Characteristics of spawning behaviour in cultured greenback flounder Rhombosolea tapirina

Aquaculture 253 (2006) 279 – 289 www.elsevier.com/locate/aqua-online

Characteristics of spawning behaviour in cultured greenback flounder Rhombosolea tapirina N.W. PankhurstT, Q.P. Fitzgibbon1 School of Aquaculture, Tasmanian Aquaculture and Fisheries Institute, University of Tasmania, Locked Bag 1340, Launceston, Tasmania 7259, Australia Received 20 September 2004; received in revised form 7 January 2005; accepted 11 January 2005

Abstract Cultured stocks of 2nd generation 2+ year old greenback flounder Rhombosolea tapirina were maintained at low density and under conditions of minimal disturbance over 4 reproductive seasons. Natural in-tank spawning began in the second year after introduction in 2 separate tank systems and continued for 3 successive seasons until the experiment was terminated. Video records showed spawning on 5 out of 34 days on which filming occurred, with spawning behaviour consisting of approach and courting of an ovulated female by a male, followed by a circular paired swim in mid-water culminating in egg release. Spawning was concentrated in austral winter and spring in all 3 study years. In the first year, spawning episodes were strongly correlated with lunar phase (new moon), whereas in years 2 and 3, initiation of substantive spawning for the season coincided with the new moon but there was little evidence of lunar synchronisation thereafter. Volumes of eggs produced and egg fertility were highly variable and not clearly related to season. Most spawning events occurred between midnight and 07:30 h, with the majority between 04:00 and 06:00 h (~2 h before sunrise). The results of the present study further emphasize the utility of low disturbance maintenance for the development of naturally spawning cultured broodstock, and suggest that wild stocks are likely to show pre-dawn spawning in winter and spring, possibly synchronised to lunar phase. D 2005 Published by Elsevier B.V. Keywords: Greenback flounder; Spawning behaviour; Spawning frequency; Egg fertility

1. Introduction

T Corresponding author. Present address: Faculty of Science, Engineering and IT, James Cook University, Townsville, Queensland 4811, Australia. Tel.: +61 7 4781 5228; fax: +61 7 4781 6844. E-mail address: [email protected] (N.W. Pankhurst). 1 Present address: School of Earth and Environmental Sciences, University of Adelaide, Adelaide, South Australia 5005, Australia. 0044-8486/$ - see front matter D 2005 Published by Elsevier B.V. doi:10.1016/j.aquaculture.2005.01.014

Domestication of new fish species for aquaculture requires development of strategies for the maintenance of broodstock so that they will successfully mature and produce viable gametes. Captive fish may mature and ovulate spontaneously; however, more commonly there is the need for some form of

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hormonal intervention to stimulate oocyte maturation and ovulation, particularly among fish collected from the wild (reviewed in Pankhurst, 1998; Zohar and Mylonas, 2001). Subsequent generations may require less intervention, with cultured stocks typically having less severe responses to husbandry stress and greater likelihood of maturing spontaneously (Cleary et al., 2000). It is less common for fish to spawn in captivity even where oocyte maturation and ovulation occur in un-manipulated fish, with spawning generally requiring duplication of some critical physical or social factor from the natural environment (Pankhurst, 1998; Zohar and Mylonas, 2001). There does appear to be some scope for endocrine manipulation of spawning behaviour with treatment of fish from a number of species with slow release forms of gonadotropin-releasing hormone analogue (GnRHa) resulting in stimulation of both ovulation and spawning (reviewed in Zohar and Mylonas, 2001). In many species; however, hormonal treatment stimulates ovulation without subsequent spawning. This appears to be the case in most flatfishes where fish ovulate but do not spawn in response to GnRHa or gonadotropin preparations (Berlinsky et al., 1996; Larsson et al., 1997; Manning and Crim, 1998; Watanabe et al., 1998; Mugnier et al., 2000). The greenback flounder Rhombosolea tapirina is currently under pilot culture in Tasmania and the early stages of domestication have been achieved. Initial culture depended on hormonal induction of ovulation and spermiation (e.g. Pankhurst and Poortenaar, 2000; Poortenaar and Pankhurst, 2000); however, subsequent cultured generations showed a consistent capacity to ovulate and spermiate spontaneously (Sun and Pankhurst, 2004). Despite this, and in common with other flatfish species, fish held in 1–4 m3 tanks failed to show spawning behaviour or spontaneous egg release resulting in serial mortality of ovulated but unspawned females (authors’ unpublished data). Anecdotal reports from pilot commercial culture suggested that mortality associated with retention of unspawned eggs can also be a significant cause of mortality under commercial culture conditions. An additional problem associated with failure to spawn is the loss of viability that occurs in eggs retained in the oviduct beyond a certain period of post-ovulatory viability, which in marine species is typically less than 10 h (Hobby and Pankhurst, 1997).

