Anti-predator response in wild and sea-ranched brown trout and their crosses

Aquaculture 253 (2006) 218 – 228 www.elsevier.com/locate/aqua-online

Anti-predator response in wild and sea-ranched brown trout and their crosses Erik Petersson a,b,⁎, Torbjörn Järvi c,d a

National Board of Fisheries, Institute of Freshwater Research, Stångholmsvägen 2, SE-178 93 Drottningholm, Sweden b Department of Animal Ecology, EBC, Uppsala University, Norbyvägen 18 D, SE-752 36 Uppsala, Sweden c National Board of Fisheries, Institute of Freshwater Research, SE-117 81 Drottningholm, Sweden d Department of Population Genetics, Stockholm University, SE-106 91 Stockholm, Sweden Received 18 May 2005; received in revised form 19 May 2005; accepted 27 August 2005

Abstract In this study we compared the anti-predator responses of hatchery-reared brown trout (Salmo trutta) juveniles having searanched or wild origins, or the reciprocal crosses between wild and sea-ranched fish. The experimental fish were exposed to a pike and a heron predator dummy. It was found that juveniles of wild origin differed from those of sea-ranched origin in their response to the predator attacks. Moreover, the wild fish also differed in this regard from the crosses between wild and sea-ranched brown trout. The responses differed mainly in terms of response to the heron dummy and response time to first attack (i.e. duration of initial inactivity when subject to first attack). However, the reciprocal crosses were not intermediate in their response patterns, canonical discriminant analysis revealing that they were closer to the sea-ranched than to the wild fish in this regard. The fish studied were reared under similar hatchery conditions and had a common genetic background, the gene flow from the sea-ranched fish to the wild segment being considerable. Thus, the differences between fish born in the wild and in hatcheries might reflect genetic differences recurrently develop within single-year classes. © 2005 Elsevier B.V. All rights reserved. Keywords: Hatchery selection; Salmo trutta; Anti-predator behaviour

1. Introduction Predation is a strong selective force in the evolution of behavioural, morphological, and life-history traits (Lima and Dill, 1990; Chivers and Smith, 1998). The ability of a prey animal to avoid being captured by a predator often requires that it be able accurately to assess current predation risk. One way to avoid predators is to hide or to avoid areas where predators may show up. A frequent consequence of such behaviour, however, is that the ⁎ Corresponding author. National Board of Fisheries, Institute of Freshwater Research, Stångholmsvägen 2, SE-178 93 Drottningholm, Sweden. Tel.: +46 8 699 06 02; fax: +46 8 699 06 50. E-mail address: [email protected] (E. Petersson). 0044-8486/$ - see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2005.08.012

feeding rate is minimised. Fish, like most other animals, must make decisions that do not simply maximise their predator avoidance but also include other ecologically important parameters, such as feeding. During foraging, anti-predator behaviour is selectively favoured when the individual optimises the trade-off between the cost, in terms of reduced growth, of performing the behaviour and the gain in terms of an increased probability of escaping the predator (Ydenberg and Dill, 1986; Bennett and Houston, 1989; Leonardsson, 1991). The ability to escape from an attacking predator, or predators that could eventually attack, is dependent upon speed, agility, vigilance, and endurance (cf. Brodie, 1989; Lima and Dill, 1990). Genetic control could play a decisive role in

