JOURNAL OF THE Vol. 32, No. 2 WORLD AQUACULTURE SOCIETY June, 2001

Sustained, Natural Spawning of Southern lethostigma Under an Extended Photothermal Regime

WADE0. WATANABEAND PATRICKM. CARROLL The University of North Carolina at Wilmington, Center for Marine Science, 7205 Wrightsville Ave.. Wilmington, North Carolina 28403 USA HARRYV. DANIELS North Carolina State University, Department of Zoology, 207 Research Station Road, Plymouth, North Carolina 27962 USA

Abstract Hormone-induced spawning of has produced substantial numbers of viable eggs, but wide variations in fertilization and hatch rates have been reported. Recently, sustained natural spawning of southern flounder broodstock, without hormone induction, has been achieved in our laboratory. Adults (average weight = 1.12 kg; N = 25), including 6 captured as juveniles in 1993 and 19 captured as adults during Septem- ber 1998, were stocked in two 4.8-m3 controlled-environment tanks in October 1998 and held under natural photothermal conditions until January 1999, when an artificial winter photo- period of 10 L: 14 D was initiated and then maintained through April 1999. Sex ratio was approximately 13 females :8 males :7 unknown. Natural spawning was observed in early December 1998 and increased in frequency to a peak in March 1999, before declining in late April. Water temperature ranged from 13.9 to 24.5 C during the spawning period. Natural spawnings over 142 d produced a total of 18.3 X 1V eggs, with a mean fertilization rate of 28.0% (range = 0-100%), yielding 4.94 x lo6 fertilized eggs. The mean percentage of eggs that remained buoyant in full-strength seawater (34 ppt) was 41.3% (0-98%), while hatching rate of buoyant eggs was 37.3% (0-99%) and survival of yolksac larvae to the first-feeding stage was 30.2% (O-l00%). Gonadal biopsies in late April identified six females from both tanks as probable spawners. A preliminary comparison suggests that natural spawning pro- duced much larger numbers of viable eggs per female, with higher egg quality (i.e., fertiliza- tion and hatching success) than hormone-induced spawning. In contrast to natural spawning, hormone-induced strip-spawning enabled timing of spawnings to be more precisely controlled. These results suggest that a combination of both natural and hormone-induced spawning of photothermally conditioned fish will help produce the large numbers of eggs required to support commercial production.

The southern flounder Paralichthys leth- Interest in the southern flounder as an ostigma is a high-valued recreationally and aquaculture candidate in the southeastern commercially harvested found in es- United States is related to its wide temper- tuarine and shelf waters of the Atlantic and ature and salinity tolerances; 50-d old ju- Gulf coasts of the United States from North veniles can tolerate salinities as low as 5 Carolina to Mexico (Gilbert 1986). With the ppt, while older juveniles can tolerate fresh- implementation of fishery quotas for the sum- water (Smith et al. 1999a). This suggests mer flounder (P. denrutus) in the early 1990s, that this could potentially be culti- landings of southern flounder have increased. vated in inland fresh and brackishwater Today, the southern flounder is the number ponds or recirculating tanks, as well as in one flatfish species landed in North Carolina, coastal areas (Berlinsky et al. 1996; Daniels and management plans are being developed et al. 1996; Jenkins and Smith 1999; Smith to protect this fishery, which is also threat- et al. 1999a). At present, reliable methods ened by overfishing (Copeland et al. 1999). for controlled breeding and production of