A similar situation appears to exist in greenback flounder where fertility is typically very low if eggs are stripped and manually fertilized any substantial time after ovulation (authors’ unpublished data). Collection of naturally spawned eggs might be predicted to obviate this problem. In the present study, cultured greenback flounder were held at lower density than in our typical maintenance regimes and left undisturbed other than for routine husbandry, over 4 reproductive seasons. Occurrence and frequency of spawning were recorded on the basis of presence of eggs in egg collectors and the timing of spawning was determined by direct video observation, and the estimated age of collected eggs. Spawning behaviour was described from video records. This study is the first report of spawning behaviour in this species, and one of the very few from Pleuronectiformes generally.

2. Materials and methods 2.1. Fish stocks and husbandry Second generation cultured fish (2 years old) produced by manual fertilization of eggs from naturally maturing and ovulating fish in 1997 were introduced into a 4-m3 Rathbun tank in early 1999 at a density of ~4 fish (200–300 g body weight) m 3. The tank was connected to an Aquahort temperature control unit and supplied with natural sea water (30–35x) recirculated through solids- and bio-filters, and a foam fractionator. Approximately 30% of the system volume was replaced with fresh sea water each week. Fish were maintained under simulated natural photoperiod on a dsummerT (December to April) temperature of 15 8C, and a dwinterT (May to November) temperature of 10 8C to approximate natural temperature conditions in eastern Tasmanian waters. Fish were fed twice daily on a mixture of marine fish pellets (Sketting., Hobart, Tasmania), chopped squid and fresh or frozen mussels. In 2000, a second tank with the same configuration was established with 2-year-old fish produced from the 1998 cohort. There were approximately equal numbers of males and females in each tank. Fish were only disturbed for weekly tank cleaning that took approximately 1 h to complete. Tank systems were main-

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tained on the same regime through to the end of 2003, with periodic addition of new 2-year-old fish to maintain stock densities at a constant level (there was a low level of periodic mortality in each tank, generally associated with females that ovulated but failed to spawn). 2.2. Spawning behaviour Observations for potential spawning behaviour were made on 34 days from 9 June to 24 August, 2001 using a QV-100 (Quality Instruments) water proof monochrome video system mounted over tank 1, with lighting provided by a sealed fluorescent light encased in dark red theatrical light filter film. The tank was permanently illuminated with the dim red light, with the simulated natural photoperiod (provided by daylight fluorescent tubes) overlaid on top. The video system was connected to a portable video recorder using standard 180- or 240-min VHS video tapes. Recordings were made from 17:00 to 24:00 h on 24 occasions, and from 24:00 to 07:00–09:00 h on 14 occasions, with filming on 8 days covering both periods consecutively. Tapes were exchanged at 3–4 h intervals as required. Tapes were then viewed and possible spawning or courting behaviours identified and correlated with the presence or absence of spawned eggs in egg collectors (see next section). Preliminary observations (and subsequent experience) showed that spawning as evidenced by egg presence never occurred during the day, and in consequence, no video records were made between 09:00 and 17:00 h. 2.3. Spawning frequency Occurrence of spawning was not recorded in 2000, although the presence of eggs on dacron solids filters at tank outlets confirmed that periodic spawning was occurring. From 2001 onwards, tank outlets were fitted with 500-Am mesh egg collectors that were checked daily during the study period. In 2001, records were made simply of whether or not spawning had occurred. In 2002 and 2003, spawns were classified as dsmallT (b10 ml eggs), dmediumT (10– 50 ml) or dlargeT (N50 ml), and fertility was estimated from examination of a sub-sample under a stereo dissecting microscope. The stage of cleavage of fertile eggs was also determined.

