Effects of predator and shelter conditioning on hatchery-reared white seabream Diplodus sargus (L., 1758) released at sea

Aquaculture 356–357 (2012) 91–97

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Effects of predator and shelter conditioning on hatchery-reared white seabream Diplodus sargus (L., 1758) released at sea Giovanni D'Anna a, Vincenzo Maximiliano Giacalone a,⁎, Tomás Vega Fernández a, 1, Antonino Maurizio Vaccaro b, Carlo Pipitone a, Simone Mirto c, Salvatore Mazzola d, Fabio Badalamenti a a

CNR-IAMC, Sede di Castellammare del Golfo—via G. da Verrazzano, 17‐91014 Castellammare del Golfo, Italy Mare e Ambiente s.r.l., Via Marchese di Villabianca, 98‐90143 Palermo, Italy CNR-IAMC, U.O. Messina—Spianata S. Raineri, 86‐98122 Messina, Italy d CNR-IAMC, Calata Porta di Massa—80133 Napoli, Italy b c

a r t i c l e

i n f o

Article history: Received 19 January 2012 Received in revised form 22 May 2012 Accepted 23 May 2012 Available online 29 May 2012 Keywords: Fish conditioning Stock enhancement Diplodus sargus VIE tag Sicily

a b s t r a c t The behavioural deficit of hatchery reared (HR) fish used for stock enhancement is the main cause of their low survival in the wild. In this study the effects of predator and shelter conditioning on survival and dispersal of HR white seabream (Diplodus sargus) released at sea were investigated. The hypotheses were that conditioned white seabream would avoid predators more efficiently and would be more capable to shelter, showing higher survival and smaller dispersal than naïve fish. Six thousand HR white seabream (6.32 ± 0.93 cm total length) were allocated in twelve plastic tanks and divided in four experimental groups: three groups were conditioned with a predator, a refuge or both, while one group was left unconditioned and used as a control. The conditioning phase lasted 30 days and was conducted using live conger eels as predators and pyramids of perforated bricks as refuges. Flight initiation distance (FID) and time to shelter (TS) were used as response variables to test the effects of conditioning in the arena, using a dummy conger eel. The effect of conditioning on post-release survival and dispersal was assessed through the monitoring of 1465 tagged seabream belonging to the four experimental groups, released at sea. Underwater visual census was used as monitoring technique. The sighting rate (SR) (sighted fish / released fish × 100) and the distance (D) of each sighted fish from the release site were used as proxies for post-release survival and dispersion, respectively. In the arena, conditioned seabream showed significantly higher FID and lower TS than naïve fish. At sea, the estimated post-release survival of conditioned seabreams (SR = 9.4%) was almost twice as much as that of naïve individuals (SR = 5.5%). The dispersal of HR seabream acclimated to refuges from the release site (D = 2.4 ± 3.1 km) resulted lower than in naïve fish (D = 3.7 ± 4 km). This study indicates that predator and shelter conditioning of HR white seabream is an effective practise to increase their post-release survival at sea, and our findings provide support for effective stock enhancement initiatives. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Human exploitation has drastically reduced the abundance of marine fish populations worldwide. Stock enhancement based on hatchery-reared (HR) fish can contribute to mitigate this impact in certain circumstances and help to reconstitute depleted stocks (Bell et al., 2008). However, stock enhancement initiatives have often failed mainly due to inadequate rearing environment (Lee and Berejikian, 2008), which has often generated fish unable to survive in the wild (Lorenzen et al., 2010; Olla et al., 1998; Salvanes and

⁎ Corresponding author at: CNR-IAMC, U.O. Capo Granitola—Via del Mare, 3‐91021 Campobello di Mazara, Italy. Tel.: + 39 092440600; fax: + 39 092440445. E-mail address: [email protected] (V.M. Giacalone). 1 Present address: CNR-IAMC, U.O. Mazara del Vallo—Via Luigi Vaccara, 61‐91026 Mazara del Vallo, Italy. 0044-8486/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2012.05.032