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such a trade-off (e.g. Magurran, 1990; Johnsson and Abrahams, 1991; Fernö and Järvi, 1998; cf. Abrahams and Sutterlin, 1999), but a learning process is most likely involved as well (e.g. Dill, 1974; Järvi and Uglem, 1993; Jachner, 2001). Growth rate has been established as a key parameter influencing foraging decisions that involve risk of predation (Abrahams and Sutterlin, 1999). In the hatchery environment, risk-taking phenotypes may be favoured as the predation pressure is relaxed or absent; this allows rapid growth in comparison with what would be expected under natural conditions (e.g. Johnsson and Abrahams, 1991). Indeed, Johnsson et al. (1996) noted that the offspring of sea-ranched anadromous brown trout (Salmo trutta) visited predator-frequented areas to obtain food more frequently than did the offspring of wild fish. Several studies have shown that hatchery selection alters risk-sensitive foraging decisions (see Johnsson et al., 1996, and references therein). However, a few studies have observed that hatchery rearing may also alter the behavioural response to an attacking predator. Fernö and Järvi (1998) observed that brown trout parr of searanched origin were more likely to swim or sink to the bottom (freeze) than were wild parr. Wild parr more often escaped by panic swimming. In their study they used a pike dummy to scare the experimental fish, finding no difference in reaction distance (i.e. the distance at which the experimental fish was observed to react on the approaching pike dummy) between wild and sea-ranched parr. As fish may respond differently to different predators (e.g. Dannewitz and Petersson, 2001), it would be interesting to investigate how wild and sea-ranched fish respond to fish vs. to bird predators. For example, in the hatchery fish are only subject to “attacks” from above, for example, hatchery staff who pick up dead fish or fill the feeders with pellets. All these “attacks” are false, and individuals that ignore them will gain an advantage over individuals that actually respond. Non-responders will, for example, use less energy by not fleeing and might gain more time for feeding. Thus, in a hatchery strain, selection might occur favouring individuals that show lower response behaviour to predator attacks from above. For fish predation the situation might be different. Hatchery fish always have other fish around, and always have to compete with them for food and space. This might help to keep up their vigilance towards movements and hence potential threats in the water. The gene flow from the sea-ranched strain to the wild segment in River Dalälven brown trout might be as high as 80% (Palm et al., 2003), making the genetic difference between the strain (based on neutral markers) almost negligible. Despite this, earlier studies have found

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several differences between wild and sea-ranched brown trout, in for example growth rate and behaviour (e.g. Johnsson et al., 1996; Lepage et al., 2000; Petersson and Järvi, 2003); but in this study we test the differences between juveniles of sea-ranched fish and juveniles of wild origin with regard to reaction—to attacking predators. The differences might be greatest in the case of reaction to attacks from above (e.g. heron predation) as compared with reaction to fish predation (e.g. pike predation). We also studied crosses between wild and sea-ranched fish, our assumption being that the crosses should show intermediate response patterns if the pure strains showed differences in any respect. 2. Material and methods 2.1. Background and strains used The study was conducted at the Fishery Research Station at Älvkarleby, central Sweden, a research station situated on the Dalälven River that flows into the Gulf of Bothnia. Diadromous fishes are prevented from following their natural migration route by the hydropower dam at Älvkarleby. Adult salmon and brown trout migrating upstream are caught in a case at the upstream end of a short fish ladder and transported to a sorting hall, where they are sorted and kept for artificial breeding. Two strains of anadromous brown trout currently occur in the river, both evolved from the same population. One strain was established in 1967, when a large number of trout were caught and used for an artificial breeding program for sea ranching. This sea-ranched strain (S) has since been kept separate from other strains. The released offspring of S trout are marked by cutting off the left pelvic fin, while wild (W) trout are identified by having both pelvic fins intact (see Petersson et al., 1996, for more details about the two strains). All the cultured fish are released as smolts (2+). The sea-ranched strain has not been actively selected for anything, i.e. all differences from a natural baseline is due to domestication. 2.2. Crossing procedure In the 1997 spawning season 40 brown trout females (20 wild and 20 sea-ranched) and 40 males (20 wild and 20 sea-ranched) were used to create 10 families each of four different cross-bred types: S female × S male, S female × W male, W female × S male, and W female × W male. The eggs of half of the families were further divided into two batches, giving a total of 60 groups. Practical problems meant that two groups had to be

