acta ethol (2011) 14:1725 DOI 10.

1007/s10211-010-0086-1

ORIGINAL PAPER

Boldness as a consistent personality trait in the noble crayfish, Astacus astacus

Anssi Vainikka & Markus J. Rantala & Petri Niemel & Heikki Hirvonen & Raine Kortet

Received: 20 January 2010 / Revised: 30 September 2010 / Accepted: 25 October 2010 / Published online: 13 November 2010 # Springer-Verlag and ISPA 2010

Abstract Consistent individual differences in behavioural responses to perceived predation risk may have extensive ecological and evolutionary implications. We studied the repeatability of boldness across time and its relation to resource holding potential in the noble crayfish, Astacus astacus L., using predator-nave immature individuals. We followed individuals shelter use both with and without exposure to the chemical and physical cues of predators, and with and without the presence of a conspecific. In addition, we examined if armament, i.e. relative chelae size, would be correlated with individual differences in behaviour. Individuals showed repeatable behaviours across time and context. Individuals that occupied the shelter in competitive dyadic tests also spent more time in the shelter during individual control observations, suggesting that boldness is a personality trait that does not necessarily relate positively to high resource holding potential in the noble crayfish. The relative size of chelae did not correlate with any of the measured behavioural variables. Our results suggest that boldness can be considered as individually
A. Vainikka (*) : P. Niemel Department of Biology, University of Oulu, P.O. Box 3000, 90014, Oulu, Finland e-mail: anssi.vainikka@oulu.fi M. J. Rantala Department of Biology, University of Turku, 20024, Turku, Finland H. Hirvonen Department of Biosciences, University of Helsinki, P. O. Box 65, 00014, Helsinki, Finland R. Kortet Department of Biology, University of Eastern Finland, P.O. Box 111, 80101, Joensuu, Finland

consistent and ecologically important personality trait in the noble crayfish. Keywords Antipredatory behaviour . Behavioural syndrome . Decapoda . Domestication effect . Life history . Repeatability

Introduction Perceived predation risk is considered as important as direct predation mortality for the function of ecological systems (Preisser et al. 2005). Perceived risk of predation affects foraging decisions and, therefore, alters the form and strength of trophic interactions in many species (e.g. Lima and Dill 1990; Lima and Bednekoff 1999; Carvalho and Del-Claro 2004; Nystrm 2005). However, individual animals show repeatable differences in their sensitivity to predation risk. This suggests the existence of individual coping styles, animal personalities or behavioural syndromes among individuals (Dall et al. 2004; Bell 2007; Sih and Bell 2008; Stamps and Groothuis 2010). By the definition of Stamps and Groothuis (2010), the term behavioural syndrome refers to behaviours that are correlated across time or contexts. Here we use term personality trait to refer to one type of behaviour that is temporally repeatable and consistent across contexts. Animal personalities, which consist of several personality traits, are potentially important in the function of food webs by inducing behaviourally mediated cascade effects (e.g. Ioannou et al. 2008). Also, individual variation in personality traits is potentially important in behavioural adaptation to predation (Dall et al. 2004; Sih et al. 2004; Rale et al. 2007). For example, consistently bold individuals may gain fitness advantages in low predation risk environments,