Braithwaite, 2006). The behavioural deficit of HR fish (Brown and Laland, 2001; Olla et al., 1994) is expressed by poor anti-predator skills and inadequate use of shelter resulting in high predation mortality and dispersal once released in the wild (Hervas et al., 2010; Odling-Smee and Braithwaite, 2003). Previous studies have demonstrated that such behavioural deficit can be reduced by proper prerelease conditioning to a variety of stimuli which may enhance the survival of HR fish in the wild (Alvarez and Nicieza, 2003; Griffin et al., 2000; Kelley and Magurran, 2003; Mathis and Smith, 1993; Olla and Davis, 1989; Suboski and Templeton, 1989). Experiments to train HR fish to avoid predators have been based on exposing fish to visual, acoustic, tactile and chemical predator stimuli using both live and dummy predators. Generally, prey fish are able to express various anti-predator strategies when encountering a predator but, irrespective of the stimulus used, a way to assess the effectiveness of anti-predator training is the distance from the

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predator at which the prey initiates its flight (Dill, 1990). It has been shown that wild fish encountering a predator keep a longer distance from it than naïve fish (Arai et al., 2007; Malavasi et al., 2004), which may approach the predator driven by curiosity (Brown and Warburton, 1999). The need to teach reared fish how to shelter assumes a particular importance when the object of fish restocking is a demersal species (Ellis et al., 1997; Fairchild and Howell, 2004). Since refuges are used by fish mainly in response to predation (Steele, 1996), the incapability of HR fish to shelter makes them more vulnerable to predators (Brown and Laland, 2001; Stunz et al., 2001; Yokota et al., 2011) and causes high dispersal from the release site with a negative effect on the efficiency of the enhancement programme (Hervas et al., 2010; Santos et al., 2006). To date, almost all predator and shelter conditioning experiments on HR fish have been tested in arenas (Olla and Davis, 1989) and only a few papers have addressed the efficiency of conditioning on fish survival in the wild (Sparrevohn, 2008). The white seabream Diplodus sargus (L., 1758) is a benthopelagic fish inhabiting the littoral rocky bottoms of the NE Atlantic and the Mediterranean from the shore to 50 m depth (Lloret and Planes, 2003). It is a commercially exploited species that gains a high market price (Harmelin-Vivien et al., 1995) and is targeted by professional and recreational fishermen (Coll et al., 2004; Reina et al., 1994). The dramatic decline of its commercial catches in the last two decades especially in the Mediterranean (Lloret and Planes, 2003) makes this species a good candidate for restocking initiatives. Juveniles of HR white seabream have been used in a pilot marine ranching experiment in the Gulf of Castellammare (NW Sicily, Mediterranean Sea) that gave evidence of incapacity to shelter, high dispersal from the release site and high mortality from predation by conger eel, Conger conger (D'Anna et al., 2004). The objective of the present study was to investigate the effects of predator and shelter conditioning on post-release survival and dispersal of HR white seabream. The hypotheses were that conditioned fish (i) avoid predators more efficiently and (ii) that these are more capable to shelter, and hence they show higher survival and smaller dispersal than naïve fish after their release at sea. 2. Materials and methods

and to sustain a high level of predation pressure throughout the experimental period. During the conditioning period no evidence of predation from conger eels was observed. Shelter conditioning was done in six tanks containing a pyramidshaped conger eel-proof refuge made of 13 perforated bricks (Fig. 1). Therefore the complete array of housing tanks represented four experimental groups, made up of three tanks each: P+R+ with predator and refuge, P+R− with predator but without refuge, P−R+ without predator but with refuge, and P− R− with neither predator nor refuge. Conditioned seabream belong to the first three groups while naïve seabream belong to the P− R− group. Both the predator and shelter conditioning lasted 30 days, which is a slightly longer period than adopted in other similar experiments (de Oliveira Mesquita and Young, 2007; Malavasi et al., 2004). 2.2. Predator avoidance and shelter capability experimental design The experimental design comprised two factors orthogonally crossed: predator detection (P), fixed with two levels (presence, P+, or absence, P− of a predator), and refuge acclimation (R), fixed with two levels (R+ and R−, as above). Since fish were held in tanks, an additional random factor tank (T), nested in the interaction P × R with three levels (T1, T2 and T3), was accounted for the potential effect of unknown factors. 2.2.1. Predator avoidance experiment Forty five predator avoidance trials for each experimental group were conducted in a separate trial tank using fifteen fish taken randomly from each housing tank. In each trial one dummy conger eel was put into the tank along the wall, then one seabream was introduced on the opposite side of the tank at about 250 cm distance. The dummy conger eel was made of a 15 cm pipe covered by a thin-walled 40 cm plastic tube stretched on the tail side, which was moved slowly towards the fish with the aid of a long handle. The distance between the seabream and the dummy predator at any time was recorded with a video camera fixed orthogonally above the tank. The response variable was the closest distance (flight initiation distance, FID in cm) between the dummy predator and the seabream before the flight reaction. FID was estimated with an accuracy of 1 cm from the video recordings with the aid of a reference scale bar placed on the tank bottom.

