Predation risk and inter-population variation in antipredator behaviour in the three-spined stickleback, Gasterosteus aculeatus L.

Anim. Behav., 1984, 32, 264-275

PREDATION RISK AND INTER-POPULATION VARIATION IN ANTIPREDATOR BEHAVIOUR IN THE THREE-SPINED STICKLEBACK, GASTEROSTEUS ACULEATUS L. BY NICK GILES* & FELICITY A. H U N T I N G F O R D Department of Zoology, The University, Glasgow G12 8 QQ, Scotland Abstract. Predation risk from fish: and bird predators was assessed at seven Scottish Gasterosteus aculeatus L. sites. Samples of adult male and female sticklebacks and fry from each site were tested with either a model heron or a live pike to measure antl-predator responses. Principal Component Analyses were then used to describe the responses, the first factor to emerge from each multivariate analysis providing art index of overall level of response toward the predator. Significant sexual and inter-population differences in behaviour are described. Sticklebacks from populations at high risk from either predatory fish or birds showed higher fright response scores than fish from low risk sites. In general, adult male sticklebacks are bolder than adult females during interactions with predators. (Hagen & Gilbertson 1972, 1973; Moodie 1972; Gross 1978). Behavioural variation in sticklebacks has also been related to differential selection of G. aculeatus phenotypes by predators (Moodie et al. 1973; McPhail 1969). Huntingford (1982) quantified conspecific aggression and the level of fright response shown toward a hunting pike, Esox lucius in sticklebacks from 12 sites including populations sympatric and atlopatric with known fish predators. Clear interpopulation differences in anti-predator responses between fish from heavily predated and unpredated populations were described. In that study the level of predation by fish was assessed crudely and no attempt was made to determine the abundance of piscivorous birds, though G. aculeatus is known to be vulnerable to such predators (e.g. herons, Arden cinerea, Giles 1981b; mergansers, Mergus serrator, Bengtson 1971; terns, Sterna hirundo, S. paradisea, Lemmetyinen 1973; divers, Gavia stellata, G. arctica, Madsen 1957; gulls, Larus ridibundus, L, canus, Fjeldsa 1975; kingfishers, Alcedo althis, Eastman 1969). Pressure from piscivorous birds may affect the fright responses of sticklebacks differently from piscine predation pressure. The study described in this paper was therefore designed to improve and extend this pilot investigation by measuring more carefully predation risk and responsiveness of various categories of sticklebacks to fish and bird predators from seven sites. The aims were to assess whether (a) anti-predator behaviour varies significantly between populations and (b) differences relate to known and potential predation risks from both fish and birds. In addition, two of the study populations are composed largely of the rare

'Predation is an ecological factor of almost universal importance for the biologist who aims at an understanding of the habits and structures of animals' (Curio 1976). The power of predation pressure in producing within-species variability has frequently been demonstrated, e.g. in the banded snail, Cepaea nemoralis (Cain & Sheppard 1954) and the peppered moth, Biston betularia (KettleweI1 1955). Local variation in predation pressure appears to affect significantly many behaviours in Trinidadian guppy Poecilia reticulatus populations. Seghers (1970, 1973) reports differences in micro-distribution, Shoaling, feeding and fright responses between guppies sympatric with the large cichlids Hoplias malabaricus and Crenicielffa alta and guppies allopatric w i t h these predators. Where large predators are common, guppies shoal tightly in the shallow stream edges, but where Only the small cyprinodont Rivulus hartii is present, guppies spread out occupying the whole stream bed= Differences in overhead fright response measured experimentally persisted in laboratoryreared young from various sites indicating that the basis of this behavioural variation may be inherited (Seghers 1974). Further evidence that differences i n anti-predator behaviour in fish may be inherited is given by Kimmel et al. (1974) in the zebra danio, Braehydanio rerio, Jackobsson & Jarvi (1976) in the Atlantic salmon, Salmo salar, and Pattern (1977) in the coho salmon, Oneorhynchus kisuteh. Within and between three-spirted stickleback populations intra-specific morphological ~ariation has been related to predation pressure *Present address: School of Life Sciences, University of Buckingham, Buckingham, MK18 lEG, England. 264

GILES & HUNTINGFORD: ANTI-PREDATOR BEHAVIOUR IN STICKLEBACKS spine-deficient morph of G. aculeatus (Campbell 1979) providing fish which appear to be morphologically vulnerable to predators and allowing an initial description of their behaviour. Methods Characteristics of Study Sites

Four of the sites are on the Scottish mainland. These are: Loch Lomond, a mesotrophic freshwater lake 25 km long and 7 km wide; the Mar burn, a small shallow, fast-flowing upland stream; the River Luggie, a comparatively eutrophic suburban site; and Lennox Castle reservoir, a small artificial still water known to have contained sticklebacks for at least the past 30 years. Three further sites are on the Outer Hebridean Island of North Uist: Loch Fada, Loch a Bharpa and Loch art Daimh are all oligotrophic peat bog lakes and are largely shallow with few dense stands of aquatic macrophytes. A full description of the sites is given in Giles (1981a). Wherever possible predation risk from piscivorous fish was assessed by stomach samples of gill-netted or trapped fish (Lomond, Luggie, Lennox, Mar). At the Mar and Lennox sites heron diet was studied by collecting food remains and pellets from beneath nesting birds (Giles 1981b). For various practical reasons which were out of our control, direct measurement of predation pressure was not carried out at all sites. In these cases the direct information was replaced by observation of the presence and