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Table 1 Relationship between egg cleavage stage and time since spawning Cleavage stage

Time since spawninga (h)

Before 1st cleavage 2 cell stage 4 cell stage 8 cell stage 16 cell stage 32 cell stage

b2 2–2.5 2.5–3 3–3.5 4–4.5 5.5

a Egg development time from Crawford (1986), and direct observation of correlation between spawning events and egg stage (this study).

2.4. Timing of spawning The approximate or exact timing of spawning was determined in 3 ways: i) Direct observation of spawning events by video, and subsequent correlation with egg presence in egg collectors. ii) By elimination, when eggs appeared in collectors but video records covering part of the scotophase showed that no spawning had occurred during the observation period. iii) Through back calculation of spawning time from the developmental age of freshly spawned eggs in egg collectors. Egg ages were based on the developmental timescale described by Crawford (1986), and validated in the present study by using the correlation between egg developmental stage and known time of spawning from events recorded on video, as shown in Table 1.

3. Results 3.1. Spawning behaviour Spawning events were observed on 5 of the 34 days on which video recording was conducted. There were eggs present in egg collectors on an additional 6 days when taping covered only part of the scotophase, but no spawning behaviour was observed in video records, i.e. spawning had occurred during the period for which there was no video record. Eggs were present in the egg collectors on all 5 of the mornings where video

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a)

b)

Fig. 1. Profiles of typical (a) male and (b) ovulated female cultured greenback flounder. Females show unilateral swelling as oocyte hydration and ovulation proceed.

circling by both fish, high frequency fin dflutterT and then the release of eggs to the water column (Fig. 2). Visible egg release generally occurred for 30–60 s after which the male broke off and dropped out of the water column with the female following soon after. A variation to the above pattern was observed on two occasions where a male moved under a female already swimming in the water column and circling behaviour and spawning then occurred. Video blind spots in the tank prevented observation of whether these events had been initiated earlier through dcourtingT by the male. Other males in the tank did not attempt to interfere with a spawning pair, nor did more than one male at a time typically attempt to court an ovulated female resting on the tank bottom.

records indicated that spawning had occurred during the previous night. Males were easily distinguishable from ovulated females which had clearly visible unilateral swelling associated with oocyte maturation and hydration (Fig. 1). Spawning activity was initiated by lateral approach of a male towards an ovulated female and adoption of a dhead raisedT posture (Figs. 1a and 2a). This was often followed by repeated nudging of the female’s ovipore by the male’s snout, continuing in some cases for several minutes. The female then either broke away and no spawning ensued, or rose off the tank bottom, allowing the male to move in closely beneath the female. This brought the latero-dorsally located male gonopore into close association with the latero-ventrally located female gonopore. The pair then rose in the water column in a slow circle with marginal fin dripplingT which in the majority of cases, ended when the female dropped back out of the water column. In a smaller number of cases, the slow paired circling advanced to rapid tight

a)

3.2. Spawning frequency Spawning activity began in May 2001, with a short burst of spawning; sporadic spawning occurred

b)

d)

c)

e)

Fig. 2. Diagrammatic representation of the behavioural events leading to spawning; (a) approach and head raised posture by males; (b) ovipore nudging by the male; (c) pairing with male beneath, and ascent into the water column; (d) slow circling in the water column with fin dripplingT; (e) rapid circling with high frequency fin dflutterT and egg release.

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through June and early July, and there were concentrated episodes of spawning activity in late July and August (Fig. 3). Subsequent shorter episodes of spawning occurred in late September, and October, respectively, and sporadic spawning continued into December. With the exception of the concentrated spawning period in late August, the beginning of all spawning episodes coincided with, or followed within a few days of the new moon (Fig. 3). Day length appeared to have little bearing on spawning activity, with spawning occurring over photophases of 9.5 to 14.9 h. In 2002, there was no spawning activity in tank 1 until September, then intermittent spawning occurred until early December (Fig. 4). The first occurrence of spawning coincided with a new moon but thereafter, there was no strong evidence of lunar synchronisation of spawning. The size of spawns was variable with no clear temporal trends in the volume of eggs spawned. In contrast to tank 1, there was spawning in June and early July in tank 2, no spawning for most of July and August and then a resumption of spawning in September that coincided with the beginning of spawning in tank 1 (Fig. 4). The initiation of spawning in both June and September occurred with