2.1. Fish housing and conditioning Twelve plastic tanks located at the CNR-IAMC research facilities in Torretta Granitola (SW Sicily) were used for fish housing and conditioning. Each tank was 250 cm in diameter and 4000 l in volume, and was equipped with an independent flow-through seawater system from a common source. Salinity was 32.4 ± 1.2 [mean ± S.D.] and temperature was 17.5 ± 2.2 °C during the whole study period. A batch of 6000 six-month old HR white seabreams (total length, TL = 6.32 ± 0.93 cm), obtained from induction of a broodstock composed by wild spawners, was randomly distributed (ca. 500 fish per tank) into the tanks in October 2008. All fish were fed a commercial diet with pellets scattered directly on the tank bottom with the aid of a plastic tube. This foraging technique was aimed at teaching the fish to search for food on the bottom (like in nature) but was not considered a conditioning factor in the experimental design. Fish conditioning started 30 days after their allocation in the tanks to ensure proper acclimatisation (TL = 7.22 ± 0.85 cm at the start of conditioning). For predator conditioning of seabream a group of 18 wild conger eels (TL = 70 ± 10 cm) caught in the Gulf of Castellammare and kept in an independent tank were used. Eels were starved for 10 days prior to introducing one of them into each of six tanks (see below) and were substituted every 10 days to prevent carry-over effects

2.2.2. Shelter capability experiment Thirty predator avoidance trials for each experimental group were conducted using ten fish taken randomly from each housing

Fig. 1. Pyramid-shaped conger eel-proof refuge made of 13 perforated bricks.

G. D'Anna et al. / Aquaculture 356–357 (2012) 91–97

tank. The fish were taken individually with a soft-tissue hand-held net and put in a trial tank that contained a shelter made by one brick like those used in the housing tanks devoted to refuge conditioning. The dummy conger eel was introduced along the tank wall and moved to simulate a slow attack. Each trial lasted 60 seconds and was video recorded as abovementioned. The response variable (time to shelter, TS) was the proportion of trial time spent by the seabream to get into the refuge after the introduction of the dummy predator. An arbitrary value of 1 was assigned to those individuals that did not shelter within the trial time, hence the theoretical proportion values ranged from 0 to 1.

2.3. Survival and dispersal at sea One thousand four hundred and sixty-five seabreams (TL = 8.72 ± 0.64 cm) were tagged with either green or red visible implant elastomer (VIE) tag (Doupe et al., 2003) applied to the caudal fin in order to identify the four experimental groups as defined above: 350 individuals were tagged with one red band (P +R−), 360 with two red bands (P +R+), 385 with one green band (P− R−) and 370 with two green bands (P −R+). An experimental design similar to that used in the arena trials was adopted to test the effect of conditioning on the survival and dispersal of HR seabream at sea. The only difference was that factor T was deleted from the design, due to the expected low probability of detection of tagged individuals in the wild as a result of the dilution of fish spreading over a large area. The study area corresponded to a 20 km stretch of the central coastline of the Gulf of Castellammare (Fig. 2), where a habitat suitable to the settlement of juvenile white seabream occurs (D'Anna et al., 2004). Nine coastal sites characterised by a shallow bottom with vegetated rocks and patches of the seagrass Posidonia oceanica in a sandy matrix were selected for our survey. All tagged fish were released in the site of Balestrate (Fig. 2) on 4 March 2009. The relative abundance of tagged fish inside each experimental group was assessed by random linear underwater visual censes, which started 1 month later due to unfavourable weather conditions and water turbidity. A total of 64 4 × 200 m linear transects were covered by divers within a period of 15 days in the nine sites. Repeated sightings of tagged fish in any one transect were unlikely since observers recorded tagged fish while proceeding forward, and it is extremely unlikely that a sighted fish left behind came back into the observer's field of vision (personal observation). Anyway repeated sightings are expected to be equally distributed across all the experimental groups; thus the comparison of their relative frequencies would remain unbiased. To estimate the relative survival of tagged seabream at sea, the sighting rate (SR) expressed as sighted fish / released fish × 100 was used as response variable.

