiating male; MF, mature female; MM, mature male For gonadal morphology: GP, gonadal primordium; OL, ovarian lumen; G, gonium; O1, primary-growth stage oocyte; O2, cortical-alveolus stage oocyte; O3, vitellogenic stage oocyte; SC, spermatogenic cyst; SS, sperm sinus a Scattered b No sperm present in sperm sinuses c Abundant d Sperm present in sperm sinuses e Including mature, spent, females with atretic O3 or muscle bundles M Liu, Y Sadovy de Mitcheson / Aquaculture 287 (2009) 191–202 Gonads and body cavities were dehydrated in ascending grades of ethanol, embedded in paraffin or resin, and sectioned at 5-6 μm or μm thickness, respectively Sections were stained with haematoxylin and eosin, or metanil yellow-periodic acid/Schiff's haematoxylin, and mounted on slides 2.3 Gonadal classification and sexual definition Each gonad was assigned to one of nine categories according to gonadal morphology of E coioides and C altivelis: undifferentiatedphase (Ud), ovarian-phase-1 (Ov-1), ovarian-phase-2 (Ov-2), ovarianphase-3 (Ov-3), bisexual-phase-1 (Bi-1), bisexual-phase-2 (Bi-2), bisexual-phase-3 (Bi-3), ovary and testis (Table 1) Sex and maturation stages were defined based on the most advanced developmental stage of oocytes, proportion of spermatogenic cysts, appearance of sperm sinuses and presence of sperm in sperm sinuses Stages were classified as undifferentiated juvenile, differentiating female, differentiating male, mature/functional female and mature/ 193 functional male (Table 1) (Smith, 1965; Grier, 1981; Wallace and Selman, 1981; Selman and Wallace, 1989; Tyler and Sumpter, 1996; Liu and Sadovy, 2004a) Mature females were those with vitellogenic stage oocytes (O3), or later stages, and mature males had sperm present in sperm sinuses The resulting sections were examined and micrographs taken under a light microscope The gonadosomatic index (GSI%) was only calculated for sexually mature females and males by GSI% =GW × (BW − GW)− × 100 Results 3.1 Biological parameters For E coioides, the relationships between SL and BW and between SL and TL were: BW = 7.0 × 10− × SL2.8362 (n = 777, r2 = 0.9927, p b 0.05) and TL = 1.2084 × SL + 1.1087 (n = 777, r2 = 0.999, p b 0.05), respectively For C altivelis, the relationships between SL and BW and between SL Fig Size (standard length in mm, SL) variation within age groups (weeks after hatching) in samples taken for gonadal histology with the calculated growth curves and their equations presented (a) Epinephelus coioides and (b) Cromileptes altivelis 194 M Liu, Y Sadovy de Mitcheson / Aquaculture 287 (2009) 191–202 Table Gonad development of Epinephelus coioides during sexual differentiation with age (weeks after hatching, wah) and gonadal morphology present (+) or absent (−) Age (wah) 12 16–30 34–82 86 104 108 113 141 Gonadal morphology GP + + − − − − − − − OL − − +/− + + + + + + SS − − − − − +a − − +a GC G O1 O2 O3 SC − + + + + +b + + + − − − + + + + + + − − − − − − + + − − − − − − − − + − − − − − + + + + +b G, gonium; GC, germ cell; GP, gonadal primordium; O1, primary-growth stage oocyte; O2, cortical-alveolus stage oocyte; O3, vitellogenic stage oocyte; OL, ovarian lumen; SC, spermatogenic cyst; SS, sperm sinus a No sperm present in sperm sinuses b Abundant and TL were: BW = 1.0 × 10− × SL3.1441 (n = 273, r2 = 0.9736, p b 0.05) and TL = 1.2252 × SL + 0.2598 (n = 273, r2 = 0.9916, p b 0.05), respectively Size variation within age groups was found in both species, indicating differences in growth rates; growth curves were presented in Fig 3.2 Gonad development during sexual differentiation 3.2.1 Undifferentiated-phase (Ud) gonad For E coioides, the paired gonadal primordia (GP) were first observed in wah larvae (April 2005) with a few somatic cells below the swim bladder in the anterior portion and kidney in the posterior portion of the body cavity (Table 2) In wah (May 2005), blood vessels were first observed within the GP and surrounded by somatic cells; the GP increased in size with age (Plate 1a-c) In 12 wah (June 2005), gonia were first observed within the GP in juveniles and the number increased with age (Table 2; Plate 1d-e) Gonadal walls protruded dorsally and ventrally