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Hatching success in the three-spined stickleback (Gasterosteus aculeatus) in relation to changes in behaviour during the parental phase
Anita. Behav., I971, 19, 532-541
HATCHING SUCCESS IN THE THREE-SPINED STICKLEBACK (GASTEROSTEUS ACULEATUS) IN RELATION TO CHANGES IN BEHAVIOUR DURING THE PARENTAL PHASE BY ROBERT BLACK* Department of Zoology, University of British Columbia, Vancouver 8, British Columbia Abstract. In the laboratory, there are consistent differences between individual three-spined stickleback males in aggressiveness during the parental phase. Changes in aggressive behaviour and territory size have similar U-shaped temporal patterns which are common to all males. Aggression is lowest and territory size smallest just before the clutch hatches when the male spends most of his time fanning, Males without clutches sometimes attack the nests of other males. Interference by these males is often responsible for hatching failure. Individuals that hatch clutches seem no more aggressive than those that do not. However, males hatching clutches have larger territories during the first part of the parental phase. This paper describes an investigation of the freshwater three-spined stickleback, Gasterosteus aculeatus L. The purpose was to describe changes in behaviour during the parental phase of the male stickleback, to determine whether reproductively successful males behave differently than unsuccessful ones, and to describe aspects of territorial behaviour in terms of site attachment and hostility (Tinbergen 1957). The approach I used was to quantify, in several ways, aggressive and other territorial behaviour in situations of increasing social contact: each way emphasized (a) the reaction of a male fish to another male, or (b) the location of these activities within the territory. A more detailed experiment examined territoriality. Because the nest is the focal point of the male's territory, I investigated the spatial relationships of males with their nests. I examined hostility closely in solitary males to describe an 'aggression landscape' about the nest (van den Assem 1967). Reproductive success is usually assessed by contribution to the next generation. In this laboratory study, such an approach was impossible. Therefore, I defined parental success as the ability to hatch a clutch of eggs. I chose this because some males hatched clutches and others failed, and the number of fry hatched from each successful clutch was similar, although variable, for different treatments of males. I attempted to find differences between sueeessful and unsuccessful males in the behavioural
characteristics which were easily measurable and seemed to have ecological significance. This approach is strictly correlative but, if a relationship exists, it provides predictions which can be tested independently. The rationale behind this approach is that a detailed analysis of interactions among animals and behaviour of individuals might give insight into processes operating at the population level. Behaviour is one expression of the way in which animals cope with their environment. Since environment includes conspecifics, the interactions between animals as well as the presence of others are likely to be involved in processes affecting numbers. The three-spined stickleback is a popular experimental animal. Tinbergen (1953) and van den Assem (1967) give accounts of its behaviour. The breeding male is territorial, builds a nest, courts a female and then cares for the eggs and fry. Methods
The sticklebacks came from the upper Little Campbell River in south-west British Columbia; they were all form leiurus. A combination of periodic collection of fish and 10~ water temperature and natural photoperiod, which tends to retard maturation, or 20~ water temperature and L6-hr day length, which tends to speed maturation, provided reproductively mature fish at most times of the year. When I needed fish for an experiment I selected the most mature fish in the warm stock tank; thus the state of maturity was not constant. However, I assume that all fish building a nest were equally mature; my experiments deal only with fish which built
*Present address: Department of Biological Sciences, University of California, Santa Baxbara, California 93106, U.S.A. 532
BLACK: HATCHING SUCCESS AND BEHAVIOUR OF STICKLEBACKS nests. As nearly as possible, the fish used in experiments experienced the same environmental conditions: day length of 16 hr and water temperatures of 16~ in winter to 22~ in summer. Food for the fish was mainly frozen adult brine shrimp; occasionally Daphnia and tubifex worms supplemented the diet of females. Fry received newly hatched brine shrimp nauplii until they could eat the adult shrimp. I fed the fish once or twice at similar times each day. The aquaria used for experiments were: (a) 17.5-, 37.5- and 49.4-1itre glass aquaria, (b) 269-1itre wooden tanks 240 x 56 x 20 cm, with one Plexiglass side, used as a single container, or, with partitions, as eight separate ones, and (e) 190 x 127 x 20-cm vinyl paddling pools. Aquaria and tanks had a layer of sand on the bottom, and artificial plastic plants or Ceratopteris sp. and Chara sp. planted or floating. The paddling pools had both sand and live plants located in fifteen uniformly distributed dishes. All containers had an air supply, most had a filtering system. Experimental males were kept under one of the following conditions: (1) Solitary in glass aquaria or wooden tanks. These fish could not see other fish in adjacent tanks, but perhaps could see fish in the tanks across the room. (2) Isolated-with-visual-contact (hereafter referred to as IVC) in 33.6-1itre compartments in the wooden tanks. The partitions were partially Plexiglass so neighbouring males could see and interact with each other at the transparent section. (3) Grouped in wooden tanks or paddling pools. There were four males with nests in the wooden tanks and six males with nests in the paddling pools. These fish could see and fight each other all the time. All males were territorial, but were unsynchronized in their reproductive cycles. This meant that some males were caring for eggs when others had none. Grouped males competed for females. Males of all treatments had free choice of nest site.