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omitted, leaving 58 groups. These groups were reared separately in 0.5 × 1-m tanks under normal hatchery conditions. The juveniles from these groups were used in the experiments. 2.3. Experimental procedure The study was conducted from June to November in 1998. The average weight of the fish was 0.816 g (min: 0.313 g, max: 1.17 g) on the first experimental day (29 June), and 8.99 g (4.38 g, 14.14 g) on the last experimental day (6 November). The experimental setup resembles that of Dannewitz and Petersson (2001). Two trays measuring 300 × 60 cm were used so that the behaviour of two brown trout could be studied simultaneously. Each tray was divided into two sections separated by a perforated transparent plastic wall that prevented the trout from swimming through but allowed the water current to pass (Fig. 1). A heron dummy (head and neck, natural size) was placed over one section, and a pike dummy (250 mm total length) was placed over the other. Predator models were made of wood (the pike dummies had some wood replaced by lead, in order to prevent it from floating), shape and colours were as natural as possible (which included eyes), and both models were manoeuvred from a separate room. The heron dummy attacked the brown trout from above, the time from initiation of the attack until the bill hit the water being 0.2 s. The pike dummy was run along a thin fishing line through a curtain of black plastic strips, at a maximum speed of ca. 70 cm s− 1, and stopped immediately before the transparent plastic wall (cf. Fernö and Järvi, 1998; Dannewitz and Petersson, 2001; Fig. 1). The curtain of plastic strips made it impossible 69 cm

for the trout to see the pike before an attack had been initiated. After an attack, the predator model was immediately returned to its original position. Two video cameras were placed over the experimental area, each camera taking in one tray, making it possible to study and record the behaviours without disturbing the fish. Groundwater (7.5–8.9 °C) was used in the experimental trays because the river water was too muddy to permit unimpeded observation of the fish. In addition, by using groundwater, the temperature and quality of the water were about the same in all trials. The brown trout were transferred to a holding tank initially containing throughflow river water two days before the anti-predator behaviour was studied. Over the course of the first day the river water was gradually replaced with groundwater, and the fish were then allowed to acclimatise for another day. After introducing the fish into the experimental tray, the initial pike attack was performed when the brown trout first held a position against the water current in front of the transparent wall (the “predator area,” Fig. 1). When the brown trout again took position within the predator patch, a heron attack was performed. Each fish stayed a maximum of 2 h in the experimental tray, and if there was time left after one pike and one heron attack, the procedure was repeated so that an individual was exposed to a maximum of three pike and three heron attacks. After the experiment, the brown trout were killed by an overdose of anaesthetic (2-phenoxyethanol), and their lengths and weights measured. The types and durations of anti-predator behaviours were later determined by analysing the videotapes. Two main types of reaction were observed, burst

83 cm

Water

Water outlet

C

D

B

A

inlet

60 cm

300 cm Fig. 1. The experimental set-up used to study anti-predator behaviour of brown trout juveniles. The trout was placed in the ‘triangular’ section (A). Water entered this section through a perforated transparent plastic wall. When the trout visited the predator area (about where the letter ‘a’ is placed in the figure), a predator attack was performed. The pike model started its attack at (B), but could not be viewed until it entered through a curtain made of plastic strips (C). The heron model was placed over the predator patch (D) and the attack was directed down towards the trout. The video camera was placed over the experimental tray and recorded section (A) and the area between the perforated plastic wall and the black curtain. For more details see text.

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swimming (fleeing) and resting motionless on the bottom (freezing). However, most individuals showed a combination of these two behaviours, either first freezing and then fleeing, or vice versa. From the videotapes we also estimated (1) the length of time until the fish first visited the predator patch for the first time, (2) reaction distance in mm from the attacking pike, (3) time until the brown trout reacted to the attacking heron (number of video frames, recalculated into milliseconds), (4) the time spent fleeing after an attack (estimated separately for heron and pike), and (5) time spent freezing after an attack (estimated separately for heron and pike). This gives seven behavioural variables that have been shown to provide good measures of risk taking (Fernö and Järvi, 1998; Dannewitz and Petersson, 2001).