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To assess the effect of conditioning on fish dispersal, the distance (D, in km) of tagged fish from the release site sighted at each site was used as response variable. 2.4. Data analysis Untransformed FID and TS data were analysed with 3-way analysis of variance (ANOVA). D data were transformed as fourth root and analysed with a 2-way ANOVA model with zero intercept (Quinn and Michael, 2002). The use of a zero intercept ANOVA model was justified because we assumed that naïve HR fish disperse more widely due to their inability to use available shelters, while perfectly skilled individuals are able to shelter, as shown by a previous tag-and-release initiative involving wild white seabream in NW Sicily (D'Anna et al., 2011). Homogeneity of variances was checked with a Cochran's C test (Underwood, 1997). Where appropriate, pairwise comparisons were performed by means of Student– Newman–Keuls (SNK) test. In order to detect differences between the SR of conditioned and naïve fish, two by two contingency tables employing the Chi-square test with Yates correction were used to explore significant relationships between each type of conditioning and the number of sighted fish. All statistics were performed by means of the STATISTICA 6.0 software package. 3. Results 3.1. Predator avoidance and shelter capability FID showed a significant response to factor P (Table 1, Fig. 3). White seabreams conditioned with conger eel escaped earlier, that is at a longer distance (FID = 47 ± 17 cm) than naïve individuals (FID = 31 ± 12 cm). No effect on FID of factors R and T and of the P × R interaction was detected. TS showed a significant effect of factor R (Table 2, Fig. 4). Seabreams acclimated in tanks with refuges took less time to get into the shelter after the introduction of the dummy predator than specimens not acclimated to refuges. No effect on TS of factors P and T and of the P × R interaction was detected. 3.2. Survival and dispersal at sea One-hundred and twenty-two tagged fish corresponding to 8.3% of released seabreams were sighted during the visual census surveys (Table 3). The value of SR for the P− R− group was 5.5% contrasting with 9.4% registered by the three conditioned groups altogether. Among conditioned fish, the highest SR value was attained by the P+R− group (11.4%), followed by P+ R+ (10%) and P −R+ (6.8%). The Chi-square test detected a significant difference between the SR value of conditioned and naïve white seabreams (χ 2 = 10.31, df = 2, p = 0.005). According to the 2 × 2 contingency tables analysis, the SR value was significantly related to P+ R− (χ 2 = 7.83, df = 1, p = 0.005) and to P+ R+ (χ 2 = 4.816, df = 1, p = 0.028) but not to P−R+ (χ 2 = 0.355, df = 1, p b 0.551).

Table 1 Summary of the ANOVA on flight initiation distance (FID).

Fig. 2. Map of the Gulf of Castellammare showing the nine underwater visual census sites (circles). RS = release site.

Source of variation

df

MS

F

p

Predator (P) Refuge (R) P×R Tank (T) (P × R) Residuals Total

1 1 1 8 168 179

11708 426.58 333.74 160.62 213.57

72.89 2.66 2.08 0.75

b0.0001 0.1418 0.1874 0.6454

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1.20

Mean TS (sec) ± S.D.

Mean FID (cm) ± S.D.

80 60 40 20 0 P+R+

P+R-

P-R+

P-R-

1.00 0.80 0.60 0.40 0.20 0.00 P+R+

Fig. 3. Mean and standard deviation (S.D.) of the flight initiation distance (FID) in centimetres (cm) of white seabream for each experimental group (P + R+, P + R−, P− R+, P − R−). See text for details.