8 Copyright by the World Aquaculture Society 2001

153 154 WATANABE ET AL high quality eggs are needed for develop- fort, North Carolina and raised to the adult ment of commercial growout systems. stage in flow-through seawater tanks. A In Japan, where commercial culture of second group (“wild-caught”) was cap- the congeneric flounder species P. olivae- tured as adults by commercial poundnet ceus (Japanese flounder) is well established, fishermen during September 1998 in Pam- commercial hatcheries rely mainly on pho- lico Sound, North Carolina. Wild-caught tothermal conditioning and natural spawn- fish were held in 20-m diameter outdoor ing of Japanese flounder broodstock to sup- concrete tanks supplied with flow through ply the large numbers of eggs needed to brackish water (12-20 ppt salinity) for 6 wk support growout operations and re-stocking before transport to CMS-UNCW. All fish programs (Iijima et al. 1986; Tsujigado et were individually tagged with internal an- al. 1989; Mihelakakis et al. 1995). In the chor tags and held for 3 wk in flow-through United States, natural spawnings of parali- seawater (34 ppt) tanks under ambient con- chthid species (including the southern ditions before stocking into a controlled-en- flounder) have been rare, and culturists vironment broodfish tank system. have therefore focused on hormone-induced The controlled-environment broodfish spawning to produce embryos for research tank system consisted of two outdoor cir- and commercial applications. Intramuscular cular fiberglass tanks (diameter = 2.46 m; implantation of a cholesterol-cellulose pel- depth = 1 m; volume = 4.76 m3). Brood- let containing gonadotropin releasing hor- tanks were insulated and provided with a mone-analogue (GnRHa) has produced re- conical fiberglass cover fitted with a timer- petitive spawning and substantial numbers controlled, fluorescent fixture, containing of viable eggs in southern flounder, but two 20-watt daylight bulbs. Average light wide variations in fertilization and hatch intensity at the water surface was 234 lux. rates have been reported (Berlinsky et al. Both tanks were supported by a common 1996). water-recirculating system, consisting of a Recently, sustained, natural spawning, high-rate sandfilter, fluidized bed biofilter, without hormone induction, of captive foam fractionator, and ultraviolet sterilizer. Southern flounder broodstock was achieved Water from each tank drained through an in photothermally-condtrolled tanks. The egg collector (diameter = 0.76 m; depth = objectives of this paper are to describe the 0.76 m; volume = 0.24 m’) before entering environmental and culture conditions and a reservoir tank (diameter = 1.54 m; depth the reproductive performance associated = 1 m; volume = 1.86 m3), from which with natural spawning. A preliminary com- water was pumped to the biofilter system. parison of natural spawning and hormone- Water flow to each tank was approximately induced spawning is also made. 38 L/min and makeup water was added continuously to the reservoir tank to pro- Materials and Methods vide an exchange rate of approximately 10% per d. Immersion heaters placed in the Natural Spawnings reservoir tank controlled water temperature. This study was conducted at the Center In October 1998, one broodtank (tank I) for Marine Science, University of North was stocked with 13 fish (2.73/m3) consist- Carolina at Wilmington (CMS-UNCW), be- ing of six “laboratory-reared’’ and seven tween December 1998 and April 1999. “wild-caught” adults with an average Adult broodfish southern flounder used in weight of 0.948 kg (2.59 kg/m3)).At stock- this study were obtained from two sources. ing, sex ratio was 7 female: 3 males: 3 un- One group (“laboratory-reared” ) was col- known. In November 1998, the second lected as juveniles at 100-105 mm standard broodtank (tank 2) was stocked with twelve length in the summer of 1993 near Beau- fish (2.52/m3), consisting of only wild- NATURAL SPAWNING OF FLOUNDER 155

-- I

-Temp ( C)