from the area of blood vessels but did not connect to each other to form the ovarian lumen (Plate 1f) Sex remained undifferentiated in Ud gonads (Table 1) For C altivelis, the paired GP were observed in 10 wah juveniles (January 2007) (Table 3) By 17 wah (February 2007), gonadal walls protruded dorsally and ventrally from the area of blood vessels but did not connect to each other to form the ovarian lumen (Table 3; Plate 1g) Gonia were scattered throughout the length of the paired lobes, and therefore there were no gonia in some sections No ovarian lumen was formed and sex remained undifferentiated in Ud gonads (Table 1) 3.2.2 Ovarian-phase (Ov) gonad There were three ovarian-phase gonads, Ov-1, Ov-2 and Ov-3 (Table 1) For E coioides, Ov-1 with the formation of the ovarian lumen first observed in 16 to 30 wah juveniles; no gonia had entered meiosis and no primary-growth stage oocytes (O1) were visible by 30 wah (Table 2; Plate 2a-b) By 34 wah (November 2004), gonads had developed into Ov-2 with the occurrence of gonial meiosis con- comitantly with the development of O1 (Plate 2c) Ov-2 lasted till 82 wah (October 2005) together with the proliferation and growth of O1, and the development of a lamellar structure (Table 2; Plate 2d) Sex remained undifferentiated Ov-3 with the development of corticalalveolus stage oocytes (O2) and without any sign of spermatogenic cysts was not observed in E coioides (Tables and 2) For C altivelis, all three ovarian-phase gonads were observed (Table 1) Ov-1 gonads were first observed in 22 wah (April 2007) with an ovarian lumen and gonia in mitosis only, and Ov-2 gonads with O1 in 30 wah (June 2007) (Table 3; Plate 2e) Ov-3 gonads with O2 were first observed in 66 wah (May 2007) The development of Ov-3 was from May to August 2007, prior to and overlapping with the female maturation season, suggesting female differentiation and entering maturation (Plate 2f) (see Section 3.2.4) 3.2.3 Bisexual-phase (Bi) gonad There were three bisexual-phase gonads recognized, Bi-1, Bi-2 and Bi-3 (Table 1) For E coioides, Bi-1 with the appearance of scattered spermatogenic cysts among O1 in ovarian lamellae was first observed in 86 wah (November 2005), indicating development from Ov-2 (Table 2; Plate 3a-c) Sex remained undifferentiated In 108 wah (April 2006), Bi-2 with the concomitant appearance of O2 and scattered spermatogenic cysts was first observed (Plate 3d) This phase was observed from April to September 2006, prior to and overlapping with the female maturation season, suggesting female differentiation and maturation via Bi-2 gonads (see Section 3.2.4) In 104 wah (March 2006), around the age of female differentiation, Bi-3 gonads with the concomitant appearance of O1 and sperm sinuses within the gonadal wall, and scattered spermatogenic cysts and proliferation of presumptive spermatogonial cysts, was first observed, with further development of different stages of spermatogenic cysts by 141 wah (December 2006) (Plate 3e-f) This suggested a male differentiation pathway (i.e primary male since develop directly from the juvenile phase) and indicated a relatively long period for male maturation compared to females in E coioides For C altivelis, no bisexual-phase gonads (i.e no concomitant appearance of any developmental stage of spermatogenic cysts and oocytes) were observed (Table 3) 3.2.4 Ovary and testis For E coioides, gonads with vitellogenic stage oocytes (O3), indicating mature/functional females, and scattered spermatogenic cysts, were observed from 113 (around two years old) to 135 wah (May to October 2006) (Tables and 2) GSI% of mature females were 1.0 ± 0.4 (n = 9, mean ± S.D.) and water temperatures at 23.9-32.4 °C The smallest and youngest mature female was obtained in May 2006 at 355 mm SL and 113 wah (Table 2; Plate 4a) Mature, spent, females with atretic O3 were observed from October to December 2006; muscle bundles, noted in other epinephelines as a possible criterion of previous female function, were also found (Plate 4b) (Shapiro et al., 1993; Fennessy and Sadovy, 2002; Liu and Sadovy, 2004a) No mature/ functional testis with sperm in sperm sinuses was obtained by 141 wah (December 