265

feeding activity of species known from the extensive literature on this subject to be predators of sticklebacks. In particular it is interesting to note that the spine-deficient G. aculeatus in Loch Fada are sympatric with brown trout, Salmo trutta which are known to predate them (Campbell 1976). Populations of spine-deficient G. aeuleatus occurring on the Queen Charlotte Islands off Vancouver are also thought to be allopatric to fish predators (Moodie & Reimchen 1976). The information gathered from these various sources is summarized in Table I. A weakness of this study is that no attempt was made to include predatory invertebrates which are known to predate G. aeuleatus (e.g. Reimchen 1980). In the light of these results the seven study sites were divided into three categories having high (abundant records of species known to take G. aculeatus), medium (frequent records) or low (occasional records) predation risks from piscivorous fish and birds. Lennox Castle was rated as zero risk from fish predators since no fish of any species other than sticklebacks were taken from the reservoir despite extended periods of netting. Collection and Keeping of Fish

Sticklebacks were caught with a hand net swept through any available weed beds within reach of the shore or within wading depth and transported to the laboratory where fish from a single population were housed communally at a density of not more than 12 fish/30 litre tank.

Table L Study Sites and Predation Risks

Study site

Habitat type

Productivity

Bird predators

Estimated risk

Lomond

Large lake

Medium

Merganser*,gulls, terns, grebes, heron

High

Pike*,perch*, trout, eels

High

Mar

Smatlt sream

Low

Heron*, gulls, merganser

High

Trout, eel, stickleback*

Low

Lennox

Smallreservoir

Medium

Gulls*,heron

High

None

Zero

Luggie

Small river

'High

Gulls

Low

Trout (few, small), stickleback*

Low

Fada

Large lake

Low

Divers, gulls, terns, heron, grebes, merganser

High

Trout*, eharr, eel

High

Daimh

Small lake

Low

Divers, gulls, terns, heron, grebes, merganser

High

Trout, eel

High

Bharpa

Large lake

Low

Divers; gulls, terns, heron, grebes, merganser

High

Trout, charr, eel

High

Fish predators

*These predators are known to take Gasterosteus at the study site, others are potential predators.

Estimated risk

266

ANIMAL BEHAVIOUR,

All fish were fed daily with either Tubificid worms or Daphnia. Cover was provided by clumps of Sphagnum moss. Adult fish (in their second summer of life, aged from otolith growth rings) tested during the summer months were kept on a light regime of 16 L: 8 D; fry (caught in the wild during the first summer of life), tested in the autumn were kept on 12 L: 12 D. Air temperature in the laboratory varied between 18 and 25~ (summer) and 12 and 19~ (autumn). Only fish which appeared healthy were used in behavioural experiments.

Anti-predator Behaviour Experiments Of the predatory fish present at the study sites, trout and perch, Perca fluviatilis actively chase their prey (Benzie 1965) whilst pike are stalking/ ambush predators. Since it is difficult to record accurately the stickleback's response to rapidly moving chasing predators, a small (30 cm) pike which had been fed with minnows until it would fixate and stalk a prey fish without striking at it was used to provide the fish predator stimulus. The stalking pike elicited a precautionary response from the tested sticklebacks and, as such, the experimental set-up was successful. However, between-test variability in intensity of pike stalking behaviour may have contributed to the variation in anti-predator behaviour shown by tested sticklebacks. This effect was minimized by using the same individual pike for all tests and by only performing experiments when the pike was actively stalking the test fish. During pike tests the stickleback was introduced into a releasing box, left for 5 min, then a trap door in the bottom of the box was lowered allowing the stickleback to swim into the open water of the experimental tank. A 10-min vocal recording of the subsequent behaviour began at this point. In most tests a sudden discernible change in the swimming behaviour of the stickleback occurred when the presence of the pike was detected. This behavioural change normally took the form of a reduction in pectoral fin beating and sudden binocular fixation upon the pike; the dorsal and ventral spines were also normally raised at this time. The point in time when the behaviour switched from normal exploratory behaviour to predator evasion was termed the moment of t r u t h (MOT). The timing of the MOT during the test varied with the degree of activity of the pike and the alertness of the stickleback. Of the piscivorous birds feeding at the study sites, herons, terns and gulls are stalking/aerial ambush predators whereas mergansers, grebes

32,

1

(Podiceps cristatus) and divers approach and chase their prey at speed underwater. A heron model was used to test the fright responses of sticklebacks to predatory birds, the major advantage of this approach being that a standard 'predator' stimulus can be presented at a precise time. During heron tests the stickleback was introduced into the test tank and left for 5 rain, the vocal commentary starting at this point. For 5 min the exploratory behaviour of the fish in the test tank was recorded (the pre-MOT period); the model heron head was then suddenly lowered over the tank allowing the bill to splash on the water surface and then raised back to the vertical position. The sudden overhead presentation of the heron model was termed the MOT and a 5 rain post-MOT period of stickleback behaviour was then recorded. Twelve adult male sticklebacks, 12 adult females and 24 fry from each population were heron-tested. Different groups of 12 adult males and 12 adult females were pike-tested. The behaviours recorded during these experiments are defined in Table II. Each experiment was split into a pre-MOT investigatory behaviour period and a post-MOT anti-predator behaviour period. The frequency of performance and total duration of all recorded behaviours were calculated for each period. Since the MOT occurred at different times in different pike tests these data were then converted to per-min scores.