Recording Procedures I made daily recordings for all my measures of behaviour, except for aggression tests, at 20 cm from the nest, of solitary, IVC and grouped males; these were made every 2 days. For this test, I placed a nuptially coloured male stickleback in a Plexiglass cylinder, 7 cm in diameter and 10.5-era high; the cylinder was usually 20 cm from the experimental male's
533
nest. During each test, I listed the behaviour patterns of the experimental male for 5 rain after he first approached the tester-male. The scores of the test were total frequencies of some of the types of behaviour: bites per 5 rain, bites and bumps per 5 min, and approaches, charges, bites, bumps, and fluttering per 5 rain. A bite was a contact by an open-mouthed male with the Plexiglass container or another fish. A bump was similar contact but the fish's mouth was closed. In an approach, the male swam slowly toward the tester-male; a charge was a rapid movement toward the other male. Fluttering was persistent swimming up and down and around the Plexiglass cylinder. In a separate experiment on solitary males in the wooden tanks, a modification of the aggression test involved a cylinder 5.5 cm in diameter filled with water to the level in the tank. Instead of making handwritten notes, I used a Rustrak four-channel event recorder to show: (a) how long the experimental male oriented to the tester-male, (b) how long the experimental male oriented to the nest and how long the male fanned, (c) the number of bumps, and (d) the number of bites. This method gave higher numbers of bites than the handwritten record because the fish could bite faster than I could write. This experiment served to describe the 'aggression landscape' during the reproductive cycle. Each day I presented the testermale at 20, 40, 60, 80, 100 and 120 cm from the nest, in a random order with the six 5-rain tests following each other immediately. IVC males attacked each other at the transparent partitions. I recorded all the behaviour patterns in each attack; but as an aggressive behaviour score, I used the number of aggressive interactions at the partitions during 15 min each day: each male could attack two neighbours. Grouped males interacted aggressively, with varying frequencies and intensities. High intensity interactions are charging, charging and biting, charging and chasing, and spine fighting. Low intensity interactions are approaching and hesitating. In chasing, one fish swam rapidly after another which tried to avoid the aggressor. Hesitating was short movements toward and then away from another male; orientation to the other male was maintained. A spine fight occurred when two fish attacked each other simultaneously. They circled around each other rapidly with raised ventral spines, each attempting and often succeeding in biting the other. For each interaction, I recorded the initiating and
534
ANIMAL
BEHAVIOUR,
receiving participants and the intensity of the encounter. I tabulated these raw data to show which males initiated and which received interactions, during the 15-min recording each day of the parental phase. After each recording session, I mapped the territory of each male. By 'territory', I mean a topographical area in which a male spends most of his time, and from which he excludes conspecifics (van den Assem 1967). The exact boundaries of the territories were clear because the fish engaged in boundary disputes, even though they occasionally went outside their territories. I made two measurements of location of activity of fish in the wooden tanks. For grouped males, I recorded the behaviour of individuals in the groups of males. Each 30-cm 2 area of the bottom had a name to describe its location. For 30 rain each day, I wrote down the behaviour pattern and location of each fish every 20 s (ninety observations per fish). For solitary males in the wooden tanks, I recorded the location of the fish every 5 s for 15 rain (180 observations). I converted these data into distances from the nest of the male stickleback. For each day of the parental phase, I calculated the mean of distances from the nest and the mean of distances from the nest squared, in two ways" I excluded the instances of the fish at the nest so the means reflect only the males' activity away from the nest, and I included the instances at the nest so the means reflect the proportion of activity at and away from the nest. I made all these behaviour recordings during the parental phase of the male stickleback. This is the period during which the male builds a nest, fertilizes a clutch of eggs, and cares for the eggs and fry. I recorded on the day of nest-building (NB), on the day of fertilization (day 0), and on each subsequent day up to about day 10. Time between NB and 0 varied from 1 day to several. Eggs hatched from day 6 to day 9, dependent on water temperature. Because males were extremely aggressive to females, I introduced the females only when they were ripe, and removed them as soon as they had spawned.