A significant effect of factor R on D was detected (Table 4, Fig. 5). The mean distance travelled by R+ fish was lower than in R− fish. No effect of factor P and of the P × R interaction was observed.

4. Discussion 4.1. Predator avoidance and shelter capability There is a general agreement that the capability to recognise and avoid predators is the most important skill which can significantly increase post-release survival of HR fish in the wild (Ferrari et al., 2005; Howell, 1994; Kellison et al., 2000). Living and model predators as well as visual, chemical and electric stimuli, and the presence of wild “demonstrators” are the main cues which have been used to improve the anti-predator behaviour of naïve fish in experimental arenas (Alfieri and Dugatkin, 2009; Kelley and Magurran, 2003; Malavasi et al., 2004). In this study we used living conger eels to train HR white seabreams to recognise a predator and a dummy conger eel to assess the anti-predator response of individual fish. Through a thirty-day training with living conger eels, seabreams were subjected to a variety of natural stimuli (i.e., visual, chemical, and mechanosensory) important in the development of predator detection and avoidance behaviour required for their survival in the wild. In spite of its efficiency, conditioning fish with direct exposure to living predators has been disapproved because of ethical reasons (Huntingford, 1984). On the other hand, the use of predator dummies has produced equivocal results (Dill, 1974; Vilhunen, 2006), mainly due to the risk of prey habituation to the stimulus of a predator model (Fraser, 1974; Kanayama, 1968; Magurran, 1990). Nevertheless, the use of dummy predator for predator recognition is well established, and it is reported that animals can learn the visual characteristics of both artificial and biologically significant stimuli, including predator dummies (Griffin et al., 2000). The use of a dummy conger eel in our study reduced the sources of variability due to a living predator (i.e., number of attacks, movement speed, odour, voracity, etc.) and limited the anti-predator response of seabream mainly to a visual stimulus although the detection of a predator is not necessarily followed by a reaction (Kelley and Brown 2011). During the simulated attack with the dummy predator, individual P+ seabream displayed an evident

Table 2 Summary of the ANOVA on proportion of time to shelter (TS). Source of variation

df

MS

F

p

Refuge (R) Predator (P) R×P Tank (T) (R × P) Residual Total

1 1 1 8 108 119

1.15 0.23 0.01 0.09 0.06

12.82 2.60 0.13 1.49

0.0072 0.1455 0.7318 0.1699

P-R+

P-R+

P-R-

Fig. 4. Mean and standard deviation (S.D.) of proportion of time to shelter (TS) spent by white seabreams for each experimental group (P + R+, P − R+, P + R−, P − R−). See text for details.

anti-predator behaviour through a faster reaction (i.e. longer FID) to the predator model than P− seabream. The flight distance is an important component of the escape response and the result of a complex trade-off that allows a fish to maintain a safe distance from a predator (Lima and Dill, 1990). Generally such distance increases with the risk but it was found to depend on many factors such as proximity to a refuge, attack speed, predator size and previous experience with predators (Dill, 1974; Domenici, 2010). However, as the anti-predator performance in our experiment was tested without refuges and with constant predator model size and attack speed, the higher FID recorded by P+ fish can be attributed exclusively to their ability to detect the predator. Our result is consistent with a number of other experiments with fish conditioned to the presence of living or dummy predators. Predatortrained cod (Gadus morhua), red drum (Sciaenops ocellatus) and Japanese flounder (Paralichthys olivaceus) kept a longer distance from predators than naïve individuals (Arai et al., 2007; Nodtvedt et al., 1999). According to Arai et al. (2007), the longer reaction distance to a predator in the arena could reflect a fear response, or increased caution, of predator-conditioned fish that could positively affect their survival in the wild. The various anti-predator defences acted by fish in nature also include their ability to shelter (Kellison et al., 2000; Sogard and Olla, 1993). In our study, HR white seabream conditioned with refuges spent less time sheltering than naïve individuals when exposed to the predator. During our test, R+ fish were able to recognise and use refuges quickly when attacked by a dummy conger eel, while R− fish mainly swam around the perforated brick instead of hiding inside. The behaviour of our R− fish is consistent with what described by Kellison et al. (2000) for HR summer flounder (Paralichthys dentatus), which took a longer time to hide when exposed to a predator than wild individuals. Still, despite the numerous demersal species reared