2006, end of this study) other than in differentiating males, indicating that first male maturation occurred later than in females (Table 1; Plate 3e-f) Plate Undifferentiated-phase gonads of Epinephelus coioides (a-f) and Cromileptes altivelis (g) All were in paraffin embedding except (d, e) that underwent resin embedding (a) (b) (c) (d) (e) (f) (g) E coioides larvae of 19 mm SL showed paired gonadal primordia (May 2005; wah) Higher magnification of the area in (a) showed blood vessels with somatic cells E coioides juvenile of 31 mm SL showed a gonadal primordium in larger size (June 2005; 12 wah) E coioides juvenile of 29 mm SL showed a single gonium in the right gonadal primordium (June 2005; 12 wah) E coioides juvenile of 72 mm SL showed proliferation of gonia in a gonadal primordium (July 2005; 16 wah) E coioides juvenile of 90 mm SL showed the unconnected ovarian lumen (September 2005; 26 wah) C altivelis juvenile of 71 mm SL showed the unconnected ovarian lumen (February 2007; 17 wah) BV, blood vessel; G, gonium; GP, gonadal primordium; GW, gonadal wall; SB, swimbladder Scale bars = 10 μm except (a) with 50 μm M Liu, Y Sadovy de Mitcheson / Aquaculture 287 (2009) 191–202 For C altivelis, the smallest and youngest mature female with O3 was obtained in August 2007 at 155 mm SL and 83 wah (Tables and 3; Plate 4c) Mature females were noted from June to October 2007 195 with a GSI% of 1.1 ± 0.5 (n = 15) and water temperatures at 24.0-29.5 °C Mature, spent, females with atretic vitellogenic stage oocytes were obtained in October 2007; muscle bundles were also found (Plate 4d) 196 M Liu, Y Sadovy de Mitcheson / Aquaculture 287 (2009) 191–202 Table Gonad development of Cromileptes altivelis during sexual differentiation with age (week after hatching, wah) and gonadal morphology present (+) or absent (−) Age (wah) 10 17 22 30 66 83 Gonadal morphology GP + + − − − − OL − − + + + + GC G O1 O2 O3 SC − + + + + + − − − + + + − − − − + + − − − − − + − − − − − − G, gonium; GC, germ cell; GP, gonadal primordium; O1, primary-growth stage oocyte; O2, cortical-alveolus stage oocyte; O3, vitellogenic stage oocyte; OL, ovarian lumen; SC, spermatogenic cyst Neither differentiating nor mature males were observed by 109 wah (end of this study) In summary, the proposed gonad ontogeny and development during juvenile sexual differentiation and adult sex-change for E coioides and C altivelis were indicated diagrammatically (Tan and Tan, 1974; Quinitio et al., 1997; Davies et al., 2006) (Figs and 3) Discussion Diandric, protogynous hermaphroditism is confirmed for E coioides with evidence of primary male differentiation derived directly from the juvenile phase as determined by gonadal histology (this study) and experimental manipulation of social factors (M Liu, in preparation), with secondary males from adult sex change of mature/functional females (Tan and Tan, 1974; Quinitio et al., 1997) (Fig 2) In this study, females and primary males differentiated around the same age of two years for E coioides Females matured soon after differentiation, but male maturation took longer, at least under mariculture conditions Protogynous hermaphroditism has been proposed for C altivelis based on body size- and age-biased sex ratios, with females smaller and younger than males (Davies et al., 2006) This study shows no evidence for primary male differentiation for C altivelis, suggesting monandric, protogynous hermaphroditism in the species if adult sex-change can be confirmed (Fig 3) In teleost fishes, primordial germ cells (PGCs) arise at extragonadal locations, migrate to the gonadal primordia then increase in number through mitosis (Timmermans, 1996; Braat et al., 1999; Devlin and Nagahama, 2002) In this study, the gonadal primordia (GP) were first observed in wah larvae containing only somatic cells for E coioides Although the migration of PGCs was not observed, the increase in the number of gonia in GP through mitosis was noted in both E coioides and C altivelis in early juvenile stages Gonad development during sexual differentiation is little studied in protogynous hermaphroditic teleosts In gonochoristic teleosts, the initial ovarian ontogeny