Natural spawning and fefl(lizat!on

caught adults, with an average weight of Canada) and 16% fat. Feeding rates of 1.28 kg (3.23 kg/m3). Sex ratio was un- broodstock averaged about 1 % body weight known at the time of stocking. per d. Gonadal maturity of individual brooders The natural reproductive period of was assessed periodically (23 November Southern flounder in North Carolina waters 1998, 1 I January 1999 and 27 April 1999) is presumed to be January (Berlinsky et al. by biopsy of anesthetized (0.3 g/L 2-phen- 1996). Fish in the outdoor tanks were ex- oxyethanol) fish, using a polyethylene can- posed to ambient light and temperature con- nula (1.57 mm 0.d. X 1.14 mm i.d.) (She- ditions until 9 January 1999. At this time, hadeh et al. 1973; Watanabe et al. 1998). timers were used to maintain a constant Ovarian samples were preserved in a solu- winter photoperiod of 1OL : 14D and heaters tion of 10% formalin in seawater. General maintained water temperature above ap- stage of oocyte development (i.e., pre-vi- proximately 14.5 C (Fig. I). tellogenic, cortical vesicle, vitellogenic, and Egg collectors were checked daily for atretic) was determined from the micro- spawned eggs. Once daily, when spawning scopic appearance, and males were identi- had occurred, eggs were siphoned from the fied by the presence of milt when pressure collector, transferred to a 15-L separatory was applied to the gonadal area. funnel in seawater (32-37 ppt) and buoyant Fish were fed to satiation once daily (ap- eggs (“floaters”) separated from sinking proximately 0900 h), a diet that consisted eggs (“sinkers”). The numbers of eggs in primarily of Atlantic silversides Menidia each fraction were estimated using volu- rnenidia supplemented with small amounts metric methods. of krill and commercially-prepared diets Floaters were transferred to 15-L airlift containing 45% (INVE Aquaculture, “in-tank” incubators placed inside the res- Grantsville, Utah, USA) and 55% protein ervoir tank or situated in an indoor labo- (Corey Feed Mills Ltd., New Brunswick, ratory. In-tank incubators were stocked at a 156 WATANABE ET AL density of 1,000 egg& while indoor in- (1999 and 2000). Adult southern flounder cubators were stocked at densities of 300- (average weight 1.2 kg) were captured by 600 eggs/L. Indoor incubators were provid- commercial poundnet fishermen during ed with 1-pm-mesh filtered seawater (ster- September 1998 in Pamlico Sound, North ilized by ultraviolet light) at 16-19 C and Carolina. Fish were stocked in September supplied with diffused aeration. Using vol- 1998 into tanks (diameter = 3.1 m., depth umetric methods, survival of embryos was = 1.0 m; volume = 7.4 m3) supplied with estimated at time of hatching (d2-d3 post- recirculating seawater (34 ppt). Broodfish fertilization) and at the first-feeding stage were exposed to artificial photoperiod and (d6-d7 post-hatching), when 100% of the temperature conditions simulating ambient, larvae possessed functional (fully pigment- reaching 9 h L:15 h D and 16 C by 15 ed) eyes, mouth, and alimentary tract. December and gradually increasing to 15 h Fertilization rate was determined as the L : 9 h D and 25 C by 2 1 June. Beginning percentage of eggs undergoing normal em- in January 1999, females with maximum bryonic development, while hatching rate oocyte diameters of 500 pm were selected was determined as the percentage of larvae for hormone-induced spawning. To induce hatched from fertilized eggs. Fertilization spawning, females were implanted with a rates and hatching rates were expressed as 95% cholesterol and 5% cellulose pellet percentages of total eggs and of buoyant (Sherwood et al. 1988) containing [D-Ala6 eggs. Survival to the first-feeding stage was Des-Gly’O] LHRH ethylamide (GnRH-a, expressed as a percentage of total and of Sigma Chemical Co., St. Louis, Missouri, buoyant eggs. Data from each tank were USA) at a dose of 100 pgkg (Berlinsky et maintained separately. al. 1996). Following hormone treatment, females Water Qualiry were either allowed to spawn volitionally in Temperature, salinity, and dissolved ox- the tanks (“tank spawning”), or spawned ygen were monitored in broodtanks daily, by applying manual pressure to the abdo- while pH, total ammonia-nitrogen, nitrite, men of ovulated females (“strip-spawn- and nitrate were monitored weekly. Mean ing”). Before strip-spawning, ovulation daily values (and ranges) were as follows: was first determined by the release of small salinity, 35.2 (32-37) ppt; dissolved oxy- amounts of eggs upon gentle abdominal gen, 7.64 (6.01-9.01) mgL; pH 8.12 (7.8- pressure (“squeeze check”). Eggs from a 8.4); total ammonia-nitrogen, 0.029 (0- single female were collected in a glass bea- 0.08) mg/L, nitrite-nitrogen, 0.022 (0.003- ker and mixed with the sperm from two 0.05 1) mgL; nitrate-nitrogen, 2.75 (0.6- males (Berlinsky et al. 1996), then left un- 9.4) mgL. Temperature, salinity, dissolved disturbed in at least 100 mL of seawater for oxygen and pH in the incubators were also 1 h. The floating eggs were separated from monitored once at the end of the incubation the sinking eggs in a 1-L separatory funnel. period. Average values (and ranges) were Embryos were incubated in a 70-L round as follows: salinity, 35.5 (34-38) ppt; dis- fiberglass tank containing 34 ppt filtered solved oxygen, 8.50 (7.59-9.27) mg/L; pH seawater at 16 C and at a maximum density 8.36 (8.2-8.5); temperature 17.4 (16-18.6) of 500 eggs/L. Fertilization and hatching C. rates were determined as described above. In 2000, an alternative method was used Hormone-Induced Spawning to monitor ovulation. Under this “light ta- Hormone-induced spawning trials were ble” method, developed by commercial conducted at the Tidewater Research Sta- aquaculturists (George Nardi, Great Bay tion (North Carolina State University) in Aquafarms, personal communication), the Plymouth, North Carolina over two seasons potential spawner was placed on a clear NATURAL SPAWNING OF FLOUNDER 157

Tank 1 (Iabontory-ruared and wildcaught broodatock)

3-DBC-98 17.Dec.98 31-Oac-98 1CJ.n-88 28-Jan-% 11-Feb-98 25Fob-9( 11 Mar-98 21Mar-98 8 Date FIGURE2. Total eggs production per day during natural spawning of laboratory-reared and wild-caught brood- stock) southern JIounder broodstock in a 4.76-m’ tank. Height of each bar represents numbers of fertilized and unfertilized eggs. while black portions of each bar represent numbers of fertilized eggs. plexiglass table illuminated from below by ceased through the remainder of December. an incandescent bulb, allowing light to be On 4 January 1999 spawning resumed in transmitted through the fish. Because of the tank 1 and increased in frequency to a peak flattened body conformation of flounder, in March 1999 before declining in mid- size and location of the gonads were visu- April (Fig. 2). Fertilized eggs were first ob- alized through the body wall. Females in served on 12 January in tank 1 (Fig. 2). which portions of the large ovarian mass In tank 2, which consisted entirely of turned from dark to translucent, due to in- wild-caught adults, onset of natural spawn- tra-ovarian oocyte hydration and ovulation, ing was delayed, with unfertilized eggs first were selected for strip spawning as de- observed in the egg collector on 5 February scribed above. 1999 (Fig. 3). Natural spawning in tank 2 increased in frequency to a peak in late Results March and early April before declining in Natural Spawnings late April. In tank 2, fertilized eggs were Gonadal biopsy on 23 November 1998 first observed on 2 March. revealed a few individuals with late yolk When data from both tanks 1 and 2 are globule stage eggs with mean oocyte di- considered (Fig. 4), consistent spawning be- ameters ranging from 437-445 pm and run- gan in early January, increased in frequency ning ripe males. On 3-5 December 1998, during the month of February to a peak in fully hydrated ova were first observed in March before declining to a low level by the egg collector from tank 1, consisting of late April. During a 142-d spawning period both laboratory-reared and wild-caught from 3 December 1998 to 23 April 1999, adults. Since none of these individuals had eggs were collected on 70 d in tank 1 (Fig. been treated with hormones, natural, voli- 2) and on 53 d in tank 2 (Fig. 3; Table 1). tional spawning had occurred. From 99,600 Total numbers of eggs collected per d from to 253,700 unfertilized eggs were released each tank ranged from 5,490 to 601,250, daily from 3-5 December, but spawning averaging 159,596 in tank 1 and 133,104 in 158 WATANABE ET AL.