Statistical Analysis of Data Single behavioural variables often form an inadequate basis for the description of complex anti-predator responses such as those performed by sticklebacks stalked by pike. In the heron tests the time taken (in seconds) for the stickleback to resume normal swimming after responding to the MOT was termed the recovery time. This proved to be a useful measure in distinguishing the level of response of sticklebacks from different populations toward the frightening overhead stimulus. Two-tailed Mann-Whitney U-tests were used to compare the recovery times of pairs of groups of tested fish. No appropriate single measure of anti-predator response was apparent within the pike test data. Multi-variate analyses were employed for both the heron and pike test data in order to describe both the level and the organization of the anti-predator behaviour of the sticklebacks. Because of the difficulties involved in predicting the form of the fright responses exhibited by sticklebacks, it was necessary to obtain a comprehensive record of a

GILES & HUNTINGFORD: ANTI-PREDATOR BEHAVIOUR IN STICKLEBACKS large n u m b e r of b e h a v i o u r patterns in order subsequently to describe fully h o w t h e Sticklebacks reacted during the experiments. This a p p r o a c h produced large data blocks which are d i N c u l t to interpret. I n addition, it is in the n a t u r e of anti-

predator responses to vary with circumstances which could n o t be fully controlled with the present experimental design, such as variations i n the activity of the pike, or the proximity of weed beds used b y the sticklebacks for coverl

Table II. Behaviours Recorded During the Pike Tegts and Heron Tests

Behaviour Position in tank *At surface

Definition Remaining at the water surface for more than 0.5 s

*At bottom

Laying upon the substrate usually still, for more than 0.5 s

*In weed

Remaining within artificial weed clump provided in experin~ental tank, for more than 0.5 s

*Open water

Remaining at least 1 cm away from the water sin:face, substrate or weed clump for more than 0.5 s

Spine movements *Dorsal spines raised *Ventral spines raised Locomotion type *Sneaky swimming Jerky swimming

Total or partial erection of one or more dorsal spines One or both ventral spines raised Smooth swimming usually along the bottom using caudal and pectoral fins with dorsal and ventral spines lowered Fast agitated swimming using pectoral fins typified by abrupt stopping and starting

*Normal swimming

Slow bouts of pectoral swimming with frequent pauses and stoppages

*Still

Remaining stationary in any part of the experimental tank for more than 0.5 s

Barrage balloon *Jump away Predator stimulus *Moment of truth

tDirect reaction to pike *Facing

Slow vertical ascent to water surface with little or no fin movements, facilitated by swim bladder expansion A rapid body flexure and stroke of the caudal fin causing a fast 'leap' through the water usually to a place of cover Heron tests: presentation of overhead stimulus Pike tests: the first detectable moment in the test when the stickleback apparently becomes aware of the presence of the pike Watching the pike from a head-oil position

*Monocular fixation

Watching pike through one eye

*Facing away

Looking directly away with tail toward pike

Bite

267

Biting the pike during a pike test

*Feed

Ingesting any food item during the test

*Approach

Any direct advance toward the pike over a distance of more than 1--2cm

*Retreat

Any direct movement away from the pike over a distance of more than t--2 cm

*Behaviours included in Principal Component Analyses. tBy definition these behaviours only occurred after the MOT.

268

ANIMAL

BEHAVIOUR,

This raises the possibility that fish from the same population with a similar degree of overall responsiveness to a predator may show different but equally effective anti-predator actions. Univariate statistics performed upon the pike test data may therefore fail to identify as significant, real differences between the populations in the strength of their response to a predator. In order to identify the relationships between these various patterns of behaviour and if possible to produce objectively-defined compound scores which summarize major components of behavioural variation between the fish, the data were normalized and subjected to Principal Component Analysis followed by Varimax rotation (Dixon 1973). This technique for simplifying complex data matrices has been applied quite frequently to behavioural data; Huntingford (1976, 1982) gives an account of the analysis. Briefly the Principal Component Analysis (PCA) replaces the original variables by new axes or components, the first of which accounts for as nmch as possible of the total variance within the data set. The second component accounts for the next highest proportion of variance, and so on, subject always to the constraint of orthogonality. Each component or factor is described in terms of a set of loadings which define the relationship with the original variables. Behaviours which are positively correlated have high loadings of the same sign on a single component, negatively correlated behaviours have loadings of the opposite sign on the component while unrelated variables load on different components. In each of the three PCAs described in following sections, Varimax rotation has been performed upon the components. This procedure seeks to maximize both the variance of the loadings of different acts upon the same component and also the loadings of the same act on different components. The multivariate analyses employed perform therefore a simplifying and descriptive role for the data sets. The PCA followed by Varimax rotation thus increases the chances of identifying biologically meaningful dimensions where these exist. The loadings on the components define the relationships between the original variables. Because the first few components may account for the majority of the total variance in the original data matrix, those components which collectively account for the remaining small proportion of variation (in the present study all but the first) cart be ignored for some purposes.