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the new moon but at other times there was not a clear relationship between lunar phase and spawning activity. The majority of spawning occurred after day length exceeded 11 h. The pattern of spawning in 2003 was similar to 2002, with the main period of spawning activity occurring in austral spring in both tanks (Fig. 5), and spawning beginning soon after the new moon. As in 2002, there was no clear correlation between spawning and lunar phase thereafter, there was an early burst of spawning in tank 1 and no clear temporal pattern to the volume of eggs produced, and the majority of spawning activity was associated with increasing day length (Fig. 5). Egg fertility in 2003 was highly variable (range 0– 100%) and not correlated with the volume of eggs produced, nor was there any clear association between date and fertility (Fig. 6). Similar effects were observed in 2002 but with fertile eggs regularly being produced only in tank 1 (data not shown). 3.3. Timing of spawning Direct observation of spawning in 2001 gave spawning times from 21:17 to 07:20, with most

2001 Daylength (hours)

16 14 12 10 8

Spawns

New moons

May

June

July

Aug

Sept

Oct

Nov

Dec

Date Fig. 3. Day length and dates on which spawning occurred in cultured fish held through 2001. Filled circles show the time of new moons.

N.W. Pankhurst, Q.P. Fitzgibbon / Aquaculture 253 (2006) 279–289 Daylength (hours)

284 16

2002

14 12 10 8 New moons

3

2

Tank 1

Spawns

1

0 3

2

Tank 2

1

0 June

July

Aug

Sept

Oct

Nov

Dec

Date

Daylength (hours)

Fig. 4. Day length, date of spawning events (1=small, 2=medium, 3=large) in 2 tanks of cultured fish held through 2002. Filled circles show the time of new moons. 16

2003

14 12 10 8 3 New moons 2

Tank 1

Spawns

1

0 3

2

Tank 2

1

0 April

May

June

July

Aug

Sept

Oct

Nov

Date Fig. 5. Spawning events in fish held through 2003. Other details as for Fig. 4.

Dec

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285

2003 Tank 1

Fertility (%)

100

100

a)

80

80

60

60

40

40

20

20

0 100

100

80

80

b)

0

60

60

Tank 2

40

40

20

20

0

0 A

M Jun Jul

A

S

O

N

D

Small

Date

Medium

Large

Size of spawn

Fig. 6. Relationship between egg fertility and (a) volume of eggs produced (see Fig. 4 and text for details), and (b) spawning date in 2003.

events occurring between midnight and 07:30 (Table 2). A similar pattern emerged from nights when spawning occurred (as evidenced by egg production) but outside the period covered by video records. Overall, there were 10 spawning events in the period midnight—09:00, and 3 events in the period 17:00— midnight. Back calculation of spawning time based on egg age at 09:00 from spawns in 2002 showed spawning to be concentrated into the early morning period (Table 3). There was no spawning before 03:00, or after 08:00 and the majority of spawning occurred between 04:00 and 06:00. During this period, day length ranged from 11.5 to 13.5 h, with

Table 2 Spawning time based on direct video observation during 2001 Observeda

Inferredb

Date

Time

Date

Time

18 21 20 21 23

06:30 03:20, 05:30, 07:20 21:17, 23:16 00:11–00:42 (5 events) 01:15

23 28 29 17 19 21

24:00–09:00 24:00–09:00 02:30–07:30 24:00–09:00 02:00–09:00 17:00–24:00

a

July July August August August

June June June July July August

Spawning observed directly. Spawning occurred but outside the period recorded on that night. b

spawning concentrated in the period 2 h before sunrise.

4. Discussion The results of the present study indicate that greenback flounder held at low density (~1 kg m 3) in moderate water volumes will undergo natural spawning, but only after an extended period of tank acclimation under conditions of low disturbance. This is generally consistent with studies on other species where induction of spawning behaviour often requires moderate to large holding volumes, and extended maintenance of undisturbed stocks (e.g. Smith, 1986; Table 3 Spawning time based on egg agea, from spawns collected during 2002 Time period

Frequency

02:00 03:00 04:00 05:00 06:00 07:00 08:00

0 4 10 6 10 5 0

a

See Table 1 for details.