Tank 2 (wildcaught broodatock)

3-Doc-88 17-k-88 31-Dsc-88 14.J.n-89 2EJm-88 11-Fek-88 25Feb-m 11-Mar.88 25M.r-88 0-Apr-88 22-Apr-88 Date FIGURE3. Total eggs production per day during natural spawning of wild-caught broodstock southern flounder broodstock in a 4.76-m' tank. Height of each bar represents numbers of fertilized and unfertilized eggs, while black portions of each bar represent numbers of fertilized eggs. tank 2. For the duration of the 1424 18,385,808 eggs from both tanks and for an spawning period, a total of 11,331,319 eggs average of 129,477 eggs per d (Table 1). As were collected from tank 1 and 7,054,489 evidenced by the various stages of embry- were collected from tank 2, for a total of onic development at the time of collection,

Tank 1 + Tank 2

Zwow

low00

0 3-Dec-88 17.De~88 31-DM-88 1CJ.n-88 2EJ.n-88 11-Fsb-88 2bFeb.88 11-Mar-88 2wSr-88 8-Apr-88 22-Apr4e Date FIGURE4. Total eggs production per day for both tank I (laboratory-reared and wild-caught broodstock) and tank 2 (wild-caught broodstock) during natural spawning of southern flounder broodstock in two, 4.76-m' tanks. Height of each bar represents numbers of fertilized and unfertilized eggs, while black portions of each bar represent numbers of fertilized eggs. NATURAL SPAWNING OF FLOUNDER 159 spawning occurred at all times of day, with coloration of the egg cytoplasm around the no diurnal pattern evident. central region (Fig. 60. This necrotic pro- cess was associated with a loss of buoyan- Egg Buoyancv, Fertilization, and Hutching CY. Fertilization rates, expressed as a per- For both tanks 1 and 2, fertilization rates centage of total naturally spawned eggs, of floaters averaged 61.8% (Table 1). varied from day to day from 0 to 97.2% Hatching rate averaged 37.3% of floaters (Fig. 4), averaging 30.6% in tank 1 and (15.4% overall). Survival of yolksac larvae 24.9% in tank 2, with an overall average of to the first-feeding stage averaged 30.2% of 28.0% for both tanks (Fig. 2; Table 1). A floaters (12.5% overall) for both tanks, pro- total of 3,470,5 16 fertilized eggs were pro- ducing a total of 2,397,206 first-feeding duced in tank 1, while 1,472,577 were pro- stage larvae. duced in tank 2, for a total of 4,943,092 for both tanks combined. Availability of fertil- Temperature Considerations ized eggs from both tanks, however, gen- At the time of the first spawnings from erally increased to a maximum in March 3-5 December 1998, water temperature av- and early April (Fig. 4). On days that eraged 20.1-21.2 C, although no fertilized spawning was observed, an average of eggs were obtained during this period. No 34,811 fertilized eggs were collected from spawnings were observed as water temper- each tank. atures declined sharply during the month of During incubation of eggs in full- December, reaching a minimum of 10.9 C strength seawater (32-36 ppt), the percent- on 27 December. From 5 January to 23 age of total eggs spawned that remained April, the period during which the majority buoyant (i.e.,"floaters") varied among of spawnings occurred, water temperatures spawnings from 0 to 99.2%, with an aver- varied over a wide range of 13.9 C to 24.5 age of 41.3% for both tanks (Table 1). C (Fig. 1). Fertilized eggs were also ob- When all egg batches for both tanks were tained under this temperature range, al- considered, a significant (P < 0.01) linear though availability of fertilized eggs in- relationship between percentage egg buoy- creased to a peak during March and early ancy and overall fertilization rate was evi- April, while water temperature averaged dent (Fig. 5a), with 61.3 % of the variance around 17.5 C. Numbers of eggs spawned in buoyancy rate accounted for by the var- decreased as water temperatures increased iation in the fertilization rate of the eggs. steadily in April. Spawning appeared to be However, when only eggs for which rate of stimulated by a change in weather condi- buoyancy was determined in the later gas- tions; a warming or cooling trend was fol- trula and neurula stages were considered lowed by an increase in egg release. (Fig. 5b), a higher percentage (81.5%) of the variance of buoyancy rate was account- Growth and Sex Ratios of Broodstock ed for by the variation of the fertilization A gonadal examination of all broodstock rate of the eggs (Fig. 5b). This was related made on 11 January 1999 revealed the fol- in part to the fact that, in some egg batches lowing sex ratios: 8 females : 3 males : 2 un- in which fertilization was determined dur- known in tank I and 5 females : 2 males : 5 ing the early cleavage stages, development unknown in tank 2. Males were identified was arrested prior to the blastula stage. by the presence of running milt. A gonadal These eggs were characterized by irregular examination of all broodstock made on 27 cleavage during early cell division (Figs. April 1999 revealed four individuals in tank 6b, d) and by a darkening of the germ cells 1 and two individuals in tank 2 with atretic, during the pre-blastula stage (Fig. 6f), fol- hydrated eggs, suggesting probable spawn- lowed a short time later by a brownish dis- ers. Assuming that these six individuals 160 WATANABE ET AL.