Table 3 Abundance of tagged white seabream sighted at each site and distance (D) in kilometres (km) from the release site (=site 1) for each experimental group (P−R−, P+R−, P− R+, P+R+). See text for details. Site

D

Number of fish sighted per treatment

(km)

P − R−

1 0.00 2 0.39 3 0.50 4 1.34 5 3.13 6 4.28 7 6.56 8 8.94 9 11.06 Total no. of fish sighted Fish released

P + R−

P− R+

Total P+ R+

1 3 1 3 5 2 0 2 4 21

5 10 1 6 9 0 0 3 6 40

2 11 1 1 5 0 0 1 4 25

6 10 2 5 9 0 0 3 1 36

14 34 5 15 28 2 0 9 15 122

385

350

370

360

1465

G. D'Anna et al. / Aquaculture 356–357 (2012) 91–97 Table 4 Summary of the zero intercept ANOVA on the distance (D) travelled by tagged fish from the release site. Source of variation

df

MS

F

p

Intercept Refuge (R) Predator (P) R×P Residual Total

1 1 1 1 118 122

149.08 0.78 0.27 0.07 0.16

911.17 4.75 1.65 0.42

b0.0001 0.0313 0.2016 0.5182

for stock enhancement purposes, few studies report the effect of refuge-conditioning on behaviour and survival of reared fish released at sea. Ellis et al. (1997), Fairchild and Howell (2004), and Sparrevohn (2008) documented the ability of several flatfishes to learn how to bury under a sandy bottom in hatchery tanks, thus increasing their survival chances in the wild. Kawabata et al. (2011) showed that HR black-spot tuskfish (Choerodon schoenleinii) utilised shelter more efficiently after being properly conditioned. Accordingly, conditioned HR black-spot tuskfish displayed a reduced post-release mortality. To further decrease post-release mortality, the same authors suggested the combined training for the recognition of both shelter and predator as a way to induce a smarter behaviour of HR fish in the wild. The last suggestion is not corroborated by our study as no significant effect of the P × R interaction on TS was detected although P+R+ fish spent a shorter time to shelter (0.68 ± 0.33) than P−R+ fish (0.79 ± 0.29). 4.2. Survival and dispersal at sea From our surveys at sea, the estimated post-release survival of conditioned seabream was twice as much as that of naïve individuals. Since we considered the sighting rate of HR seabreams as a measure of their survival, the efficiency of the tagging method used is crucial. Several studies have demonstrated that VIE tags (i) do not alter the vulnerability of fish to predation (Astorga et al., 2005), (ii) have a high detection and retention up to 6 months (Brennan et al., 2007; Willis and Babcock, 1998) and (iii) are effective when used for monitoring the behaviour of juvenile fish with underwater visual methods (Willis and Babcock, 1998). The only notable issue related to the use of VIE in our case was the unfavourable weather conditions and consequent high water turbidity, which forced us to start the monitoring survey 1 month after the fish release. Many laboratory experiments have tested the effectiveness of conditioning on HR fish survival but very few studies have been conducted at sea. Sparrevohn and Stottrup (2007) found the mortality of conditioned turbot, Psetta maxima, released at sea to be half as much as that of naïve fish. Similar results were obtained by Svåsand et al. (1998), who estimated a higher mortality rate in naïve HR cod released at sea when compared to wild individuals.

Mean D (km) ± S.E.