2.4. Data sets extracted from the observations A total of 533 fish were tested for anti-predator behaviour. Initially eight individuals from each group (i.e. 464 fish) were to have been tested. However, some fish had to be excluded because they failed to respond to at least two predator attacks, or did not move at all for several hours. Those fish were replaced with new fish from the same group. During subsequent video analyses we noted that some fish that were thought not to have reacted had actually reacted, and these were included in the analyses. Others turned out to have left the predator area before the predator model was released, and these fish were excluded. In total we ended up including 509 fish in the analysis. This data set (full data set) consisted of all fish that had been exposed to at least one heron and one pike attack, but only the two first attacks (one pike and one heron) for each individual were included in the full data set. A subset of the 509 individuals could be exposed to six attacks (three heron and three pike). This data set (repeated data set) were used for estimating habituation (see below). Another subset (tank data set) consisted of the 20 families that were split and raised in two different tanks. This subset was used for checking if the individuals from the two tanks differed in terms of anti-predator response (i.e. tank effect). In the tank data set only the two first attacks were included. Finally, during the first two weeks some additional fish (pilot data set) were tested to investigated whether or not the response to the first attack by one type of predator differed if the fish had previously experienced an attack by the other type, which also

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was regarded as a possible way for the fish to habituate. 2.5. Statistical procedure 2.5.1. Adjusting data For one variable (reaction time to heron dummy) there was a significant effect of date (p = 0.026) and for fleeing time after pike attack there was a significant effect of the size of the fish (p = 0.037). However, according to a MANCOVA there was no overall effect of date and/or size (date: Wilks' λ = 0.709, p = 0.175; length: Wilks' λ = 0.944, p = 0.904), therefore size and date have not been included in the subsequent analyses. The differences in freezing and fleeing time between different behavioural combinations (i.e. freeze–fleeing, fleeing–freeze). In order to simplify the analyses and reduce the number of variables all data on freezing and fleeing time were merged for each individual. Prior to merging the values were adjusted for by standardising the data (adjusted value = (x − mean) / std). The adjusted mean values for each tank, variable, and predator attack were then calculated and used in subsequent analysis. All analyses were made using SAS statistical software. Because the 509 individuals used in this study were sampled from 58 tanks the individual observations were not independent datapoints. This was adjusted for in two ways. The nested ANOVA (tank data set) and G-tests (full data set) have been adjusted for multiple sampling, using Šidák correction (Šidák, 1967), and the remaining analyses (repeated data set, pilot data set, and full data set) were calculated on group means (see above). 2.5.2. Tank effect Using the tank data set we checked, using nested analysis of variance (ANOVA) (tank nested with family), whether the individuals from the two tanks differed in terms of anti-predator response. 2.5.3. Habituation I The possible effects of habituation were analysed using repeated ANOVA on six of the variables (time to first attack excluded) on the repeated data set. As habituation seemed likely (see below) and in order to increase the sample sizes (or rather the number of observations used to calculate the mean values), only the first pike and the first heron attack are considered below. In addition, the sexes of young brown trout have been shown to differ in terms of anti-predator behaviour, not in the first attack by a heron dummy, but in response to the second attack (Johnsson et al.,

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Table 1 The results from the pilot study showing the adjusted values (see text) for fish that were attacked first with one type of dummy compared with those that were attacked first with that same type of dummy only after previously having been attacked with the other type of dummy Attack Mean ± std sequence

No. obs. t-value P-value

Pike dummy Reaction 1 distance 2 Freezing 1 time 2 Fleeing 1 time 2

− 0.030 ± 0.235 − 0.032 ± 0.109 − 0.088 ± 0.699 0.727 ± 0.292 − 0.041 ± 0.468 −0.112 ± 0.276

20 12 13 5 17 12

0.020

0.983

1.600

0.134

0.350

0.732

Heron dummy Reaction 1 time 2 Freezing 1 time 2 Fleeing 1 time 2

0.012 ± 0.029 0.002 ± 0.076 0.571 ± 0.111 0.061 ± 0.677 − 0.175 ± 0.374 0.075 ± 0.448

6 17 5 14 8 15

0.240

0.812

1.030

0.319

0.900

0.383

For example, for the pike dummy, 1 indicates that the first attack was done with the pike dummy, while 2 indicates that the second attack was done using the heron dummy. No. obs. shows the number of individuals showing that type of response; for example, for the heron dummy only six individuals were possible to analyse regarding reaction time. The total number of fish used was 22 when pike dummy was the first attack, and 20 when the heron dummy was used first.