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whereas consistently shy individuals may have superior fitness in high predation risk environments (Sih et al. 2004). Boldness traits have been found to be heritable in some (Sinn et al. 2006; Brown et al. 2007; Dingemanse et al. 2009) but not all species (e.g. Riesch et al. 2009). Questions pertaining to the development of behavioural syndromes become particularly interesting when intraspecific behavioural interactions, such as dominance relationships, are considered. The most well-known behavioural syndrome is the aggressivenessboldness syndrome in which individuals that behave boldly under the risk of predation are also more aggressive towards conspecifics (Huntingford 1976; Riechert and Hedrick 1993; Kortet and Hedrick 2007). Such syndromes can be especially important in socially hierarchical species such as crayfishes. For example, dominance hierarchies affect food uptake levels of individual crayfish (Ahvenharju and Ruohonen 2006). In addition, crayfishes are dependent on the availability of burrows, which provide shelter, and often compete aggressively for them (Ranta and Lindstrm 1993; Garvey et al. 1994; Figler et al. 1999). When several behaviours in different contexts promote high food uptake rates, behavioural syndromes may become heritable through pleiotropy or genetic correlations (Stamps 2007; Biro and Stamps 2008). Therefore, high food uptake rates in crayfish, if promoted by both boldness and aggressiveness, would be expected to result in the development of an aggressiveness boldnessbehavioural syndrome. Supporting results have been obtained in the signal crayfish Pacifastacus leniusculus Dana, in which aggression, voracity and boldness were all positively correlated (Pintor et al. 2008). However, it is not known if shelter possession, potentially achieved through aggressive behaviour (e.g. Figler et al. 1999), would be coupled with boldness in the noble crayfish, Astacus astacus L. Many aquatic organisms, including crayfishes, can detect the presence of predators through chemical cues (Appelberg et al. 1993; Blake and Hart 1993; Kats and Dill 1998). The noble crayfish is a species native to Finland and has a long co-evolutionary history with fish predators such as the Eurasian perch, Perca fluviatilis L. Predator-nave juvenile noble crayfish increase shelter use and reduce feeding activity when exposed to the chemical cues from predatory fishes such as the perch, the pike, Esox lucius L., the burbot Lota lota L. or the European eel, Anguilla anguilla L. (Appelberg et al. 1993; Hirvonen et al. 2007). In this study, perch odour and physical disturbance were used to induce/ mimic predation risk. Large armaments may facilitate the establishment of a strong position in dominance hierarchies (Berglund et al. 1996). Relative size of the chelae is known to affect the success of crayfish in aggressive encounters (Rutherford et al. 1995; Bywater et al. 2008), and a large difference in

chelae size between males helps to resolve dominance relationships without engaging in fights (Schroeder and Huber 2001). However, little is known about how personality traits, such as boldness or aggressiveness, relate to the size of the chelae. For example, large armaments could increase resource acquisition of an individual and favour bold behaviour and consequently fast growth by reducing the frequency of costly fights (cf. Stamps 2007). This could in turn maintain a positive correlation between armament size and bold behaviour via a positive feedback loop on resource acquisition (Luttbeg and Sih 2010). In this study, we focused on the temporal repeatability and across-context consistency of individual shelter use that we considered as a measure of boldness or resource holding potential, depending on the context. We assumed that in the absence of conspecifics but in the presence of food outside the shelter, low shelter use reflects high boldness. On the contrary, we assumed that in the presence of predation risk and a conspecific but in the absence of food, high shelter use reflects high resource holding potential and involves interference competition. However, we did not study aggressiveness or explicit dominance relationships between the individuals. Therefore, even in the latter case, low shelter use may reflect high boldness. We performed experiments in two contexts in order to understand the role of boldness as a potentially evolvable personality trait. In addition, to evaluate if the size of armaments relates to behaviour, we explored correlations between the relative size of the chelae and individuals behaviour. Based on comparative data on other crayfish species (Pintor et al. 2008), we predicted that bold behaviour should be consistent across time, and with and without predator stress, i.e. form a behavioural syndrome according to the definition of Stamps and Groothuis (2010). We predicted that individuals with relatively larger chelae would be bolder but also have a better ability to possess the shelter under competition compared to individuals with relatively smaller chelae.

Materials and methods Study animals Two- to three-year-old immature noble crayfish, raised in a predator-free environment, were obtained from a commercial crayfish producer in Southern Finland in August 2008. The farmed population had been cultured for several generations and originated from a large number of wild noble crayfish specimens. Upon arrival in the laboratory, the crayfish were individually numbered using a white marker pen (Textmark 250) and then moved to individual containers measuring 105 mm (width)145 mm (length)