32,

1

Because the components are mutually uncorrelated they serve as a series of independent indices which can be used to describe the original data matrix economically. One PCA was run on the post-MOT heron test data (336 sticklebacks tested). Two PCAs were run on the pike test data (168 sticklebacks tested) taking the pre-MOT and post-MOT data sets separately. In each case the variables which were composed largely of zero scores were omitted (see Table II). For each stickleback there was a score on each principal component; these are termed factor scores. As only the first factor (component) from each of the three PCAs is described and discussed in this paper these are termed factor 1 scores. For the heron-tested fish the post-MOT factor 1 scores for the 12 adult males and 12 adult females were tabulated and group means calculated. The scores of individual fish minus the group mean were then arranged as deviations from the means and grouped at intervals. The percentage cumulative frequency of the deviations were then plotted on probability paper and approximated to a straight line. This line was compared with the theoretical normal distribution line based upon the overall group variance and no significant difference was found (P > 0.05, KolmogorovSmirnov test); these data are therefore approximately normally distributed. The pike test factor 1 scores for both the pre-MOT PCA and the post-MOT PCA were similarly analysed. The pre-MOT scores differed significantly from normality, having a moderate left hand skew (Kolmogorov-Smirrtov test, P < 0.05), the postM O T scores showed no significant deviation from a theoretical normal distribution (KolmogorovSmirnov test, P > 0.05). The fright responses (factor 1 scores) of the sticklebacks to both the heron model and the live pike are therefore best analysed with parametric significance tests. Two-way analyses of variance with replication (Sokal & Rohlf 1973) followed by two-tailed t-tests between pairs of groups were used. For the pre-MOT pike test factor 1 scores the two-way analysis of variance was again used; the consequences of non-normality of error are not too serious, as only very skewed distributions have marked effects upon the level of significance of the F test or on the efficiency of the design (Sokal & Rohlf 1973). However paired comparisons of groups of pre-MOT pike test factor 1 scores were made using (non-parametric) twotailed Mann-Whitney U-tests.

GILES & HUNTINGFORD:. ANTI-PREDATOR BEHAVIOUR IN STICKLEBACKS Results H e r o n Tests

During the performance of the experiments, clear differences in reaction to the stimulus were observable between individual sticklebacks. The response at the M O T varied from a jump away with a long recovery time (Mar, Lomond, Lennox fish) to a complete disregard for the overhead stimulus (e.g. several male Luggie fish). Fish frightened by the heron model usually assumed a sigmoid posture with spines raised at the bottom of the tank, ready to take further evasive action if necessary. When recovery times were compared between groups of fish (Table III) many significant differences were found. A total of 27 paired comparisons were made. There were no significant differences between Mar, Lomond and Lennox males. Mar males had significantly longer recovery times than Bharpa males (P < 0.002), Fada males (P < 0.002) and Luggie males (P < 0.002). Lomond males had significantly longer recovery times than Daimh, Fada, Table IH. Heron Test Recovery Times (s) Means and Standard Deviations of Adult Males, Females and Fry from Study Sites

Study site Luggie Daimh Bharpa Fada Lennox Lomond Mar

Adult male

Adult female

Fry

68• 212!88 125• 102• 105 398• 427 • 166 436• 146

386• 86• 244• 148 236• 170• 483 • 160 5674-69

162-4-121 2564- 166 59zk49 211 -t-138 304• 473 • 135 338 • 158

*Figures given are mean • SD. +1.oo o.75 0.50 0.25

Bharpa and Lennox males (P < 0.002in all cases). Lennox males had significantly longer recovery periods than Luggie males (P < 0.002). Daimh males had longer recovery periods than Fada and Luggie males (P < 0.002) and Bharpa males (P < 0.02). There were no significant differences between male Fada and Bharpa fish and male Fada and Luggie fish. Amongst adult females, there were no significant differences between Mar and Lomond fish, or between Lomond and Luggie fish. Mar females had significantly longer recovery times than Bharpa, Lennox, Fada and Daimh females (P < 0.002 in all cases) and Luggie females (P < 0.02). Both Bharpa and Fada females took significantly longer to recover than Daimh females (P < 0.02 in both cases). There was no difference between Bharpa and Fada females. Among the heron-tested fry (sexes grouped) Lomond fry had significantly longer recovery times than Mar fry (P < 0.002), Luggie fry than Lennox fry (P < 0.05), Daimh fry than Bharpa fry (P < 0.002) and Fada fry than Bharpa fry

(e < 0.002). The first factor to emerge from the PCA of these tests accounted for 27% of the total variance; the main loadings on this component are shown in Fig. 1. Fish which scored positively on this factor performed many bouts of postM O T open water swimming, pausing frequently to feed, having recovered quickly from the frightening overhead stimulus at the MOT. Such fish possess a poorly-developed fright response. Fish scoring negatively on factor 1 had a long recovery time, remaining still for long periods either at the bottom or in weed during the postM O T phase of the experiments, i.e. they

Frequeocy normal FrequencyDuration normal Duration swimming still swimmingFrequencyin open Frequency feeding water in open Frequency water at bottom Poorly developed fright response

o _J

-0.2~ -0.5( -0.75 -1.00

269

I Highly developed fright response

Duration at bottom Duration

still

Recovery

time

Fig. 1. Main loadings on the first factor from the PCA of the heron test data. Height of line represents size of loading.

ANIMAL

270

BEHAVIOUR,

possessed a highly-developed fright response. Importamly, pre-MOT behaviour scores are loaded similarly to post-MOT behaviour scores indicating that fish which reacted strongly to the predator stimulus also tended to show initial caution in the unfamiliar environment of the experimental tank. Factor 1 scores provide a good compound measure of overall fright response with which to compare experimental groups of fish. Factor 1 accounts for 27 ~ of the variation in the heron data, a considerable proportion remembering that 31 components were produced during the analysis. The aim of this paper is to provide a broad comparison of fright response between groups of fish tested; the other components produced, together with those from the pike test PCA will be described in more detail elsewhere (in preparation). The second and subsequent components from both the heron and the pike test data do not allow differentiation of responses between populations but may reflect individual patterns of anti-predator evasive behaviour in sticklebacks. Table IV gives the mean and standard deviation for factor 1 scores for fry, adult males and females from all seven sites. Fry LomoRd fish had significantly lower factor 1 scores (i.e. higher fright response) than Bharpa, Luggie or Fada fry (P < 0.002 in all cases, twotailed Mann-Whitney U-test). Mar fry had significantly lower scores than Luggie fry (P < 0.002, U-test). A total of 21 paired comparisons are possible between populations for these tests; all except those above were non-significant. Lomond and Mar fry differ from those from other sites in having better-developed fright responses. In general, however, the behaviour of the fry within and between populations was similar. Factor 1 scores for the adult males and females from all seven populations were sub-