Results Changes in Behaviour I measured behaviour during the parental phase by six methods. The aggression test at 20 cm from the nest, the mean aggression test from those at six distances from the nest, partition
19,
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interactions, and initiations of interactions provide indices of aggressive behaviour. Territory size was a direct measure of territory size; mean distance from the nest provides an index of utilized area. The daily changes in these scores during the parental phase have a similar U-shaped temporal pattern for five of them (Fig. lb to f). Analysis of variance indicates that among days the scores are significantly different. The aggression test scores at 20 cm from the nest have a temporal pattern which is constant with time rather than U-shaped. Figure l a shows that the scores are different between treatments of males, but each treatment has scores constant during the entire experiment. The comparison of measures of aggressive behaviour and territory size is complicated because the days of hatching are different. This is due to differences in water temperature and seasons of the year. However, if the time scale is adjusted so that biologically significant events, day of fertilization and day of hatching, occur in phase and the intervening time is scaled accordingly, the correspondence improves greatly. Figure 1 shows only the mean levels of the measured behaviour. Table I gives the analysis of the individual's scores answering two questions: (a) are an individual's scores consistently higher or lower than scores of other males and (b) do the changes in frequency of response of each individual follow the same temporal pattern? The sample size for mean bites per 5 min and mean of distances from the nest were too small for such an analysis to be worthwhile. Visual examination of plots of these for each individual suggest that patterns were similar and levels were different. For the other measures, the analysis of Table I shows this to be generally statistically true. Scores of individuals are consistently different from the others. A temporal pattern common to all males is present for only paritition interactions, initiations of interactions and territory size of males hatching clutches. I examined the relation of scores made by different methods on the same day on the same fish by correlation analysis. There was no significant correlation between aggression test scores and initiations of interactions of eleven grouped males. Partition interactions and aggression test scores were significantly correlated in only a few eases (six of twenty-eight).
BLACK: HATCHING SUCCESS AND BEHAVIOUR. OF STICKLEBACKS
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Fig. 1. Measures of aggressive behaviour and territory size during the parental phase. NB is day of nest-building and 0 is day of fertilization. (a) Aggression test (handwritten recording) scores (bites plus bumps per 5 rain) with tester-male 20 cm from the nest, for solitary (S), isolated-with-visual-contact (IVC), and grouped (G) males. N=ten fish for each treatment of males. (b) Mean aggressiontest fish scores (Rustrak recording, bite per 5 rain) for six distances from the nest for solitary males in the wooden tanks. Mean day of hatching is 7.0. N=three fish. (c) Partition interactions of IVC males. Mean day of hatching is 6.0. N=twelve fish. (d) Initiations of interactions of grouped males. Mean day of hatching is 8.5. N=nine fish. (e) Territory size in m2 for grouped males which do hatch ((3) and which do not hatch (Q) clutches. Mean day of hatching is 8.0. N= Four fish for both kinds of males. (f) Index of utilized area (mean distance from nest) for solitary males in the wooden tanks N--two fish. Spearman rank correlation between mean territory size and mean initiations of interactions per neighbour is 0.501 (P<0.05, N = 9 ) .