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230 mm (height of water) built in 300-l tanks. Crayfish were fed ad libitum with carrot, alder leaves and periodically fresh fish and shrimps. The lightdark rhythm (lights on 07:0017:00) and water temperature (101C) were kept constant. Carapace length (to the tip of the rostrum, to the nearest 0.1 mm), mass (to the nearest 0.1 g), and length and width of the chelae (to the nearest 0.01 mm) of the study animals were measured on the 10th of February 2009. No moults occurred during the course of experiments, which were conducted between March and June of 2009. Overview of tests First, each crayfish was individually observed for both latency to emerge from a shelter and total activity levels. This was done for a period of 3 days in an environment with no predators or conspecifics (Fig. 1a). Second, crayfish were stratified by sex and then randomly allocated between three treatments: (1) control, (2) predator odour and (3) predator odour + general stress. Individuals were

exposed to their respective treatments for a period of 3 days, during which behavioural observations were made. Third, the crayfish were divided to sex- and size-matched pairs, and observed for shelter possession for 3 days (Fig. 1b). Between the boldness tests and shelter possession trials, the crayfish were treated according to the group assignments for additional 18 days. At the end of this period, the crayfish were let to encapsulate a 6-mm-long, 0.20 mm in diameter nylon implant placed through a small puncture in the first joint of the right cheliped for 7 days. However, the results of the encapsulation test, assumed to have no importance for later behavioural tests, are not reported here. Boldness tests In order to examine both the effects of perceived predation risk on the behaviour of crayfish and the consistency of individual behaviour, 46 females [body mass 7.23.7 g (meanSD), carapace length 30.34.6 mm] and 35 males (body mass 8.63.5 g, carapace length 32.24.0 mm) were used. Each trial period lasted for a total of 6 days. On day 1, nine crayfish were randomly placed into individual housing compartments (105 145 mm, water depth 210 mm), including a gray plastic tube 75 mm in length and 36 mm in inner diameter and a piece of carrot within the control tank (Fig. 1a). The control tank had a separate closed water circulation. After 3 days of individual housing, the crayfish were randomly allocated in groups of three (two females and one male or two males and one female) and then transferred to their respective treatment tanks (Fig. 1a). The two tanks used for the predator stress treatments were connected to a tank with 23 wild-caught perch (mean total length 177 mm, SD 26.6 mm, fed regularly with frozen shrimps), which functioned as the source of predator odour. Constant water flow between the tanks was 60 lmin1. The control crayfish were transferred to other individual housing compartments within the separate control tank to guarantee equal levels of stress induced by the handling procedure in all treatments. Physical disturbance treatments involved catching and holding the crayfish individuals above the water level for 10 s every 2 h (8:0016:00) over five successive days (MondayFriday). Crayfish had access to an excess of food (carrot) during the trials (Fig. 1a). The crayfish were videotaped using infrared cameras for 3.5 h starting at 16:30, i.e. a half an hour before the lights went off. During daytime, crayfish most often burrow themselves in the shelter and emerge after darkness falls. Therefore, video recordings were analysed for the latency to emerge from the shelter (time until emergence) and for the total time the crayfish spent outside the shelter during the dark period (total exposure time). Both of these measures reflect boldness but are not interchangeable since, on average, individuals emerged (and correspondingly went

Fig. 1 Schematic presentation of the boldness tests (a) and shelter possession tests (b). First, nine crayfish were observed for 3 days without a predator odour and then distributed to new tanks and compartments corresponding the treatments (control, predator odour, odour+disturbance). All individuals had access to a shelter (rectangle) and a piece of carrot (circle). In shelter possession tests (b), there was a shelter for only one crayfish at time, and the two individuals placed in one arena competed for the shelter when exposed to the predator odour