varies with species and is first indicated either by the appearance of the ovarian lumen or by germ cell meiosis (Nakamura et al., 1998) For Cichlasoma dimerus (Cichlidae), Oryzias latipes (Adrianichthyidae) and Anguilla anguilla (Anguillidae), oogonia enter meiotic division and primary-growth stage oocytes (O1) develop prior to the formation of the ovarian lumen; therefore, the earliest recognizable sign of female differentiation is the occurrence of O1 (Satoh and Egami, 1972; Colombo and Grandi, 1995; Meijide et al., 2005) For Paralichthys olivaceus and P lethostigma (Paralichthyidae), Oreochromis niloticus and O aureus (Cichlidae), on the other hand, meiotic division of oogonia occurs after the formation of the ovarian lumen, which can be used as the earliest recognizable sign of female differentiation (Tanaka, 1987; Nakamura et al., 1998; Luckenbach et al., 2003) For Carassius auratus (Cyprinidae), Cottus bairdii (Cottidae) and O mossambicus (Cichlidae), the formation of the ovarian lumen occurs coincidentally with the appearance of the meiotic division of oogonia (Nakamura et al., 1998) Detailed examination of early gonad development in E coioides and C altivelis revealed, for the first time in the epinephelines, that the meiotic division of gonia and the development of O1 did not occur prior to the formation of the ovarian lumen (i.e Ov-1 and Ov-2), as in gonochoristic P olivaceus, P lethostigma, O niloticus and O aureus Ov-1 and Ov-2 are the gonadal phases that all juveniles pass through during sexual differentiation for E coioides and C altivelis (Figs and 3) Therefore, careful histological examination of gonad ontogeny is important for understanding sexual pattern, and the occurrence of an ovarian lumen in epinepheline males cannot be used as an indicator of a previous functional female phase and hence, sex-change from female to male Bisexual-phase gonads (Bi-1) with scattered spermatogenic cysts and O1 with an ovarian structure have been commonly reported in Plate Ovarian-phase (Ov) gonads of Epinephelus coioides (a-d) and Cromileptes altivelis (e-f) All were embedded in paraffin (a) (b) (c) (d) (e) (f) E coioides juvenile of 79 mm SL showed Ov-1 with the completion of the ovarian lumen and scattered gonia (August 2005; 22 wah) E coioides juvenile of 130 mm SL showed Ov-1 with the completion of the ovarian lumen and proliferation of gonia (October 2004; 30 wah) E coioides juvenile of 180 mm SL showed Ov-2 with gonia in meiosis and the growth of primary-growth stage oocytes (December 2004; 38 wah) E coioides juvenile of 275 mm SL showed Ov-2 with the proliferation of primary-growth stage oocytes (October 2005; 82 wah) C altivelis juvenile of 118 mm SL showed Ov-2 with the proliferation of primary-growth stage oocytes (June 2007; 30 wah) C altivelis differentiating female of 156 mm SL showed Ov-3 with the growth of cortical-alveolus stage oocytes (August 2007; 78 wah) BV, blood vessel; G, gonium; GW, gonadal wall; LA, lamellae; O1, primary-growth stage oocyte; O2, cortical-alveolus stage oocyte; OL, ovarian lumen; PO, gonium in meiotic prophase Scale bars = 50 μm Plate Bisexual-phase (Bi) gonads of Epinephelus coioides All were embedded in paraffin (see plate on page 198) (a) (b) (c) (d) (e) (f) E coioides juvenile of 315 mm SL showed Bi-1 with the concomitant appearance of primary-growth stage oocytes and spermatogenic cysts (November 2005; 86 wah) E coioides juvenile of 208 mm SL showed Bi-1 with the concomitant appearance of primary-growth stage oocytes and spermatogenic cyst (December 2005; 91 wah) E coioides juvenile of 325 mm SL showed Bi-1 with the concomitant appearance of primary-growth stage oocytes and spermatogenic cysts (April 2006; 108 wah) E coioides differentiating female of 335 mm SL showed Bi-2 with the concomitant appearance of cortical-alveolus stage oocytes and spermatogenic cysts (April 2006; 108 wah) E coioides differentiating primary male of 301 mm SL showed Bi-3 with the presence of sperm sinuses (arrows) and the proliferation of presumptive spermatogonia in cysts (March 2006; 108 wah) E coioides differentiating primary male of 368 mm SL showed Bi-3 with the presence of sperm