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Garratt, 1991; Leu, 1994; Tucker et al., 1996; Okumura et al., 2002). An exception appears to be Pacific herring Clupea harengus pallasi, where wild fish spawned in large volume tanks but very soon after capture as mature fish (Stacey and Hourston, 1982). In greenback flounder, the critical factor appears to be low holding density as other stocks held under similar conditions but at higher density (up to 10 kg m 3) matured but failed to show spawning behaviour (authors’ unpublished observations). Spawning episodes in greenback flounder only ever included a single male and female with a sequential occurrence of approach, apparent courting or testing by the male followed by the close association of the pair during the circular swimming behaviour and subsequent egg release. Close association of male and female is characteristic of the other flatfishes where spawning behaviour has been described (Konstantinou and Shen, 1995; Smith et al., 1999) and is consistent with the generally low milt volume found in greenback flounder and other flatfishes (Pankhurst and Poortenaar, 2000). This is in contrast to the higher milt volumes found in group spawning species such as sparids where competing males release large volumes of milt around dispersing egg masses (Smith, 1986; Leu, 1994; Domeier and Colin, 1997). Male greenback flounder generally nudged the vent of ovulated females early in a spawning sequence. Similar behaviour is reported for wild bothid flounders (Konstantinou and Shen, 1995) and a wide taxanomic range of non-pleuronectids (Brawn, 1961; Hara et al., 1986; Smith, 1986; Garratt, 1991; Tucker et al., 1996). It is unclear whether nudging behaviour is dcourtingT that stimulates female responsiveness, olfactory testing of female readiness, or a combination of both. The widespread occurrence of hormonal pheromones released in the urine and ovarian fluid of freshwater fishes, and detected by males (reviewed by Kobayashi et al., 2002; Stacey, 2003) suggests that olfactory testing by males might be occurring here. There is less information on the occurrence of sexual pheromones in marine fishes but in at least one species (Pacific herring) olfactory detection of a pheromonal component of milt stimulates spawning behaviour in both sexes (Carolsfeld et al., 1997). In the case of greenback flounder, the response of females to nudging by males was

relatively passive. A female response generally only occurred when the male attempted to move beneath the female. This suggests a process whereby the male tests the female for spawning readiness through an olfactory cue that in turn elicits a behavioural response in the male. The female then responds or not presumably on the basis of her endocrine state. A model for similar behaviour exists in cyprinids where post-ovulatory release of prostaglandin F2a (PGF2a) to the water by females acts as an olfactory stimulant of male spawning behaviour. Female spawning behaviour is in turn centrally mediated by elevated plasma levels of PGF2a (reviewed in Kobayashi et al., 2002; Stacey, 2003). Zohar and Mylonas (2001) suggest that the failure of captive fish to spawn results from a combination of endocrine dysfunction associated with husbandry stress, and the lack of natural spawning environment, and that this can be overcome by treatments that elevate plasma luteinising hormone (LH) levels. This certainly appears to be the case in sea bass Dicentrarchus labrax (Fornie´ s et al., 2001), marbled grouper Epinephelus microdon (Tamaru et al., 1996) and gilthead seabream Sparus aurata (Zohar and Mylonas, 2001) but as noted earlier, not in flatfishes (Berlinsky et al., 1996; Larsson et al., 1997; Watanabe et al., 1998; Bengtson, 1999; Mugnier et al., 2000). Assessment of the phenomenon is not aided by the incorrect description of ovulation as dspawningT in a number of studies (e.g., Head et al., 1996; Smith et al., 1996; Tan-Fermin et al., 1997; Watanabe et al., 1998; Mugnier et al., 2000). The failure of many GnRHatreated stocks to spawn after hormonally induced ovulation appears to confirm the dual necessity of elevated plasma LH levels and appropriate social or physical conditions (Pankhurst, 1998). Greenback flounder in the present study showed strong evidence of lunar synchronisation in 1 year and correlation of initiation of major spawning episodes with lunar phase in the other 2 years. Lunar synchronisation of spawning is common among marine species and arguments for its function include; off-reef dispersal of eggs and larvae, synchronisation of reproductive readiness of adults, and predator swamping of potential predators of both eggs and adults (Robertson et al., 1990). In some groups such as tropical rabbitfishes, reproductive endocrine cycles are very closely tied to lunar rhythms, and spawning