Parental Success and Behaviour From general observations on solitary, IVC and grouped males it became apparent that, in my aquaria at least, the effect of other male sticklebacks on the fate of the clutch was great. Since in nature males are grouped, I include here a description of the influence of other males. Van den Assem (1967) reported similar effects. Each male in a grouped treatment engages in aggressive encounters in order to maintain his territory. Males repulse not only attacks on themselves, but also on their nests. Each male behaves as if he recognizes his neighbours, and knows the exact location of their nests.
Males raid each other's nests, not only when the owner is absent, but also when he is present. These attacks are of two kinds. One results in the theft of nesting material, the other in loss of well-developed eggs which are rarely adopted, but usually eaten. The attacking male takes little notice of the resident male; he does not flee, but persists in boring into the nest, keeping his body vertical and tail waving above the nest. At the same time, the resident male attacks the raider energetically. This activity seems to attract other males if it lasts long enough. I f other males come to the nest, the owner cannot keep them all away, and they destroy the nest and steal or eat the eggs. Loss of eggs usually occurs just before they hatch. I observed enough raids resulting in loss of clutches to feel assured that this was the usual cause for failure to hatch
536
ANIMAL
BEHAVIOUR,
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Table I. Coefficients of Concordance for tile Aggressive Behaviour and Territory Size Measurement
Treatment of males
Measurement
No. of males
Consistency of individual's level of response W
P
Common tempora pattern W
P
Aggression test
Solitary IVC Grouped
10 I0 10
0.334 0-310 0.372
0"02 0.02 0.01
0.089 0.111 0.067
0.50 0-40 0.60
Partition interactions
IVC
12
0-716
0.001
0-266
0-05
8
0-841
0"001
0.381
0-001
Grouped hatching clutch
4
0.550
0"001
0.493
0.05
Grouped not hatching clutch
4
0.861
0.001
0-355
Initiations of interactions Grouped Territory size
<0.20 >0-10
A significant coefficient indicates that temporal changes in the size of the measurements are common to all males or that the males are consistent in their scores relative to the other fish in the treatment. [See Siegel 1956 for the coefficient of concordance (W).] clutches. These attacks did not occur as frequently as van den Assem (1967) reports that sneaking and egg-robbing occurred in his experiments, although it is likely that we are referring to the same phenomena. A n analysis o f variance shows the trend o f the means o f the tests over successive days, the data being repeated observations on each subject each day. The only scores which vary from d a y to day are partition interactions, for twelve fish, initiations o f interactions, for nine fish, and territory size, for eight fish. The same analysis o f variance shows that only territory size is different for males hatching and not hatching clutches (Figure le, Table II). This difference occurs only during the first part o f Table II. Differences in Mean Territory Size of Males Hatching and Males Not Hatching Their Clutches
Mean territory size (m2) 4- sE Day
Hatch
Not hatch
t
NB
0.42 4- 0.095
0.33 -4- 0.071
0-758
0
0.62 -l- 0.095
0.32 4- 0.063
2.643
1
0.67 ::k 0.093
0-32 4- 0.069
3.026
2
0.68 ::[:0"092
0.40 4- 0"064
2.489
3
0.49 4- 0.101
0"46 • 0'054
0"261
t -- 0.05, 7 df = 2.365 Sample size for each class of fish is four.