sinuses (arrows) and the proliferation of spermatogenic cysts (December 2006; 141 wah) BV, blood vessel; G, gonium; GW, gonadal wall; LA, lamellae; O1, primary-growth stage oocyte; O2, cortical-alveolus stage oocyte; OL, ovarian lumen; pSG, presumptive spermatogonial cysts; SC, spermatogenic cyst Scale bars = 50 μm M Liu, Y Sadovy de Mitcheson / Aquaculture 287 (2009) 191–202 juveniles in the epinephelines, including Cephalopholis, Epinephelus and Plectropomus species (Smith, 1965; Tan and Tan 1974; Siau, 1994; Sadovy and Colin, 1995; Chan and Sadovy, 2002; Fennessy and Sadovy, 197 2002; Adams, 2003; Liu and Sadovy, 2004a) The developmental significance of Bi-1 was proposed to be the gonadal phase that all juveniles passed through prior to female and male sexual Plate 198 M Liu, Y Sadovy de Mitcheson / Aquaculture 287 (2009) 191–202 Plate (see caption on page 196) M Liu, Y Sadovy de Mitcheson / Aquaculture 287 (2009) 191–202 199 Plate Ovaries of Epinephelus coioides (a-b) and Cromileptes altivelis (c-d) No mature male testis was found for either species All were embedded in paraffin (a) (b) (c) (d) The smallest and youngest mature female of E coioides with 335 mm SL (May 2006; 113 wah) Mature, spent, female of E coioides with 372 mm SL (December 2007; 141 wah) The smallest and youngest mature female of C altivelis with 155 mm SL (August 2007; 83 wah) Mature, spent, female of C altivelis with 205 mm SL (October 2007; 106 wah) aO3, atretic vitellogenic stage oocyte; GW, gonadal wall; MB, muscle bundle; O1, primary- growth stage oocyte; O2, cortical-alveolus stage oocyte; O3, vitellogenic stage oocyte; OL, ovarian lumen; SC, spermatogenic cyst Scale bars = 100 μm differentiation in the bi-directional sex-changer Cephalopholis boenak (Liu and Sadovy, 2004a,b; Sadovy de Mitcheson and Liu, 2008) The pathway of juvenile sexual differentiation via Bi-1 was also found in the diandric E coioides in this study (Fig 2) However, bisexual-phase gonads were not observed in C altivelis during juvenile sexual differentiation, suggesting monandric, protogynous hermaphroditism, if adult sex-change occurs, and underscoring the variation of juvenile sexual differentiation within the epinephelines There is significant value in developing E coioides and C altivelis hatchery and mariculture to increase production of the species for high market demand especially if this reduces pressure from wild populations E coioides is listed on the IUCN Red List as ‘Near Threatened’ due to the large numbers of wild juveniles that are estimated to be caught annually for grow-out mariculture and adult fisheries for food, and mangrove (key juvenile habitat) degradation and loss (Sadovy, 2000; Sadovy et al., 2003; Cornish and HarmelinVivien, 2004) C altivelis is listed as ‘Vulnerable’ due to its natural rarity and, perceived declines in abundance and increase in harvest due to its high value in the live food fish trade (Sadovy et al., 2003; Sadovy et al., 2008) A major constraint for some epinepheline grouper hatcheries is a shortage of males to fertilize females since larger fish (usually males) are difficult to procure from the wild in heavily fished 200 M Liu, Y Sadovy de Mitcheson / Aquaculture 287 (2009) 191–202 Fig Gonad development during juvenile sexual differentiation and adult sex change for diandric, protogynous, hermaphroditic Epinephelus coioides MB, muscle bundle; O1, primary-growth stage oocyte; O2, cortical-alveolus stage oocyte; O3, vitellogenic stage oocyte; OL, ovarian lumen; SC, spermatogenic cyst The arrow indicates the increase of body size and age Three horizontal lines (dash, long dash and dash dot) indicate juvenile sexual differentiation, first sexual maturation and adult sex change, respectively areas because size-selectivity has tended to target larger fish which are often males Broodstock management in grouper hatcheries by obtaining males through androgen hormone manipulation is a commonly used technique (Chen, 1979; Yeh et al., 1986; Chao and Chow, 1990; Liao et al., 2001) However, maleness achieved in this way is often temporary with males changing back to females after the termination of androgen treatment and can increase