TABLE1. Summarized data on natural spawning of southern flounder broodstock in two 4.76-m3 tanks (3 Dee. 1998 to 23 April 2000). Each tank was stocked with 12-13 adults. Data are presented for each tank and for both tanks combined.

Fertilization No. of days Fertilization rate (% of eggs Total eggs spawned No. of floaters Floaters rate (% overall) floaters) Tank no. observed (no. spawned per day) (no. per day) (%) (range) (range) (range)

~ ~ 1 70 I133 1.3 19 5,737,902 46.6 30.6 61.3 (5.490-601,250) (0-412.750) (0-97.5) (0-99.0) (0-100) 2 53 7,054,489 2,199,867 34.2 24.9 62.3 (22,750-4 19,000) (0-244,Ooo) (0-96.5) (0-95.2) (0-100) 1 t- 2 123 18,385,808 7.937,769) 41.3 28.0 61.8

I Estimated as hatching rate (% overall) = floaters (Yo) X hatching rate (% of floaters). Estimated as survival to first-feeding (% overall) = floaters (%) X survival to first-feeding (% of floaters). participated in spawning, an average of od” to monitor ovulation produced 3,064,301 eggs was released per female 1,658,000 eggs, of which 466,000 (28%) (approximately 2,760,632kg) during the were floaters, with 61% of these floaters study period. fertilized (19% overall). Hatching rate of During the period 11 January to 27 April, floaters averaged 38%. average weight and biomass density of fish For both seasons (1 999-2000) combined, in both tanks increased slightly from 1.09 57 hormone-induced spawning trials pro- kg and 1.86 kg/m3 to 1.12 kg and 2.94 kg/ duced 2,759,000 eggs, of which 1,170,000 m3, while average TL remained unchanged (42%) were floaters, with 40% of these at 45.3 cm. On 27 April, males averaged floaters fertilized (17% overall). Hatching 43.9 cm and 1,009 g, not significantly dif- rate of floaters averaged 26%. ferent from females, which averaged 45.1 cm and 1,134 g. Discussion Hormone-Induced Spawnings In this study, commercially significant In 1999, 14 tank spawnings following quantities (i.e., 4.94 X lo6) of viable em- hormone induction produced 345,000 eggs, bryos of southern flounder were produced of which 55% were floaters, but no fertil- through sustained, natural spawning of cap- ization was obtained under this method (Ta- tive broodstock. Previous attempts to obtain ble 2). Seventeen strip-spawning trials us- natural spawning and fertilization of this ing the “squeeze check method” to monitor species had limited success. Using photo- ovulation yielded 756,000 eggs, of which periods and temperatures which simulated 68% were floaters, with 40% of these float- natural seasonal changes, Arnold et al. ers fertilized (27% overall). Hatching rate (1977) obtained natural spawning from 3 of of floaters averaged 30%. Of the 17 strip- 6 females for 13 consecutive days in 30-m3 spawning trials, only five produced fertil- tanks producing 120,000 eggs with a fertil- ization rates of >70%. These spawns were ization rate of 30-50% and a hatching rate associated with relatively high rates of of 6-35% of fertilized eggs. In a later study buoyancy (78%), fertilization (80% of float- (Henderson-Arzapalo et al. 1988), con- ers, 62.4% overall) and hatching rate (77% trolled photoperiod and temperature were of floaters). also effective in stimulating release of In 2000, 26 hormone-induced strip- 200,000 eggs from 5 females over 2 spawn- spawning trials using the ”light table meth- ing seasons, but no fertilization was ob- NATURAL SPAWNING OF FLOUNDER 161