2001). As the youngest fish used in this experiment were very small (see above) and hard to sex correctly, there is a second argument for only including the first attacks. 2.5.4. Habituation II The data from the pilot data set were used for comparing fish that were attacked first with one type of dummy compared with those that were attacked first with that same type of dummy only after previously having been attacked with the other type of dummy. The data were analysed using t-test. 2.5.5. Analyses of the full data set To analyse any eventual differences between the crosses in terms of anti-predator response, MANOVA was used; each variable was post hoc analysed using a pair-wise t-test on least-square means. In order to analyse the overall response between the four cross types, a canonical discriminant analysis was performed. The relations between the cross types were measured using the Malahanobis squared distances between groups. Intercorrelations between the variables were analysed using pooled, within cross-type correlations. The frequencies of different types of reaction in relation to type of predator dummy were analysed using G-test.

3. Results 3.1. Tank effect No significant effects were found for any of the seven variables examined in this study, and the nested multivariate analysis of variance (MANOVA) emphasised this (Wilks' λ = 0.155, F259, 763.75 = 0.92, P = 0.797). Therefore the tank effects were regarded as negligible. 3.2. Habituation I In a pilot study we investigated whether or not the response to the first attack by one type of predator differed if the fish had previously experienced an attack by the other type. No such effects were noted (Table 1). 3.3. Habituation II As can be seen in Table 2 (based on the repeated data set), the cross types differed in terms of one of the variables (time freezing after a heron attack). Time effect, or habituation, was noted for four of the variables, but reaction time for the heron dummy and time freezing after a pike attack were not affected by repeated attacks. In addition, there was no interaction Table 2 F-values and levels of significance for between cross-type effects and within cross-type effects (repeated ANOVA) Between cross-type effect (df = 3, 52)

Pike dummy Reaction distance Time freezing Time fleeing

Heron dummy Reaction time Time freezing Time fleeing

Within cross-type effects Time (df = 2, 104)

Time × cross-types (df = 6, 104)

F = 0.77, P = 0.516 F = 0.96, P = 0.419 F = 0.39, P = 0.761

F = 4.29, P = 0.016 F = 2.06, P = 0.133 F = 3.87, P = 0.024

F = 1.01, P = 0.423 F = 1.12, P = 0.356 F = 0.22, P = 0.970

F = 1.15, P = 0.338 F = 3.65, P = 0.018 F = 0.65, P = 0.567

F = 0.35, P = 0.706 F = 3.32, P = 0.040 F = 4.99, P = 0.009

F = 1.11, P = 0.362 F = 1.32, P = 0.256 F = 0.73, P = 0.626

The analysis was performed on a subset of the data set, i.e. only those brown trout juveniles that were subjected to six predator dummy attacks (three heron attacks and three pike attacks).

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Fig. 2. Frequency of threes types of responses of brown trout juveniles to attacks by pike and heron dummies. Four different brown trout crosses were used: sea-ranched ( ), sea-ranched · wild (▴), wild · sea-ranched (▾), and wild (●). Solid symbols represent values for the pike dummy and open symbols represent values for the heron dummy.

▪

between time and cross types, i.e. the cross types showed no difference in habituation pattern.

was also the case after freezing–fleeing, the least frequent type of reaction, had been excluded from the analysis (G = 70.50, P < 0.001).

3.4. Analyses of the full data set 3.4.1. Type of predator dummy The fish reacted differently to the pike and heron dummies (Fig. 2). For example, fleeing followed by freezing was more frequent after a heron attack than after a pike attack (G-test: G = 70.67, P < 0.001). This

3.4.2. Intercorrelations The pooled, within cross-type correlation coefficients revealed that no variables were correlated. However, a tendency was noted between reaction time to heron dummy and time freezing after a heron attack (see Table 3).