6

95

In large-scale releases, the estimated survival has been generally expressed as recapture rate of released non-conditioned HR fish. Return rates smaller than 5% have been reported for restocking initiatives involving grey mullet (Mugil cephalus), red seabream (Pagrus major), cod, gilthead seabream (Sparus aurata) and sea bass (Dicentrarchus labrax) (Grati et al., 2011; Leber et al., 1996; Ottera et al., 1999; Sanchez-Lamadrid, 2002). The overall returns of tagged seabreams in our experiment (8%) is higher than in other studies and we consider it a conservative value as it was estimated 1 month after release on a wide study area. The SR values of our P+ fish resulted two and four times higher than the recapture rate of HR white seabream recorded in southern Portugal (Santos et al., 2006) and in NW Sicily (D'Anna et al., 2004), respectively. Another factor that may negatively affect the efficiency of stock enhancement programmes is an excessively wide dispersal of HR fish from the release site (Hervas et al., 2010). Tagged seabreams in our experiment were sighted up to about 11 km from the release point. Although such distance may be considered limited, it nevertheless allowed to detect substantial differences between R+ and R− fish. In particular, D values of R+ fish were lower than those of R− fish, indicating a weaker tendency to disperse in refuge-conditioned fish than in naïve ones. Even though the significant effects of a zero intercept ANOVA model should be interpreted with some caution (Neter et al., 1996), differences were large enough to safely conclude that they were an effect of refuge-conditioning. The dispersion pattern of R− individuals in this study is comparable with that of naïve HR seabreams released in a previous experiment in the same area (D'Anna et al., 2004). However, even if 52% of our R+ seabream was sighted within 1 km from the release site (Table 3), the remaining 48% spread along the coast up to a distance of 11 km from the release site. In contrast, the dispersion pattern of R+ fish was similar to that of wild individuals. In particular, R+ individuals appear to be reluctant to cross large portions of bare sand, possibly due to the lack of adequate shelter against predation (D'Anna et al., 2011; MacPherson, 1998; Vega Fernández et al., 2008). A wide dispersal of naïve HR white seabream was also observed along the southern Portugal coast (Santos et al., 2006) where more than 80% of recaptures occurred at a distance between 18 and 37 km from the release site. The authors explained the dispersal pattern with the incapacity of naïve fish to perceive environmental stimuli useful for their settlement, such as the presence of refuges and food resources. In our experiment white seabreams were fed with pellets scattered directly on the tank bottom in order to force fish to overcome the general tendency of HR fish to search for food in the water column. However a feeding behaviour deficit can cause starvation in released HR fish contributing to post-release mortality (Brown and Day, 2002; Brown et al., 2003; Suboski and Templeton, 1989), although no evident effect on dispersal has been demonstrated. La Mesa et al. (2008) found HR grouper not conditioned with a refuge to exhibit a wide dispersal in spite of their pre-release conditioning with live prey. Such dispersal pattern was explained with the limited carrying capacity of the artificial reef where they had been released.

5 5. Conclusions

4 3 2 1 0 P+R+

P+R+

P-R+

P-R-

Fig. 5. Mean and standard error (S.E.) of the distance (D) in kilometres (km) from the release site of white seabreams for each experimental group (P − R+, P+ R+, P+ R−, P − R−). See text for details.

This study indicates that predator and shelter conditioning of HR white seabream is an effective practise to increase their post-release survival at sea. The adopted conditioning procedures resulted in an effective training of fish to recognise predators and to use refuges efficiently. Our hypotheses were confirmed by the results of the experiments in the arena and received further strength from postrelease monitoring at sea, where a higher survival and a shorter dispersal of conditioned fish were observed. Restocking plans over a wider scale would benefit from some practical aspects of our conditioning protocol, such as the use of one single live predator and of

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a few refuges to train 500 fish. Our findings provide potential for future stock enhancement initiatives with white seabream, considering that an increased survival of released fish would result in an increased number of fish reaching their adulthood. However, many gaps remain in our knowledge of post-release behavioural performance and survival of HR white seabream. Among them, the density of fish to be released in relation to the carrying capacity of the environment and the impact of released fish on the wild fish assemblage. Acknowledgements We wish to thank the following: Gaspare Barbera of “Ittica Acquazzurra” hatchery farm; Amelia Roccella for volunteer collaboration; Paolo Evola, Andrea and Antonino Di Maria for fishing the conger eels. We are also grateful to Giovanni Cicchirillo for his precious help through the whole conditioning phase at Capo Granitola. References Alfieri, M.S., Dugatkin, L.A., 2009. 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