TABLEI. Extended. 100 ,- n

60 50 Survival to first- Survival ooeo O 20 0 No. of Hatching feeding to first- 2 10 fertilized rate (% of Hatching stage feeding 0 eggs floaters) rate (% (%of stage (% c 0 20 40 60 80 100 (range) (range) overall)' floaters) overall)? 3,470,516 41.9 19.5 34.2 15.9 (0-177.300) (0-87.1) (0-100) 100 r 1,472,577 31.9 10.9 26.6 9.1 0 90 . (0-233.996) (0-99.1) (0-84. I ) 80 . B 4,943,092 37.3 15.4 30.2 12.5 70 . 60 . 50 . 40 . tained due to lack of male participation in spawning. In this study, an average of 3,064,301 0 20 40 60 80 100 eggs was released per female (2,760,632/ Buoyancy (%) kg) during the study period, comparable to what Smith et al. (1999b) reported for the FIGURE5. Relationship between overall fertilization rate (y, %) and egg buoyancy (x, %)for naturally- southern flounder under hormone-induced spawned eggs of southern flounder. When data for tank spawning (5,927,333/female; all egg batches (N = 116) were used (Fig. 5A), re- 1,924,459kg). This is also similar to what gression analysis defined this relationship as fol- was reported for the Chilean flounder P. mi- lows: y = 0.746 x - 3.2314 (R = 0.6127: P < crops, which produced an average of 0.01). When only data for which buoytrncy was de- termined during the gastrula through neurula stages 2,400,000 eggs per kg female during natu- (N = 32) were used (Fig. 5B),this relationship was ral spawning in tanks. These fecundity lev- dejined as follows: y = 1.0727 .X - 14.396 (R2 = els are low when compared to the Japanese 0.815, P < 0.001). flounder, which produce up to 25 million eggs per female over a 6-mo period (Smith et al. 1999b). Relatively large numbers of stock held in captivity for at least 1.5 yr and southern flounder broodstock may be re- receiving photothermal conditioning for a quired by commercial hatcheries to achieve minimum of 12 wk prior to spawning equivalent levels of production. (Smith et al. 1999b). The results of this study demonstrate that The natural spawning period for southern wild-caught southern flounder adults, con- flounder is believed to be December and ditioned through photothermal manipula- January (Henderson-Arzapalo 1988; Ber- tion for only 6 mo, can be spawned suc- linksy et al. 1996; Smith et al. 1999b). In cessfully without hormone induction during this study, viable embryos were produced their first season in captivity. This is im- from January through late April 1999, in- portant to avoid a prolonged period of ac- dicating that the extended winter photope- climation to captivity. In wild-caught riod regime was effective in prolonging the Scopthalmus maximus, efficient spawning spawning season by at least 2 mo beyond occurred only after 2 yr of habituation to the natural spawning period for this species. captivity (Devauchelle et al. 1988). In Using a combination of photothermal con- southern flounder, only females >2 kg ditioning and GnRHa implants, southern spawned naturally in captivity (Arnold et al. flounder broodstock spawned volitionally in 1977), and successful hormone-induced tanks over an extended period of 99 d from tank spawning was attained with brood- January to late April (Smith et al. 1999b). 162 WATANABE ET AL.

FIGURE6. Normal (left side) and abnormal embryos (righi side) of southern flounder during the 8-cell stage (A,B), 32-cell stage (B.C), and mulii-cell stage (D,E). Necrotic eggs at the multi-cell stage were characterized by (I durkening of the germ cells (E, botiom left)followed by a gradual brownish discoloring of the cytoplasm fE, top).

Sustained, natural spawning and fertiliza- dence of disease throughout the study pe- tion of southern flounder in this study was riod. In addition to the simulated winter probably promoted by a number of inter- photothermal conditions, which maintained acting factors. Fish were received from fish- fish in a reproductive state from December ermen in good health, and water quality pa- through April, other factors such as tank rameters in broodtanks were maintained size, color, and low illumination levels ap- within optimal ranges throughout the study. parently minimized stress and were condu- Fish fed well and showed little or no evi- cive to natural spawning. The results dem- NATURAL SPAWNING OF FLOUNDER 163

TABLE2. Summarized data on hormone-induced spawnings of southern flounder (Paralichthys lethostigma) during the I999 and 2000 seasons, including eggs collected following tank and strip spawnings. Average percent hatch (range) was determined 12-24 h after jrsr observed hatch for floating eggs only.