the parental phase; at this time, those males which later fail to hatch clutches have smaller territories. The activity data on grouped males are for four fish only; two that hatched clutches, and two that did not. The conclusions drawn f r o m this experiment must be tentative. O n any 1 day, only one fish appeared in some o f the 30-cm 2 areas. I n that case, that fish was an 'exclusive occupant' o f that square. The n u m b e r o f squares which a male occupied exclusively is one measure o f territory size. A n o t h e r measure is the n u m b e r o f squares in which a male appeared at least 75 per cent o f the time that any fish was in that square. Analysis o f variance shows that for selected measures o f territory size and activity, males hatching clutches are similar to males not hatching clutches in the area o f exclusive occupancy, the mean distance f r o m the nest at which aggressive encounters occur, and the p r o p o r t i o n of activities which are aggressive. However, males not hatching clutches spend less time at the nest, have almost significantly greater m e a n distances f r o m the nest, and larger (but not significantly so) areas o f 75 per cent occupancy d u r i n g m o s t o f the reproductive cycle (Fig. 2). Site Attachment and Hostility Components o f Territoriality
The object o f this section is to examine h o w fish use the space a r o u n d their nest, and how this use and their aggressive behaviour at different
BLACK: H A T C H I N G SUCCESS A N D B E H A V I O U R O F STICKLEBACKS 75%
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Fig. 2. Behavioural measures during the parental phase from data on activity comparing grouped males hatching ( 9 and not hatching ( e ) clutches. N = t w o fish for both kinds of males. (a) Territory size as numbers of 30 cm2 units occupied exclusively by the male. (b) Territory size as numbers of 30-cm2 units in which the male occurs at least 75 per cent of the observations in those units. (c) Utilized area (mean distance from the nest of all activities not at the nes0. (d) Mean distance from the next of aggressive activities. (e) Proportion of observations of the fish at the nest. (f) Proportion of observations of the fish in which they were engaged in aggressive activities.
distances from the nest change during the parental phase. Analysis of variance showed the following. The means of distances from the nest (excluding occurrences at the nest) during the reproductive cycle were no different for three solitary or four grouped males (Table I I I A ) . The means of distances from the nest squared, however were greater for the grouped males (Table III B). Neither set of means showed changes with time during the reproductive cycle, although there is an indication of this in the solitary males. The means of distances from the nest (including occurrences at the nest) for solitary males have a U-shape pattern during the reproductive cycle (Table III C). The results of the aggression tests at 20 cm from the nest for ten solitary and ten grouped males show that little change occurs during the reproductive cycle in the frequency of the response (Fig. la). Even though the 95 per cent
confidence intervals of the points for solitary and grouped males do overlap, the response of solitary males is consistently higher. A far more detailed analysis of this aggressive behaviour towards a tester-male was possible from the Rustrak recording for tests at six distances from the nest done on three solitary males. Figure 3 shows each of the measures described in the methods in two ways: (a) by distances, averaged over the 14 days for each distance, and (b) by days, averaged over the six distances for each day. There are two patterns of change with increasing distance from the nest. Bites, seconds oriented to tester, mean bout oriented to tester, and bites per second oriented to tester all decrease; seconds oriented to nest, mean bout oriented to nest, seconds of fanning, and mean bout length of fanning all increase. Of the last four measures, all but fanning tend to level off at the two greatest distances. There are three general patterns in the changes
538
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with time. Bites per 5 min has a definite U-shape with the trough at days 6 and 7 just before hatching of the clutch on day 8. If bites per 5 min are plotted for each distance separately, those at 20 cm from the nest are fairly constant during the entire cycle, while at all the other distances there is the U-shape pattern. Seconds oriented to the tester and the mean bout length oriented to the tester both decrease with time, while bites per second oriented to tester increases. All t h e measures related to the nest have an
inverted U-shape with highest values on day 6or7. The analyses of variance for these measures show,, in all cases, the variables are significantly different for distances and days. The interactions are not significant. So these behaviours measured by the aggression tests change with time and distance, and we can try to interpret the significance of these changes in relation to the biology of the fish. One relationship which appears is the inverse