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back to the tube) 2.7 times during the observation period (an individual does not stay out once emerged). Due to a bug in the recording software (InterVideo WinDVR5, InterVideo Inc.), we lost data for six observation periods (see Results section). However, individuals with missing data were still used for analyses deploying data from successfully observed periods (in correlation analyses). Shelter possession trials In order to study if boldness was related to shelter possession, the remaining crayfish were assigned to weight-matched pairs and then subjected to a resource possession trial. There were 14 female pairs and nine male pairs, with a mean weight difference of 5.6% between paired individuals. Past exposure treatment was not taken into account when assigning pairs (11 pairs had the same past exposure history). Either the left or right chela was painted white with a water-insoluble ink for identification. Each pair was placed in 290 mm105 mm210 mm (water level) testing arena for 4 days (Fig. 1b). As shelter, the arena contained one grey plastic tube 75 mm in length and 36 mm in inner diameter in one end of the arena, placed in a way so that only one of the crayfish could use it at a time (Fig. 1b). A similar kind of arena has been used previously to study the resource holding potential of signal crayfish (Ranta and Lindstrm 1993). The arenas were build in one 300-l tank that received water (60 l/min) from another tank containing 26 perch (the same as in boldness tests, complemented with three small individuals, length 172.2 28.4 mm, meanSD). The crayfish were not fed during the trials. Each arena was videotaped from 7:00 to 9:00 (the time crayfish naturally return to a shelter) at low light conditions for 3 days using digital infrared cameras. The crayfish inside the tube and outside the tube were later identified using the recordings. At day 4, prior to the removal of crayfish, we noted identities of the resource holder and the individual outside of the tube via visual inspection. The individual found outside of the tube more often (>50% of occasions) than the other was judged to be the potentially subordinate individual within each pair (Ranta and Lindstrm 1993). Statistical analyses The three tanks containing the individual compartments were equivalent to the three treatments that were applied during the boldness test. Common holding conditions (potential chemical cues between individuals) may induce statistical dependence between individuals. We controlled for this by entering week (corresponding to the group effect, as groups were changed every week) as a nesting factor in the repeated measures ANOVA (RM-ANOVA),

which was used to analyse the effects of predation risk treatments on the behaviour of crayfish. Period (control observation vs treatment period, each lasting for 3 days) and day within the period were included as repeated measures (within-subject factors). Interactions between these were omitted in order to avoid overparameterisation of the model. Treatment, nested by week, sex and the interaction sextreatment were entered as between-subject factors. Two models were run: in the first model, time until emergence was the dependent variable; in the second model, the total time spent out of the tube was the dependent variable. Test statistics were based on Wilks , and Bonferroni post hoc comparisons were used to determine the source of variation using an all-pairwise comparison approach. In order to study the consistency of individual behaviour, an analysis of repeatability was used (Krebs 1999; Bell et al. 2009). Statistically significant repeatability of individual behaviour implies that it is a property of an individual and not entirely dependent on the situation (Bell et al. 2009). For a correlation analysis between trait expression in morphological and behavioural characters, we calculated individual means for the three behavioural measurements during the control and the treatment period. These means were used as estimates of individual boldness and were examined for correlations with the relative (linear to carapace length) size of the chelae. Correlations were explored using Pearsons correlation analysis (correlation coefficient as r) and partial Pearsons correlation analysis controlling for the effect of carapace length (correlation coefficient as rc). RM-ANOVAs were used to test for sexspecific differences between the shelter possessor and the loser. Pearsons 2 test was used to test if shelter possession was related to the past exposure to the predation risk treatments. Normality of the studied parameters was checked using KolmogorovSmirnov test and the requirement of homoscedasticity was confirmed using Levenes test. All GLM, 2 and correlation analyses were performed in SPSS 16.0.1 (SPSS Inc., USA).

Results Boldness tests According to multivariate RM-ANOVA results, only the day within the treatment period explained variation in the time until emergence (Table 1). Univariate within-subject effects did not differ from multivariate effects. However, between-subjects effects of sex, treatment group (nested by week) and sextreatment group (nested by week) were significant (Table 1, Fig. 2). There was a distinct difference between the sexes in emergence time across the control and

acta ethol (2011) 14:1725 Table 1 Results of repeated measures ANOVA for the time until emergence from the shelter and the total exposure time Source of variation F df P value 2

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Time until emergence from the shelter MV Period 1.85 MV Periodsex 1.85 MV Periodtreatment 1.23 MV Day 4.14 MV Daysex 0.88 MV Daytreatment 0.86 MV Daysextreatment 0.61 B-S Sex 9.23 B-S Treatment by week 3.06 B-S Sextreatment by week 2.27 Total exposure time MV Period 3.53 MV Periodsex MV Periodtreatment MV Day MV Daysex MV Daytreatment MV Daysextreatment B-S Sex B-S Treatment by week B-S Sextreatment by week 0.46 1.23 4.38 0.12 0.84 0.99 0.20 1.10 0.98

1, 21 1, 21 20, 21 2, 20 2, 20 40, 40 38, 40 1, 21 20, 21 19, 21 1, 21 1, 21 20, 21 2, 20 2, 20 40, 40 38, 40 1, 21 1, 20 1, 19