32,

1

jected to a two-way analysis of variance with replication, with sexes cast in rows and populations in columns. For populations F = 8.14, df= 6, 154, P < 0.001; for sexes F = 7.4, df= 1, 154, P < 0.01; for interaction effects F = 8.34, df= 6, 154, P < 0.001. Since the significance of the interaction complicates the interpretation of the results, comparisons were made between pairs of tested fish groups. In general, within populations, adult male sticklebacks scored higher on factor 1 (i.e. lower fright response) than females. This is true for all populations except Dailnh and Lennox. Comparing like sexes between populations with two-tailed t-tests, breeding males from Mar (high risk from herons) had significantly lower factor 1 scores than those from Luggie (P < 0.001), Bharpa (P < 0.01) and Fada (P < 0.01). Lomond males (high risk from mergansers) had significantly lower factor 1 scores than Luggie (low risk) (P < 0.001), Fada or Bharpa males (P < 0.05 in both cases). There were no significant differences between Lomond and Daimh, or Lennox and Bharpa. A total of 21 possible paired a posterior• comparisons could be made between tested groups of adult males; 12 statistical tests were actually performed. In fact, the statistical comparisons made were at least partly decided before the execution of the experiments because of the differences in estimated predation level at the sites, and might therefore be regarded as planned or a priori tests. Amongst adult female sticklebacks, Mar fish (high risk from herons) had significantly lower scores than Fada (P < 0.05), Lennox (P < 0.05), Bharpa (P < 0.05) and Daimh (P < 0.01) fish. There were no significant differences between Mar, Lomond and Luggie females. Luggie females, despite their estimated low risk from avian predators, had significantly lower scores thart Daimh (P < 0.01) females. There was no significant difference between Fada and Daimh

Table IV. Factor 1 Scores from Principal Components Analysis of Heron Test Data

Study site

Luggie Daimh Bharpa Fada Lennox Lomond Mar

Adult male 1.7 --0.7 0.3 0.3 --0.42 --0.5 --0.8

• 1.4" 4- 1.14 4- 0.7 4- 0.67 4- 1.01 4- 1.15 4- 0.42

Adult female

Fry

--0.68 • 0.82 0.53 4- 1.07 0.49 4- 0.9 0.01 • 0.9 0.06 4- 0.59 0.5 4- 0.42 --0.17 4- 0.79 0.54 4- 0.7 -;0.05 4- 0.39 0.07 q- 0.96 Z0.75 -t- 0.64 --0.54 • 0.55 --0.9 • 0.51 --0.3 4- 0.94

*Figures given are mean 4- SD of scores for all tested fish.

Predation risk from

birds

Known low Estimated high Estimated high Estimated high Known high Known high Known high

GILES & HUNTINGFORD: ANTI-PREDATOR BEHAVIOUR IN STICKLEBACKS fish. Nine paired comparisons (two-tailed t-tests) were made. Pike Tests Again marked population differences were obvious during the course of the experiments in that most fish from high-risk sites (typically Lomond, Fada, Daimh, Bharpa) were frightened at the MOT and subsequently avoided the pike whilst, for example, Lennox fish (zero risk from fish predators) often approached the pike with naive curiosity.

Pre-MOT Data For the pre-MOT data, the first factor to emerge from the PCA accounted for 25 % of the total variance; loadings on this factor are shown in Fig. 2. Note that the negatively-loaded durations of 'still', 'in weed' and 'at bottom' have small loadings and are included to illustrate those behaviours most closely associated with a late MOT in a pike test. This factor is interpretable as a measure of exploratory (positively loaded) behaviours versus timid (negatively loaded) behaviours. The late M O T is interpreted as being associated with timid fish because cautious sticklebacks usually remained still and in cover for long periods after leaving the releasing box. These fish tended not to be noticed by the pike as quickly as sticklebacks that swam in open water, and therefore a late sticklebackpike first interaction (i.e. late MOT) tended to occur when unadventurous fish were tested. As in the case of the heron tests, this first factor was thought to be an adequate summary of general cautiousness in a potentially-dangerous environ-

-H.O00.75" 0.50'

o, 0.25:

.E

o

0--0.25

-0.50 -0.75

271

ment. The other factors from the pre-MOT pike test PCA will be discussed elsewhere (in preparation). Table V gives the factor 1 scores, means and standard deviations for the 168 sticklebacks which were pike-tested. A two-way analysis of variance with replication with sexes in rows and populations in columns gives, for sexes: F = 0.56, dJ'= 1,154,P > 0.05; for populations: F = 3.41, df = 6,154, P > 0.05; and for interactions: F = 1.07, df= 6,154, P > 0.05. Paired M a n n Whitney U-tests failed to show arty significant differences between groups of tested fish. As in the heron tests, there is a trend in the data for males to be less timid than females in exploring the novel environment of the test tank, for all populations except Daimh. In general then, the exploratory behaviour shown by fish from all populations in the pike tests was similar. Post-MOT Data The post-MOT pike test data describes the anti-predator behavioural strategies adopted by the tested fish after sensing the presence of the pike. The structure of factor 1, which accounted for 27 % of the total variance is given in Fig. 3. This factor is interpretable as an index of open water activity where fish swam around in front of the pike, often facing away from the predator and sometimes pausing to feed (positive scores) versus remaining absolutely still, usually at the bottom of the test tank (with all spines raised) hiding from the stalking pike (negative scores). Individual fish tended to score similarly on factor 1 in both the pre-MOT and post-MOT periods. As with the heron test data this indicates

Frequency Frequency normal Frequencyopen swimming still water Frequency dorsal spine raising Duration normal swimming

] ~

I

I

Duration Duration Duration in weed still at bottom

Late

M.O.T.