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occurs to a regular lunar cycle (e.g. Rahman et al., 2000). In other groups such as damselfishes, lunar periodicity is often present early in the spawning season but breaks down as the spawning season progresses (Tyler and Stanton, 1995). Greenback flounder seem to show the latter pattern, although whether the observed spawning periodicity reflects that shown by wild fish is not known. It does not appear that lunar cycles entrain reproductive development in cultured greenback flounder, with fish showing population asynchrony in ovarian development, and ovulated fish being present in small numbers over an extended part of the year (Sun and Pankhurst, 2004). In most temperate species and possibly many tropical species as well, photoperiod exerts a dominant effect on the phasing of reproductive development and spawning (Pankhurst and Porter, 2003). Greenback flounder mature and ovulate over a wide range of photophase length (Sun and Pankhurst, 2004), and in the present study, spawning occurred over photophases of 9.5 to 15 h, suggesting that reproductive development is not tightly regulated by day length, with the proviso that, in two out of the three study years the majority of spawning events occurred as day length increased in austral spring. Temperature was held constant over this period, eliminating temperature as a possible cue in the present study. However, the ubiquity of temperature–photoperiod interactions in regulating reproductive cycles in temperate fishes generally (Pankhurst and Porter, 2003) strongly suggests that temperature will have a role in regulating spawning among greenback flounder in the wild. The limited data available suggest that ovulated fish occur year round in the wild but consistent with the present study, occur more commonly in late winter and spring (Barnett and Pankhurst, 1999). Spawning in the present study was concentrated in the scotophase with the majority of events occurring ~2 h before dawn. In the only report of flatfish spawning in the wild, bothid flounders spawned 20–30 min either side of sunset (Konstantinou and Shen, 1995), and captive southern flounder Paralichthys lethostigmata also spawned in the late afternoon or early evening (Smith et al., 1999). Evening spawning has also been reported in captive silver bream Rhabdosargus sarba (Leu, 1994), New Zealand

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snapper Pagrus auratus (Smith, 1986), cod Gadus callarias (Brawn, 1961), Nassau grouper Epinephelus striatus (Tucker et al., 1996), and red spotted grouper Epinephelus akaara (Okumura et al., 2002). In contrast, santer Cheimerius nufar spawned just before sunrise (Garratt, 1991). These studies show that spawning times can be variable even among closely related taxa, but also that there is a general pattern of spawning during the scotophase when visual predators of eggs are either absent or less effective. Exceptions to this are strongly diurnal tropical reef species where spawning often occurs in daylight but here also, spawning is usually displaced towards the beginning or end of the photophase (Domeier and Colin, 1997). Fertility of naturally spawned eggs in the present study was highly variable, ranging from 0 to 90%, with no clear correlation with season, or the volume of eggs released on a particular night. The source of the variability is not known but may result from the habit of paired spawning and typical involvement of only a single male in the spawning event. Greenback flounder milt volumes are small and vary from male to male (Pankhurst and Poortenaar, 2000), with the result that fertilization effectiveness might also vary among males. Alternatively, females may contribute to low fertility by not spawning immediately after ovulation. Periodic checks of swollen ovulated females in the present study showed that spawning was sometimes delayed for several days after natural ovulation. Given the short period of post-ovulatory egg viability in most marine teleosts (Hobby and Pankhurst, 1997), this probably results in low fertility. A similar phenomenon has been observed in red spotted grouper where natural spawns gave fertility of ~20% compared with in excess of 80% for manual stripping (Okumura et al., 2002). However, later studies on grouper showed that fertility improved with an increase in tank volume, suggesting that low fertility can be an artefact of holding volume (Okumura et al., 2003). Despite the fact that not all greenback flounder spawns resulted in high fertility, ongrowing of eggs from high fertility spawns consistently produced viable and robust larvae and juveniles (Shaw et al., 2003). The present study provides further evidence that spawning in un-manipulated broodstock is facilitated by low disturbance maintenance at low holding density, and has the capacity to provide an extended

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supply of eggs for larval rearing. Further, captive stocks offer the opportunity to observe spawning behaviour in species where due to the location or timing of natural spawning, observation of spawning behaviour is unlikely to be possible.

Acknowledgements This study was supported by Infrastructure and Large Grants from the Australian Research Council. Thanks are extended to Ryan Longland, Tish Pankhurst and Gavin Shaw for assistance with fish husbandry and egg collection.

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