Fertilization Fertilization No. Hatching Method No. Total eggs Floaters rate rate (% of fertilized rate (% of Yr of spawning trials spawned Floaters (%) (% overall) floaters) eggs floaters) 99 Tank 14 345,000 190,000 55 0 0 00 Strip’ 17 756,000 514,000 68 27 40 206,000 30 00 Strip2 26 1,658,000 466,000 28 19 61 284,000 38 99-00 Tank or strip 57 2,759,000 1,170,000 42 17 40 490,000 26 ’ Ovulation determined by “squeeze check”. * Ovulation determined using a light table. onstrated that, under proper conditions, ings of 12 females over a 2-wk period, but wild-caught southern flounder adults can be fertilization rates varied considerably (7- spawned successfully, without hormone in- 95%) between females and between spawns duction, during their first season in captiv- from individual fish. As an alternative to ity. strip-spawning, photothermal conditioning In this study, a temporary cessation of coupled with GnRHa implants have also re- spawning throughout the latter part of De- sulted in successful tank-spawning of cember was likely related to the inhibitory southern flounder (Smith et al. 1999b). effects of low water temperature, which fell From a broodstock consisting of 3 females, to 10.9 C in late December. This is consis- these workers collected eggs on 64 d during tent with an earlier report that a decrease in a 99-d period. On days that spawning oc- water temperature from 17 to 14 C inhibited curred, mean number of eggs collected was spawning in southern flounder, which re- 277,844 for a total of 17,782,000 of which sumed when temperature were returned to 32.8% were fertilized (range = 0-82%), al- 17 C (Smith et al. 1999b). Based on the though survival through hatching was not observation in this study that egg release reported. A combination of photothermal increased following a change in weather conditioning and GnRHa implants appar- conditions, we hypothesize that pulsatile ently reduced stress and resulted in higher temperature conditions from January egg production and spawning over an ex- through April may have been an important tended period (Smith et al. 1999b). stimulus for sustained natural spawning. Results of this study demonstrate that Gentle raising or lowering of water tem- photothermal conditioning of southern perature stimulates spawning in red drum flounder can produce natural spawning, (Roberts 1990). No clear diurnal pattern of without the use of hormones, resulting in spawning periodicity could be discerned. In relatively high spawning success in terms turbot, ovulatory spawning rhythms of total egg production, egg quality and the showed a distinct diurnal rhythmicity which duration of the spawning period. Natural was constant for any one female, but varied spawning of southern flounder broodstock among females (McEvoy 1984). produced a total of 18.4 million eggs, from Sustained-release GnRH-a pellet im- 6 females, with an overall fertilization rate plants are highly effective in inducing ovu- of 28% and a hatching rate of 37.3% of lation of successive batches of eggs in the floaters. In comparison, 57 hormone-in- southern flounder, allowing repetitive strip- duced spawning trials produced 2,759,000 spawning (Berlinksy et al. 1996). These eggs, with lower fertilization and hatching workers produced substantial numbers (1.6 rates of 17% overall and 26% of floaters, X lo6) of eggs from multiple strip-spawn- respectively. While spawning success var- 164 WATANAEE ET AL ied widely under both natural and hormone- (6.5-15.7 C), but highest spawning success induced spawning, the data suggest that nat- was attained at 10 C during the end of ga- ural spawnings generally resulted in higher metogenesis, with no fertilization obtained egg quality. Natural spawning was also at high temperatures within this range (De- much less labor intensive and time consum- vauchelle et al. 1988). In Japanese flounder, ing than hormone-induced spawning, pro- spawning was obtained over a 113-d period ducing large numbers of viable embryos from February to June during which water with little or no handling of broodstock. temperature showed a general increase from Higher egg quality under natural spawn- 9.1 to 19.8 C and was correlated with a de- ing (as opposed to hand-stripping) has also cline in egg size (Mihelakakis et al. 1995). been reported in turbot (Bromley et al. Studies are needed to determine the effects 1986) and was attributed to reduced stress of thermal regime on vitellogenesis, spawn- and risk of physical damage to brooders ing, and egg quality in southern flounder. when allowed to spawn naturally. In addi- In this study, egg batches with high fer- tion, proper timing of strip-spawning in re- tilization and hatching rates were character- lation to ovulation markedly influences egg ized by transparent, spherical eggs with a yield, fertilization and hatching rates in flat- single oil droplet, regular cell cleavages and fish and other marine batch spawners high buoyancy. Non-viable (albeit fertil- (Kjorsvik and Holmefjord 1995). Strip- ized) eggs showed irregular cell cleavages spawning can disturb the ovulation process with development stopping by the blastula when performed prematurely, cause a rapid stage, and with necrotic eggs showing a dis- loss of egg viability inside ovary after ovu- coloring of the cytoplasm and a loss of lation if stripping is delayed (overripening), buoyancy. These characteristics are identi- or be subject to low milt production and cal to those reported in non-viable turbot quality in males (McEvoy 1984; Devau- eggs (Devauchelle et al. 1988). Addition- chelle et al. 1988; Bromage 1995; Berlinksy ally, under