-1.00

Fig. 2. Main loadings on the first factor from the PCA of the pre-MOT pike test data. Height of line represents size of loading.

272

ANIMAL

BEHAVIOUR,

Table V. Factor 1 Scores from Principal Component Analysis of Pre-MOT Pike Test Data

Study site

Adult male

Lennox Luggie Mar Bharpa Daimh Fada Lomond

--0.5 4-1.2" 0.19 ~ 0.55 0.21 4- 0.52 0.35 :t= 0.98 --0.04 i 1.11 0.7 :t= 0.79 0.24 • 0.9

Adult female --0.24 ~= 0.96 --0.34 • 1.02 --0.53 -E 0.45 0.047 :t= 0.67 0.43 :t: 1.97 0.028 ~ 0.99 --0.37 -4- 0.72

*Figures given are mean • SDof scores for all tested fish. that fish which are cautious within the novel environment of the test tank are the individuals which exhibit the most highly-developed antipredator responses. Once again this first factor provides an adequate summary of general responsiveness to a predator and is used in the subsequent analysis. The means and standard deviations for the groups of fish tested are given in Table VI. A two-way analysis of variance with replication gives: for sexes, F = 11.4, df = 1,154 P < 0.001 ; for populations, F = 52.4, df = 6,154, P < 0.001 ; for interactions, F = 0.41, df= 6,154, P > 0.05. Again, in general, females exhibited higher fright responses than males. Amongst adult males Lennox, Luggie and Mar fish (all with few or no fish predators) scored positively on average (i.e. low fright response); Lomond, Fada, Bharpa and Daimh fish (with fish predators) scored negatively (high fright response): the pattern was similar for females. When faced by a hunting pike the cautious behaviour of Lomond, Daimh, Bharpa and Fada males was similar; there were no significant differences

+1.00 0.75 0.50 =~ 0.25 "o

8

_J

o -0.25 -0.50

32,

1

between these groups. Luggie males (low risk from predators) scored significantly higher on factor 1 than Mar (P < 0.01), Lomond, Fada, Bharpa and Daimh males (P < 0.001 in all cases). Lennox males (zero risk from fish predators) scored higher than Luggie (P < 0.01), Mar, Lomond, Fada, Bharpa and Daimh males (P < 0.001 in all cases). Amongst adult females the pattern of significant differences between populations was the same as that for adult males. There were no significant differences between Mar, Bharpa, Lomond, Daimh and Fada fish, all of which scored negatively. Luggie females scored significantly more highly on factor 1 than Mar females ( P < O . O 1 ) and Bharpa, Lomond, Daimh and Fada females (P < 0.001 in all cases). Lennox fish (no risk from fish predators) scored significantly higher on factor 1 than Luggie fish (P < 0.01) and female fish from all other populations (P < 0.001). Discussion

Table VII summarizes the information available on predation risk and anti-predator responses from the seven study sites. In the heron tests the samples of fry from Mar and Lomond showed significantly greater alarm responses than fry from the other five populations. Herons are known to predate adult Mar G. aculeatus and experiments have shown (Giles 1984a) that the overhead fright response develops with age and/ or body size in this population. In Loch Lomond, red-breasted mergansers (a specialist stickleback predator in freshwaters) patrol stickleback breeding areas extensively during the summer

Frequency Frequency Frequency Duration Frequency Frequency normal, facing monocular normal Frequency dorsal still swtmmmgaway fixation swimrn" Frequencybinocular spine Duration mg of retreat fixation raising facing Frequency open away water

I

II

Duration Duration still at bottom

-0.75 -1.00

Fig; 3. Main loadings on the first factor from the PCA of the post-MOT pike test data. Height of line represents size of loading.

GILES & HUNTINGFORD: ANTI-PREDATOR BEHAVIOUR IN STICKLEBACKS Table VI. Factor 1 Scores from Principal Component Analysis of Post-MOT Pike Test Data

Study site

Adult male

Adult female

be at high risk. Luggie males had the highest scores and have little or no avian predation risk. Luggie females had a low score but may have nothing to lose by being cautious; a possible basis for the sexual difference in level of fright response shown by G. aculeatus may involve the need for males to return quickly to reproductive territories after predator encounters (Giles, 1984b). The low score of Lennox males agrees with the known high risk from black-headed gulls at this site. Lennox females tested were less cautious than males in this population. This apparently anomalous result is explicable as all 12 females were found upon dissection to be heavily infected with Schistocephalus solidus larvae (1-8 parasites per fish). Only three of the 12 Lennox males tested were found to be parasitized. S. solidus larvae have subsequently been shown to adversely affect the overhead fright response of G. aeuleatus (Giles 1983a). Despite the abundance of avian predators on North Uist, both male and female sticklebacks from Lochs Fada, Bharpa and Daimh showed moderate fright responses towards the heron model. Inspection of the raw data reveals that at