mortality rates The influence of social factors on juvenile sexual differentiation and adult sex-change has been demonstrated in the epinephelines, and could be applied in broodstock management in grouper hatcheries by obtaining males through social control instead of through hormone manipulation (Quinitio et al., 1997; Okumura, 2001; Mackie, 2003; Liu and Sadovy, 2004b) In C boenak, for example, the number of males differentiating directly from the juvenile phase was influenced by the number of juveniles in a social group (Liu and Sadovy, 2004b) In this species, all isolated, single, juveniles differentiated directly as males, and in social groups of two to four juveniles, all larger/largest individuals were males and the sex ratios of males to females did not differ significantly from 1:1 In the diandric E coioides, one juvenile differentiated as a primary male in pairs, and one or two as primary males in quartets (M Liu, in preparation) As in the diandric, protogynous, hermaphroditic Thalassoma bifasciatum (Labridae), in which one juvenile differentiated as a primary male in groups of three (Munday et al., 2006) These examples suggest an alternative to hormones in male production in such species that could be applied in grouper hatcheries In this study, E coioides and C altivelis were maricultured under natural environmental conditions in floating cages and outdoor tanks, respectively; except during larval and nursery periods when they were cultured in indoor tanks with water temperatures controlled for optimum survival and growth rates Water temperatures showed large variations year-around in this study and reflected weather conditions in Hong Kong waters Both species survived at low water temperatures of 12-14 °C in winter There is evidence showing that E coioides matured younger at age of 90 wah in indoor mariculture conditions with water temperatures above 20 °C year-round (M Liu, in preparation) In this study, the age of first sexual maturation for the youngest C altivelis females was about 86 wah (one and half years old), was half a year younger than previously reported (two years old in Australian waters) (Davies et al., 2006) The M Liu, Y Sadovy de Mitcheson / Aquaculture 287 (2009) 191–202 201 Fig Gonad development during juvenile sexual differentiation and adult sex change for possible monandric, protogynous, hermaphroditic Cromileptes altivelis MB, muscle bundle; O1, primary-growth stage oocyte; O2, cortical alveolus stage oocyte; O3, vitellogenic stage oocyte; OL, ovarian lumen; SC, spermatogenic cyst The arrow indicates the increase of body size and age Three horizontal lines (dash, long dash and dash dot) indicate juvenile sexual differentiation, first sexual maturation and adult sex change, respectively influence of water temperature on sexual maturation of epinephelines under mariculture conditions merits further investigation Acknowledgements We thank Mr James Hui and Ms Rachel Wong (University of Hong Kong, Hong Kong), Prof Irani Quagio-Grassiotto, Mr Antonio Salvador and Mr Rafael Henrique (Universidade Estadual Paulista, Brazil), and Dr Kazue Asoh (Ochanonizu University, Japan) for histological processing, and Dr Harry Grier (Florida Marine Research Institute, USA) and Dr Fabiana Lo Nostro (University of Buenos Aires, Argentina) for gonadal examination We also thank Guangdong Dayawan Fishery Development Center (mainland China), and Mr So Fat Kei, Mr Brian Ho and Mr Michael Templeton (mariculturists, Hong Kong) for providing different stages of groupers We are thankful to Dr Ashley J Williams (James Cook University, Australia) for providing published and non-published materials We are grateful to two anonymous reviewers for constructive comments This 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Size variation within age groups was found in both species, indicating differences in growth rates; growth curves were presented in Fig 3.2 Gonad development during sexual differentiation 3.2.1... first sexual maturation to better elucidate its sexual pattern, and of C altivelis juveniles during sexual differentiation until first sexual maturation to provide information on early sexual development