0.188 0.188 0.320 0.031 0.432 0.686 0.937 0.006 0.007 0.036 0.074 0.505 0.319 0.025 0.887 0.713 0.511 0.659 0.412 0.518

0.081 0.081 0.540 0.293 0.081 0.461 0.366 0.305 0.744 0.672 0.144 0.021 0.540 0.307 0.012 0.455 0.485 0.009 0.512 0.469

treatment period: males emerged significantly later than females over all treatments, with the largest difference occurring in the predator+disturbance group (Fig. 2). With the exception of the period between days 1 and 2, there was a general and significant increase in the mean time until emergence after each transfer of individuals [mean time at emergence12.96.3 min (meanSE), 23.35.7 min and 39.46.6 min for days 1, 2 and 3, respectively]. Similarly, variation between days was the only factor contributing to the total exposure time (Table 1). Again, univariate within-subject effect results were in line with the multivariate results. None of the between-subjects effects was significant (Table 1). There was a general decrease in the exposure time after each transfer to a new location, indicating reduced activity, although only the difference between days 2 and 3 was significant [102.96.5 min (meanSE), 102.67.7 min and 82.46.5 min, respectively for days 1, 2 and 3]. Mean exposure time decreased slightly from the control period to the treatment period in all treatment groups, although this difference was not significant (Fig. 3). Consistency (repeatability) of individual behaviour Since the treatments did not influence individual behaviour relative to individual behaviour during control observations, the treatment groups were pooled for subsequent analyses. Time until emergence was repeatable between the days of the control period (R =0.21, N =72, P =0.001), the treatment period (R =0.17, N =70, P =0.009) and across the whole experiment (R =0.16, N =62, P <0.001), indicating that there are consistent individual differences in this behaviour. Also, the total exposure time was repeatable during the control period (R =0.28, N =72, P <0.001) but not during the treatment period (R =0.00, N =70, P =0.467). However, across the whole period, the total exposure time was weakly repeatable indicating individual consistency in activity (R =0.10, N =62, P =0.004).
Total exposure time S.E. (min)
180 160 140 120 100 80 60 40 20 0

Test statistics are based on Wilks . MV refers to a multivariate effect and B-S refers to a between-subject effect. Partial 2 refers to the
proportion of the total variance that is attributable to an effect. Period (including 3 days) refers to the effect of treatments compared to control observations (first three control days, then 3 days under treatments). If treatments have an effect on behaviour, interaction between period and treatment is expected to be significant

Time until emergence S.E. (min)

70 60 50 40 30 20 10 0

Females Males

Before After

Control

Predator odour

Odour + disturbance

Predator odour Odour + disturbance Treatment

Control

Fig. 2 Time until emergence from the shelter in female and male noble crayfish in different treatment groups (control, predator odour, predator odour and disturbance) across control and treatment periods (estimated marginal means from the RM-ANOVA)

Fig. 3 Total exposure times during the control and treatment period (before/after the introduction of predator risk) in each treatment group (estimated marginal means from the RM-ANOVA). An observation period lasted 180 min

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Correlations among behaviours and relative chelae size The longer it took for the crayfish to come out from the shelter, the less time it spent outside of the shelter during the control period (r = 0.72, N =72, P <0.001) and during the treatment period (r = 0.65, N =79, P <0.001). However, the mean times until emergence between the control and treatment periods were not correlated across treatments (r = 0.20, N =71, P =0.090). The mean exposure times between the control and the treatment periods were also uncorrelated across treatments (r =0.21, N =71, P =0.076). Relative length or width of the chelae were not related to the behavioural variables measured during the control or the treatment period (rc = 0.0730.066, N =68, P 0.546, effect of carapace length partialled out). Shelter possession trials In 16 trials out of the 23 total trials, the same individual of the two crayfish was outside the tube at every observation. The individual that was out of the tube during a greater proportion of the observations was denoted as the loser of the resource holding competition. In two of the trials, an individual was found dead outside of the tube and was subsequently classified as a loser. On three occasions, both of the crayfish had burrowed themselves in the tube, and the winner and loser could not be identified. Only the behaviour during the control observations was related to the outcome of the shelter possession trial (Table 2). The individual that monopolised the shelter had a shorter exposure time and emerged later from the shelter when observed during the control period. There were no sex-related effects in shelter possession (Table 2). 2 test did not indicate any effects arising from the exposure to the different predation risk treatments (2 =1.90, df =4, P =0.754).