both natural and hormone-in- et al. 1996). duced spawning, some batches of eggs were Even under natural spawning, consider- buoyant, but non-fertile. For example, of 3 1 able variability in egg quality was observed hormone-induced spawning trials in 1999, in this study, with an average of 62% of 14 tank spawnings yielded 345,000 eggs, eggs being non-viable. Production of rela- 55% of which were buoyant, but unfertil- tively large proportions of non-viable eggs ized (Table 2). Buoyancy has been used by has been observed during natural spawning some workers as an indirect measure of fer- of other flatfish. In turbot, 53-66% of nat- tilization success. Present results show that, urally spawned eggs were non-viable since high rates of buoyancy can occur in (Bromley et al. 1986) and in spawning unfertilized eggs, as well as eggs of poor in tanks, 49% of the eggs produced were viability, buoyancy may not necessarily not fertilized (Houghton et al. 1985). In provide a good indication of egg viability Chilean flounder, 67-92% of naturally if determined prior to the blastula stage of spawned eggs were non-fertile (Silva development. In , fertilization rates 1994). In Japanese flounder, proportions of were not reduced, but hatching rates were non-viable eggs collected during the spawn- significantly lowered when eggs were ing season varied from 47-93% (Tsujigado stripped and fertilized prematurely (Kjors- et al. 1989; Mihelakakis et al. 1995). vik and Holmefjord 1995). In this study, temperature regime during While natural spawning minimizes labor, the 142-d period of natural spawning varied handling stress and may improve egg qual- over a relatively broad range of approxi- ity, reliability and control are of great con- mately 15 to 24.5 C. In turbot, vitellogen- cern to the commercial culturist. Hormone- esis occurred over a wide temperature range induced spawning, on the other hand, en- NATURAL SPAWNING OF FLOUNDER 165 ables spawnings to be timed to suit the hancement in North Carolina. North Carolina Sea needs of the culturist. Culturists may there- Grant, North Carolina State University, Raleigh, fore opt to compensate for lower egg qual- North Carolina, USA. Daniels, H. V, D. L. Berlinsky, R. G. Hodson, and ity by hormone-induced strip-spawning of C. V. Sullivan. 1996. Effects of stocking density, multiple females concurrently. A combina- salinity and light intensity on growth and survival tion of both GnRHa-induced and natural of southern flounder Puralichthys lethostigma lar- spawnings of photothermally-conditioned vae. Journal World Aquaculture Society 27: 153- fish may help supply the large numbers of 159. eggs necessary to support commercial Devauchelle, N., J. C. Alexandre, N. Le Corre, and Y. Letty. 1988. Spawning of turbot (Scopthulrnus hatcheries. To improve and minimize vari- maximus) in captivity. Aquaculture 69: 159-1 84. ability in egg quality, additional research is Gilbert, C. R. 1986. Species profiles: Life histories needed on hormone-induced spermiation in and environmental requirements of coastal fishes males, and on hormonal, nutritional and en- and invertebrates (south Florida)-southem, gulf vironmental effects on egg quality. and summer . Biological Report 82 (1 1.54). US. Fish and Wildlife Service, Washing- Acknowledgments ton, D.C., USA. Henderson-Arzapalo, A., R. L. Colura, and A. F. We thank Dr. John Burke for donating Maciorowski. 1988. Temperature and photoperi- “laboratory-reared’’ southern flounder od induced maturation of southern flounder. Man- broodstock, and Joanne Harcke, Christo- agement Data Series No. 154, Texas Parks and Wildlife Department. Austin, Texas, USA. pher Woolridge, Andrew Rhyne, and Todd Houghton, R. G., J. M. Last, and P. J. Bromley. Guerdat for technical assistance and Dr. 1985. Fecundity and egg size of sole [Solea solea Ron Hodson and Mr. Robert Wicklund for (L.)]spawning in captivity. Journal du Conseil, helpful advice. This research was supported Conseil International Exploration de la Mer, 42: by the U.S. Department of Agriculture, Co- 162-165. operative State Research Education and Ex- Ijima, T., T. Abe, R. Hirakawa, and Y. Torishima. 1986. Early spawning of Japanese flounder Par- tension Service (Grant No. 98-38854-6009) alichrhys olivaceus with elongated day treatment. and by the North Carolina Sea Grant (Grant Saibai-giken, 1557-62. Nos. RMG9804 and NA86R003). Jenkins, W. and T. I. J. Smith. 1999. Pond nursery production of southern flounder (Paralichthys Literature Cited lethostigma) and weaning to commercial diets. Aquaculture 176:173-1 80. Arnold, C. R., T. D. Williams, A. Johnson, W. H. Kjorsvik, E. and I. Holmefjord. 1995. Atlantic hali- Bailey, and J. L. Lasswell. 1977. Laboratory but ( hippoglossus) and (Gadus spawning and larval rearing of red drum and morhua). Pages 169-196 in N. R. Bromage and southern flounder. Proceedings Southeastern As- R. J. Roberts, editors. 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Photoperiod/temperdture Science Ltd., Oxford, UK. control in the commercial production of red drum Bromley, P. J., P. A. Sykes, and B. R. Howell. 1986. (Sciaenops ocellutus) eggs. Pages 35-43 in G. W. Egg production of turbot (Scophthalmus maximus Chamberlain, R. J. Miget and M. G. Haby, editors. L.) spawning in tank conditions. Aquaculture 53: Red Drum Aquaculture, Texas A&M University 287-293. Sea Grant College Program No. TAMU-SG-90- Copeland, B. J., J. Miller, and E. B. Waters. 1999. 603, Texas A&M University, College Station, The potential for flounder and red drum stock en- Texas, USA. 166 WATANABE ET AL.

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