Table 3 Pooled within cross-type correlation coefficients and p-values for the seven variables examined in this study of anti-predator behaviour in brown trout juveniles

Time to first attack Reaction time (H)

Reaction time (H)

Time freezing (H)

Time fleeing (H)

Reaction distance (P)

Time freezing (P)

Time fleeing (P)

0.031 P = 0.822

0.046 P = 0.741 − 0.242 P = 0.075

− 0.001 P = 0.995 0.078 P = 0.570 − 0.077 P = 0.576

0.203 P = 0.138 − 0.008 P = 0.955 0.103 P = 0.452 0.103 P = 0.455

− 0.022 P = 0.872 − 0.142 P = 0.302 0.044 P = 0.751 − 0.188 P = 0.170 0.043 P = 0.754

0.041 P = 0.764 0.078 P = 0.569 − 0.078 P = 0.571 0.177 P = 0.197 0.028 P = 0.839 − 0.191 P = 0.163

Time freezing (H) Time fleeing (H) Reaction distance (P) Time freezing (P) H = heron dummy; P = pike dummy. Sample size = 58 in all cases.

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a) F3,54=3.07; p=0.035

500 b a

a

a

100 50

c) F3,54=1.27; p=0.293 Pike: freezing time (s)

15.0 12.5 10.0 7.5 5.0

a 0.20

a ab

b

15.0 12.0

d) F3,54=1.40; p=0.254

9.0 6.0

3.0

1.5

2.5

e) F3,54=3.06; p=0.036

15.0 12.0

3.5

10.0 8.0 6.0

c

bc

4.0 a

ab

2.0

Pike: fleeing time (s)

Pike: reaction distance (m)

0.25

0.15

10

Heron: freezing time (s)

b) F3.54=2.57; p=0.064

0.30

Heron: reaction time (s)

Time to first attack (s)

1000

f) F 3,54=0.03; p=0.994

3.0 2.5 2.0 1.5 1.0

Heron: fleeing time (s)

g) F3,54=2.91; p=0.043 3.5 3.0 2.5 2.0

a ab

ab b

1.5

1.0 Fig. 3. The outcome of analysis of the seven variables associated with anti-predator behaviour in juvenile brown trout. The graphs show mean values and standard deviations (log-transformed values), the dots next to the means being the raw data points. Means denoted with the same letter were not different at the 5% level (pair-wise t-test). Symbols are similar to those used in Fig. 2, except that all symbols are filled.

E. Petersson, T. Järvi / Aquaculture 253 (2006) 218–228 Table 4 Statistics from the canonical discriminant analysis: Malahanobis pairwise squared distances between cross types (D2), F-value (ndf = 7, ddf = 48), and significance value for each comparison

S·S

S·W

W·S

S·W

W·S

2

2

D = 0.913 F = 0.808 P = 0.585

D = 1.863 F = 1.648 P = 0.145 D2 = 1.533 F = 1.460 P = 0.204

W·W D2 = 2.994 F = 2.648 P = 0.022 D2 = 3.030 F = 2.886 P = 0.014 D2 = 2.702 F = 2.574 P = 0.025

3.4.3. Anti-predator responses of the crosses Overall differences were observed for three of the seven variables examined in this study (Fig. 3): time to first attack, reaction time to heron dummy, and time freezing after a heron attack. In the first case, wild fish waited longer before entering the predator area than did the three cross types (Fig. 3a). Wild fish also displayed the shortest reaction time to the heron attack, although they were not significantly different from the SW fish in this regard (Fig. 3b). Wild fish also displayed the longest freezing time after a heron attack, although they were not significantly different from the WS fish in this regard (Fig. 3e). The WS fish displayed the shortest fleeing time after a heron attack (not significantly different from wild and SW fish) (Fig. 3g). The MANOVA revealed an overall difference between the cross types