Discussion Despite large between-days variation, individual crayfish showed repeatable behaviours within and across periods of varying risk of predation, which suggests that boldness can be considered as a personality trait in the noble crayfish. In addition, behaviour observed during the control period was consistent with the behaviour observed in the shelter possession trials: individuals behaving shyly in the absence of predation risk possessed the shelter more often than their rivals. This suggests that resource holding potential, defined as shelter possession under interference competition (Ranta and Lindstrm 1993), and bold behaviour are negatively associated in the studied population of the noble crayfish. Since we did not observe the formation of shelter possession, it is unclear if shelter possession required dominance or was associated with aggressiveness. Consequently, we cannot either assess if aggressiveness or dominance would form a behavioural syndrome with boldness in the noble crayfish (cf. Huntingford 1976; Riechert and Hedrick 1993; Kortet and Hedrick 2007). Behavioural syndromes and animal personalities have been observed in a wide variety of taxa (e.g. Huntingford 1976; Dall et al. 2004; Wilson and McLaughlin 2007; Bell et al. 2009). One potential mechanism promoting the evolution of behavioural syndromes is the coupling between growth rate and mortality (Stamps 2007). This mechanism implies that behaviours that promote high food intake rates may become genetically coupled over evolutionary time, and that the temporal consistency in personality traits is maintained by the cost of deviating from intrinsic growth trajectories (Stamps 2007; Adriaenssens and Johnsson 2009; Biro and Stamps 2008). This mechanism should favour the development of an aggressivebold behavioural syndrome in crayfishes since both aggression and boldness are likely to increase resource acquisition

Table 2 Comparison of trait means between the shelter possessor (In) and the crayfish that stayed outside the shelter (Out) according to multivariate results of RM-ANOVA, where sex was entered as a between-subject factor Trait In Out Shelter possession F Time until emergence (min) Total exposure time (min) Tr.p. time until emergence Tr.p. total exposure time Relative length of chelae Relative width of chelae 37 79 21.4 79.0 0.28 0.67 6.3 110 36.6 90.5 0.28 0.66 8.74 4.77 0.76 0.67 0.60 0.52 df 1, 1, 1, 1, 1, 1, 21 21 20 20 21 21 P 0.008 0.041 0.393 0.421 0.448 0.478 Sh. possessionsex F 0.62 0.00 0.013 0.31 0.21 0.001 df 1, 1, 1, 1, 1, 1, 21 21 20 20 21 21 P 0.439 0.990 0.911 0.582 0.650 0.972

The pairs were size matched by weight. Length and width of the chelae are relative to carapace length. Tr.p. refers to treatment period. F tests based on Wilks