Predation risk from fish

Lennox Luggie Mar

1.89 q- 1.45" 1.38 • 0.86 Known zero 0.93 4- 0.7 0.49 4- 0.64 Known low 0.017 4- 0.46 --0.29 • 0.19 Estimated low Bharpa --0.45 4-0.33 --0.6 • 0.34 Estimated high Daimh --0.5 4- 0.32 --0.64 4- 0.4l Estimated high Fada --0.43 4- 0.3 --0.7 4- 0.19 Known high Lomond --0.33 4- 0.25 --0.62 4- 0.28 Known high *Figures given are mean :5 SDof scores for all tested fish. months and this, together with the known heavy perch predation may have led to a heightened awareness of predators in the young fish. Amongst adult fish, the heron test PCA factor 1 scores agree well with the assessments of natural predation risk. Mar and L o m o n d male and female sticklebacks had the lowest average scores (i.e. highest response) and are known to

Table VH. Predation Risk at file Study Sites and Anti-predator Responses of Tested Sticklebacks

Predation risk from Study site

Fish

273

Birds

Lomond

Known High

Known High

Mar

EstimatedLow

Known High

Lennox

Known Zero

Known High

Luggie

Known Low

Known Low

Fada

Known High

EstimatedHigh

Daimh

EstimatedHigh

EstimatedHigh

Bharpa

EstimatedHigh

EstimatedHigh

Behavioural response to Live pike Heron model High ~

High

High ~

High

Medium c? High c? High ~

High

Low 4

High d~

Low ~

Low

Low c~

Low c~

Low ~

High

High d~

Low d~

High ~

Medium

High c~

Mediumc~

High ~

Low

High c~

Low c~

High ~

Medium

274

ANIMAL

BEHAVIOUR,

the M O T these fish often jumped away to open water (rather than to cover) and recovered relatively quickly when compared with Mar and Lomond fish. A possible explanation for this consistent response from the three Hebridean populations may lie in the low-visibility peatybrown waters of their origin. A short jump away may be sufficient for these small individuals, which blend inconspicuously with their background, allowing a reduced recovery time and a lowering of the overall response to bird predators. Cook (1978) has timed the feeding behaviour of grey herons on the Ythan estuary (Scotland), finding that adult birds strike at prey once every 9.66 min on average (50 % success rate); juvenile birds strike once every 3.97 min (about 30% success rate). If these data are characteristic of herons in freshwater habitats the long recovery times shown by Mar males, females and fry correlate well with a known high risk from a predator that pauses for considerable periods between strikes at the prey. The heron test data support the thesis that high natural risks of predation lead to highly developed anti-predator behaviour with significant inter-population effects. In the pike tests the post-MOT factor 1 scores also agree well with the known and potential risks from fish predators. Lomond, Daimh, Fada and Bharpa males all scored negatively (i.e. cautious behaviour); all are at risk in the wild. Mar males were intermediate and have no known fish predators (but high heron predation), although there are two or three possible potential fish predators. Luggie and Lennox males have low and zero risks of fish predation and both groups scored high positive factor 1 values. This p a t t e r n was also observed amongst pike-tested female sticklebacks sampled from the study sites. The pike-test data, therefore, also support the argument for adaptive behavioural responses to varying predation pressures at the population level in G. aeuleatus. The Fada and Bharpa populations are particularly interesting since they contain spine-deficient morph sticklebacks which lack almost completely protective dorsal and ventral spines. These fish show no specialized anti-predator behaviour to compensate for their lack of morphological protection. Bell (1974) suggested that spine-deficient Gasterosteus may be better streamlined and thus more able to escape from underwater predators than normally spined individuals; no evidence was discovered to support this theory during the present study. An hypothesis proposing that low environmental

32,

1

calcium levels may have led to the evolution of skeletal reduction in North Uist G. aculeatus populations has recently been suggested (Giles 1983b). In summary, this study has demonstrated that (1) in general, within each population, and in response to both fish and bird predators, adult female sticklebacks have better developed fright responses than adult males; (2) in both sexes there are significant differences between populations in response to both fish and bird predators; and (3) these differences relate to known and estimated predation risks at the study sites. This correlation between responsiveness and risk seems of obvious adaptive significance. It could arise experientially within the lifetime of the individual fish, or be the result of natural selection acting upon inherited differences, or indeed may be dependent upon both of these processes. Benzie (1965) has demonstrated that experience of a predator does influence antipredator responsiveness in sticklebacks. However, the overhead fright response of Mar and Lomond stickleback fry develops normally in fish reared predator-naive in the laboratory (Giles 1984a). Clearly further evidence is required on this point. Acknowledgments This research was funded by an S.R.C. grant. REFERENCES Bell, M. A. 1974. Reduction and loss of the pelvic girdle in Gasterosteus (Pisces): A case of parallel evolution. Nat. Hist. Mus. Los Ang. Cty Contrib. Sci., 257, 1-36. Bengtson, S. 1971. Food and feeding of diving ducks at Lake Myvatn, Iceland. Ornis Penn., 48, 77-92. Benzie, V. L. 1965. Some aspects of anti-predator responses of two speciesof stickleback.D.Phil. thesis, University of Oxford. Cain, A. J. & Sheppard, P. M. 1954. Natural selection in Cepaea. Genetics., 39, 89-116. Campbell, R. N. B. 1976. Comparisons between the diet of two Hebridean loch populations of Chart (Salvinus alpinus), one alloptric the other sympatrie with brown trout (Salmo trutta). B.Sc. thesis, University of Aberdeen. Campbell, R. N. 1979. Sticklebacks (Gasterosteus aeuleatus (L) and Pungitius pungitius (L)) in the Outer Hebrides, Scotland. tIebridean Nat., 3, 8-15. Cook, D. C. 1978. Foraging behaviour and food of Grey Herons, Ardea cinerea on the Ythan estuary. Bird Study, 25, 17-22. Curio, E. 1976. The Ethology of Predation. Berlin: Springer Verlag. Dixon, W. J. (Ed.). 1973. B;M.D. Biomedical Computer Programs. Berkeley: University of California Press. Eastman, R. 1969. The Kingfisher. London: Collins.