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when all seven variables were examined (Wilks' λ = 0.352, F33, 130.3 = 1.68, P = 0.0218). The canonical discriminant analysis also revealed that the cross types differed in anti-predator response (Wilks' λ = 0.473, F21, 138.4 = 1.96, P = 0.0113). The pairwise comparisons showed that wild fish differed significantly from all other cross types (Table 4, cf. Fig. 4). There were no significant differences between the other three cross types (Table 4, cf. Fig. 4). 4. Discussion This study shows that juvenile brown trout of wild origin differed both from juveniles of sea-ranched origin and from crosses between wild and sea-ranched brown trout in terms of their response to predator attack. It should, however, be noted that the two types of dummies used differ in many respects (i.e. the way they were presented to the fish), which makes a direct comparison between the dummies problematic. On the other hand, a comparison of the response of the crosses to each of the two type of dummies probably is more meaningful. The differences in response were mainly found for the responses to the heron dummy and time to first attack (see Fig. 3). However, the SW and WS cross types were not intermediate in response pattern, canonical discriminant analysis revealing that they were closer to the sea-ranched cross type. For the WS and SW cross types to produce an overall intermediate pattern, most of the variables included in this study would have to have mean values as in Fig. 3d and e, but this was not the case.

Fig. 4. The outcome of the canonical discriminant analysis of the seven variables associated with anti-predator behaviour in juvenile brown trout. The first axis explained 57.9% of the variation and had especially high positive loading for time to first attack and freezing time after heron attack. The second axis explained 29.2% of the variation and had especially high positive loading for reaction time to heron attack and freezing time after heron attack, and high negative loading for fleeing time after heron attack. The length of the axis is proportional to its explanatory power. Symbols are similar to those used in Fig. 3, except that the symbols for sea-ranched fish are white, crosses are grey, and wild are black. The class (cross type) means for the first and second canonical axis, respectively, are as follows: SS: − 0.484, − 0.470; SW: − 0.542, −0.241; WS: − 0.181, 0.807; WW: 1.143, − 0.159.

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The sea-ranched and wild fish did not differ in type of response, about equal proportions reacting with fleeing, freezing, and fleeing–freezing (Fig. 2). This contrasts to an earlier study performed on the same strains, but examining only “pure” cross types (Fernö and Järvi, 1998). This study found that wild fish, when attacked by a pike dummy, fled more frequently than did searanched fish, and that wild fish froze less often than did sea-ranched fish. However, the present study takes a much more conservative approach. The present study focused on how the fish responded to a predator attack, not on how they would attempt to avoid an attack. Obviously, both types of behaviours are important for fish survival in a wild habitat. We still do not understand how these two behavioural processes relate to each other, or their relative importance to the survival probabilities of an individual. A study of the inanga (Galaxias maculatus) showed that while fish may not be able to learn the daily pattern of predation risk, they are capable of learning the daily pattern of food availability (Reebs, 1999). This indicates that a suitable response to a sudden and unexpected predator attack is necessary; an avoidance strategy, though important, might not be good enough on its own. Fish use many tactics, such as shoaling and reduced movement, in response to predation risk. Daily habitat shifts could be rooted in the avoidance of predation, but instead of being the direct result of learning could mostly be innate (cf. Reebs, 1999). Released hatchery fish often show lower survival rates than do wild conspecifics. Einum and Fleming (2001) noted that hatchery fish consistently experienced reduced survival, and in 15 of 16 reviewed studies wild fish were found to do better. The lower success of released hatchery-reared fish most likely stems from several factors besides just anti-predator behaviour, factors such as feeding behaviour (e.g. Johnsen and Ugedal, 1990), aggressiveness (e.g. Hedenskog et al., 2002), morphological characteristics (e.g. Riddell and Leggett, 1981), and physiological characteristics (e.g. Fleming et al., 2002). In addition, some of these characteristics have been shown to change after hatchery-reared fish are released into the wild. For example, L'Abeé-Lund and Langeland (1995) found that while the diet of released brown trout initially differed from that of wild trout, within the first summer the released fish had adopted a similar diet. This shows that released hatchery fish possess the ability to learn what to eat. Similarly, anti-predator conditioning can improve post-release survival, as predator recognition and avoidance behaviour in juvenile salmonids improves in fish exposed to