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rates in these socially hierarchic animals (Ahvenharju and Ruohonen 2006). In accordance with the mechanism of state-dependent safety generated by high foraging rate (Luttbeg and Sih 2010), a positive correlation would have been expected between boldness and the size of morphological armaments signalling fighting ability (safety), i.e. size of the chelae. Against expectations, the relative size of the chelae did not relate to behavioural measures despite having been repeatedly reported to relate to dominance rank in crayfishes (Snedden 1990; Usio et al. 2001). Resource value is a subjective concept. Therefore, it might be possible that shy individuals perceived the value of shelter higher than bold individuals did. Therefore, the coupling of shelter possession and boldness could indeed be negative, and conflict with the idea that aggressiveness and boldness should be positively correlated (see above). Since the mean difference in the relative chelae size was rather small and non-significant between the rivals (7.2% in length and 7.6% in width), it is unlikely that shelter possession would have been readily resolved by armament size. However, we did not study aggressiveness or dominance over a food resource. A recent study in the signal crayfish suggests that the aggressivenessboldness behavioural syndrome could be a result of resource acquisition dynamics (Pintor et al. 2008). Therefore, more direct tests of aggressiveness, in relation to competition for food, would be needed to evaluate the coupling between resource intake rates, facilitated by bold behaviour in the presence of predators, and aggressive behaviour in the presence of intraspecific competition for food. Consequently, the presence of an aggressiveness/dominanceboldness behavioural syndrome in the noble crayfish cannot be excluded in the context of food acquisition. However, an alternative mechanism may be proposed to explain why shelter possession, which in general is thought to indicate dominance in crayfishes (Figler et al. 1999), was inversely related to boldness. In our study, predator odour did not induce changes in the behaviour of crayfish, and was thus not likely experienced as a threat either in the shelter possession trials. Therefore, bold individuals could have been allocating their time to exploring the arena, potentially in the search of food, rather than engaged in fights for the shelter, which in the absence of perceived predation risk could have had low resource value. Anyhow, shelter use during predation risk treatments did not relate to the shelter possession, suggesting that the coupling of shy behaviour and shelter possession cannot be generalised over all contexts. The lack of response to predator odour contrasts earlier studies in the noble crayfish (Appelberg et al. 1993). Further, in contrast to an earlier study on Ocronectes propinquus (Stein and Magnuson 1976), females emerged earlier than males in our study. In the wild, females are

predicted to be more vulnerable to fish predation due to their smaller chelae (Stein and Magnuson 1976). In our study, crayfish were clearly older than the crayfish in previous experiments (Appelberg et al. 1993). Therefore, they could have habituated to a predator-free environment and lost their responsiveness to the predator odour by learning. In addition, the previously documented responses were rather weak and slightly stronger only when perch were starved (Appelberg et al. 1993). We fed the perch that functioned as the source of predatory fish odour regularly with shrimps, which might have contributed to the absence of a detectable effect. Also, the perch used in this study were probably too small to impose a real threat on the size of intermoult crayfish used in this study. Whether larger perch would induce a stronger response only by odours remains to be examined. Another potential explanation for the lack of clear response relates to the normally nocturnal activity of the noble crayfish: usually shelter use increases in daytime and the most pronounced predator-induced shifts in behaviour are observed in darkness (Appelberg et al. 1993; Hirvonen et al. 2007). In this study, several crayfish stayed outside the shelter also during daytime, which reduced variation in the behavioural variables studied after switching off the lights. This made it more difficult to detect a significant effect. However, handling associated with transfer of crayfish to their treatment tanks induced an increase in exposure times, indicating that human-induced disturbances may increase predation risk for disturbed individuals. This can occur, for example, during and after release of undersized individuals in crayfish harvesting. Response of prey to predator cues may also be dependent on the temporal presence of predator cues (Lima and Bednekoff 1999; Sih and McCarthy 2002). In this study, the predator odour was constantly present for several days. Presence of perch led to a considerable predation mortality and shifts in the behaviour of A. astacus in the experiment of Sderbck (1992). However, the behavioural responses did not differ from those of non-native Pacifastacus leniusculus, indicating that the role of learning might also be considerable. Thus, it is possible that the crayfish in our study could have rapidly learned that the predators caused no danger even though their odour was continuously present. In conclusion, the noble crayfish did not clearly respond to predator odour, but instead showed individually repeatable hiding behaviour both with and without predator odour and a size-matched competitor. Therefore, we propose that boldness can be considered as a personality trait in the noble crayfish and has the potential for having extensive ecological and evolutionary implications depending on the predation risk and intensity of intraspecific competition. Finally, we would like to promote studies that couple

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behaviours and behavioural responses more tightly with morphological and life-historical traits.
Acknowledgements This research has been supported by the Academy of Finland (project #127398) and by the Emil Aaltonen Foundation. We thank the staff of the Experimental Unit of the Department of Biology, University of Oulu for the maintenance of the crayfish and help in the practical execution of the experimental procedures. We thank Aki Puhka for his help in the experiments and two anonymous referees for the very valuable comments on the manuscript. We also gratefully acknowledge Nick DiRienzo for his valuable linguistic revision of the manuscript. The perch in this experiment were held in laboratory conditions with the permission ESLH-2009-06035/Ym-23 from ELLA (Finnish board for the use of animals in experiments).

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