GILES & H U N T I N G F O R D : A N T I - P R E D A T O R BEHAVIOUR I N STICKLEBACKS Fjeldsa, J. 1975. A census of black-headed gull Larus ridibundus and arctic tern Sterna paradisea in the Lake Myvatn area of Northwest Iceland. Dansk. OrnithoL Fovenings Tidsskrift, 65, 65-72. Giles, N. 1981a. Predation effects upon the behaviour and ecology of Scottish Gasterosteus aculeatus L. populations. Ph.D. thesis, University of Glasgow. Giles, N. 1981b. Summer diet of the grey heron. Scot. Birds, 11, 153-159. Giles, N. 1983a. Behavioural effects of the parasite Schistocephalus solidus (Cestoda) on an intermediate host, the three-spined stickleback, Gasterosteus aculeatus L. Anim. Behav., 31, 1192- 1194. Giles, N. 1983b. The possible role of environmental calcium levels during the evolution of phenotypic diversity in Outer Hebridean populations of the three-spined stickleback, Gasterosteus aeuleatus L. J. ZooL, Lond., 199, 535-544. Giles, N. 1984a. Development of the overhead fright response in wild and predator-naive three-spined sticklebacks, Gasterosteas aeuleatus L. Anita. Behav., 32, 276-279. Giles, N . 1984b. Implications of parental care of offspring for the anti-predator behaviour of adult male and female three-spined sticklebacks, Gasterosteus aculeatus L. In: Fish Reproduction: Strategies and Tactics (Ed. by G. W. Potts), pp. 275-289. London: Academic Press. Gross, H. P. 1978. Natural selection by predators on the defensive apparatus of the three-spined stickleback, Gasterosteus aculeatus L. Z. Zool. Syst. Evol., 15, 252-278. Hagen, D. W. & Gitbertson, L. G. 1972. Geographic variation and environmental selection ~ Gasterosteus aculeatus L. in the Pacific Northwest America. Evolution, 26, 32-51. Hagen, D; W. & Gilbertson, L. G. 1973. Selective predation and the intensity of selection acting upon the lateral plates of three-spined sticklebacks. Heredity, 31, 75-84. Huntingford, F. A. 1976. An investigation of the territorial behaviour of the three-spined stickleback (Gasterosteus aculeatus) using Principal Components Analysis. Anita. Behav., 24, 822-834. Huntingford, F. A. 1982. Do inter- and intraspecific aggression vary in relation to predation pressure in sticklebacks? Anim. Behav., 30, 909-916. Jakobsson, S. & Jarvi, T. 1976, Anti-predator behaviour of two-year-old hatchery-reared Atlantic salmon, Salmo salar, and a description of the predatory behaviour of Burbot, Lota Iota. ZooL Revy., 38, 57-70.

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Kettlewell, H. B. D. 1955. Selection experiments on industrial melanism in the Lepidoptera. Heredity, 9, 323-342. Kimmel, C. B., Patterson, J. & Kimmel, R. O. 1974. The development and behavioural characteristics of the startle response in the Zebra fish. Dev. Psychobiol., 7, 47-60. Lemmetyinen, R. 1973. Feeding ecology of Sterna paradisea, Pentopp. and S. hirundo L. in the archipelago of Southwest Finland. Ann. Zool. Fenn., 10, 507-525. Madsen, F. J. 1957. The food of diving ducks in Danish fjords. Danish Rev. Game Biol., 3, 19-83. McPhail, J. D. 1969. Predation and evolution in a stickleback. J. Fish. Res. Bd Can., 26, 3183-3208. Moodie, G. E. E. 1972. Morphology, life history and ecology of an unusual stickleback (Gasterosteus aculeatus) in the Queen Charlotte Islands, Canada. Can. J. Zool,, 55, 398-404. Moodie, G. E. E., McPhail, J. D. & Hagen, D. W. 1973. Experimental demonstration of selective predation on Gasterosteus aculeatus. Behaviour, 47, 95-105. Moodie, G. E. E. & Reimchen, T. E. 1976. Glacial refugia, endemism and stickleback populations of the Queen Charlotte Islands, British Columbia. Can. Field Nat., 87, 173-175. Pattern, B. G. 1977. Body size and learned avoidance factors affecting predation on Coho salmon, Onchorhyncus kisutch fry by Torrent sculpin Cottus rhotheus. Fish. Bull. U.S. Dept. Commer. Natl Oceanic. Atmos. Adm. Natl Mar. Fish. Serv. Seattle, 75, 457-459. Reimchen, T. E. 1980. Spine deficiency and polymorphism in a population of Gasterosteus aculeatus: an adaptation to predators? Can. J. ZooL, 589 1232-1244. Seghers, B. H. 1970. Behavioural adaptations of natural populations of the Guppy, Poecilia reticulata to predation. Am. ZooL, 10, 489-490. Seghers, B. H. 1973. An analysis of geographic variation in the anti-predator adaptations of the Guppy, Poecilia reticulata. Ph.D. thesis, University of British Columbia. Seghers, B. H. 1974. Geographic variation in the responses of Guppies (Poecilia reticulata) to aerial predators. Oecologia (BerL), 14, 93-98. Sokal, R. R. & Rohlf, F. J. 1973. Introduction to Bitstatistics. San Francisco: W. H. Freeman. (Received 27 August 1982; revised 7 June 1983; M S . number: 2282)