predators (Potter, 1977; Olla and Davis, 1989; Bejerikian, 1995) or to odours from injured conspecifics (Brown and Smith, 1998; Bejerikian et al., 1999). Most of the differences between wild-born and released hatchery-reared fish might be due to their different rearing environments. However, this study shows that there also seems to be a genetic component. In conclusion, the anti-predator response differed between sea-ranched and wild (naturally produced) brown trout. The results of this and previous studies (e.g. Johnsson et al., 1996; Lepage et al., 2000; Petersson and Järvi, 2003) indicate that the differences between wild and sea-ranched fish exist despite the unidirectional gene flow from the sea-ranched to the wild fish. Under such circumstances the populations are expected to remain almost identical (Ford, 2002). There might be two explanations for this. First, the observed differences represent nongenetic maternal effects caused, for example, by differences in the size (cf. Jonsson et al., 1996), juvenile growth (McAdam and Boutin, 2003) or quality (cf. Lombardi, 1996) of eggs laid by wild vs. sea-ranched females. Previous experiments have not been designed to allow discrimination between this and other sources of variation. In this study, the overall effect revealed by the discriminant analysis (Table 4) showed a confusing pattern. Nevertheless, SS fish were closest to SW than to any other cross type, and WW fish were closest to WS fish than to any other cross type. Thus, this explanation cannot be ruled out. Second, the differences reported in previous studies might reflect genetic differences between fish born in the wild and in hatcheries. These differences recurrently develop within single-year classes, but are counteracted by a strong gene flow preventing cumulative differentiation over many generations (cf. Palm et al., 2003). Assuming additive genetic variance for the traits studied and a marked difference in selective regime between wild and hatchery environments, some degree of genetic divergence may be generated from the egg to adult stage within a single-year class, even when a majority of the wild trout have parents born in the hatchery. In this way comparison of offspring derived from returning searanched and wild adults may yield trait differences in spite of a high level of gene flow. Indeed, Crozier (1998) showed that divergence in genetic composition could be derived in just one generation. However, as can be seen in Fig. 4, the variation is large and it is not known to what extent the differences noted in this study actually affect the survival of hatchery-reared fish when they are released. It is also not known how these genetic effects relate to environmental effects. For example, if hatchery-reared fish of both wild and sea-ranched origin are released the

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differences in survival might be hard to detect (Dannewitz et al., 2003). The large effects in just one generation might be hard to accept, but several other studies have noted significant changes in different trait in just one generation. For example (Mazzi et al., 2004) noted that female sticklebacks being inbred for one generation exhibited stronger preference for symmetric male models than an outbred control. In two species of Darwin finches showed significantly changes in bill shape and body size from one generation to the next, due to climatic conditions (Grant, 2003). Finally, Salonen and Peuhkuri (2004) noted that one generation hatchery history was enough for significantly reducing levels of aggressiveness in European grayling. Thus, the effects noted in our study might be expected when the wild and hatchery environment differs greatly in their respective selective regimes. Acknowledgements We would like to thank Bengt-Åke Jansson and Anna-Carin Löf for their assistance throughout the study. They did over half of the time-consuming task of waiting for the fish to respond, and Anna-Carin Löf also analysed the video recordings. Lars Helling made the pike and heron dummies as well as constructing most of the experimental set-up. Bjarne Ragnarsson and his staff at the Fishery Research Station reared the fish, all the way from milt and unfertilised eggs to plucky small fry. Three anonymous referees gave valuable comments on the first submitted version. Stephan Sanborn at Proper English AB made linguistic improvements on an earlier version of the manuscript. The work was carried out with financial support from the Swedish Council of Forestry and Agricultural Research and from the Commission of the European Communities, Agriculture and Fisheries (FAIR) specific RTD programme, CT-97-3498, Performance and Ecological Impacts of Introduced and Escaped Fish: Physiological and Behavioural Mechanisms. The present research does not necessarily reflect its views and in no way anticipates the Commission's future policy in this. The experimental work, conducted under licence no. 34 3632/92, complied with the standards and procedures stipulated by the Swedish Ministry of Agriculture. References Abrahams, M.V., Sutterlin, A., 1999. The foraging and anti-predator behaviour of growth-enhanced transgenic Atlantic salmon. Anim. Behav. 58, 933–942. Bejerikian, R.A., 1995. The effects of hatchery and wild ancestry and experience on the relative ability of steelhead trout fry (Oncor-

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