539
BLACK: HATCHING SUCCESS AND BEHAVIOUR OF STICKLEBACKS Table HI. Area Utilized by Solitary and Grouped Males
Day of parental phase Treatment
1
2
3
4
5
6
7
47.0 41-0
54.5 35.1
38.8 34.8
44.7 28-7
44.1 27.4
63.4 27.7
45.8 22.5
43.3
B. Mean distances from the nest squared Grouped 4 Solitary 3
3598 2290
4346 1737
2727 1626
3390 1132
3546 1060
4263 1056
3778 590
2641 2528
C. Means of distances from the nest Solitary 2 24-8 7 7.5
22.6 9 18-7
24-8 10 19.8
16"3 11 31"5
10"6 12 61.0
9.5
9.1
A. Means of distances from the nest Grouped Solitary
N
NB
0
4 3
19"7 8 11.5
8
38.8
The raw data for each fish are distances from the nest. For each day of the parental phase for each fish the data analysed are the means of distances from the nest or means of distances from the nest squared. In A and B, the instances when the fish was at the nest are excluded from the calculation of the means. In C, means were calculated including distances at the nest. trends for biting, and nest orientation and fanning. The respective correlation coefficients are --0.44 and --0-29; both are significantly different from zero ( N = 2 5 2 - - t h r e e fish x six tests/day x 14 days). A decrease in biting is associated with an increase in nest orientation and fanning. A further analysis of the characteristics of the 'aggression landscape' was one in which the aggressive behaviour scores for the three fish are regressed on the six distances from the nest. The initial analysis of variance shows a difference in the level of aggressive behaviour scores between fish. The final analysis o f variance indicates that the linear regression accounts for a very large proportion of the variance, and even though there is some heterogeneity among the slopes of the regression lines for the individual fish (--0.65, --0.97 and --1.3), the intercepts for each fish are very different (141.3, 105.0 and 208.0 bites). The pooled regression equation is bites=151-5 -- 0.99 cm from nest. A plot of bites on distances from the nest for each fish separately shows that the curvature indicated in the analysis of variance comes from only one of the fish. This fish has the smallest mean level of bites per 5 rain and the smallest intercept. His aggressive behaviour at 80 cm and beyond is almost identical; that is, his response to a testermale at 80 cm and beyond is minimal. Discussion Changes in Bchaviour
The essence of the results of this section is
that, if we think in terms of a biological time scale, rather than a daily one, most measures of aggressive behaviour and territory size indicate similar changes in intensity at corresponding biological times. One of the measures, the testermale score at 20 cm from the nest, does not conform to this temporal pattern. The explanation for non-conformity of one measure and the close correspondence of the others may be the same. A tester-male presented at 20 cm from the nest for 5 rain is a rather unnatural situation. Most natural intruders rarely get that close to the nest; and, if they do, the defending male chases them off or they flee almost immediately. It seems likely that my tests at 20 cm from the nest produced a maximum response from the solitary males. The decreased level of response in grouped and IVC males (Fig. la) was probably due to stimuli from other males, because IVC males attacked their neighbours at the partitions, and neighbours distract grouped males. However, for all three treatments of males the proximity to the nest left day to day levels unchanged during the reproductive cycle. All the other aggressive behaviour measures are related to the activity within the territory. Partition interactions occur at the artificial boundaries of the transparent partitions. Initiations of interactions are strictly boundary defence. The mean of aggression test scores for six distances from the nest is certainly influenced by the low intensity of attacks at distances far from the nest. The territory size measurement was based on the interacting system of a male and
540
ANIMAL
BEHAVIOUR,
his neighbours and was a resultant of the male's activity within his territory. Besides aggressive behaviour, the males must perform parental behaviour, i.e. fanning of the eggs in the nest. The amount of fanning reaches a peak just before the clutches hatch (van Iersel 1953). The mean distance from the nest (Fig. lf) is art index of parental care because a large number of occurrences at the nest (distance:0) lowers the mean. The changes in amount of parental care necessitate changes in the location of activities about the nest (mean distance from the nest). This is reflected in changes in the measured territory size (Fig. l e) and aggressive behaviour, especially that expressed far from the nest (Fig. 3, mean bites per 5 min); yet aggressive behaviour close to the nest remains great (Fig. 1a). These experiments are purely descriptive, and leave the question of the causal relationship between parental activities, aggressive behaviour and territory size unanswered. They have shown, however, that five measures of behaviour change in the same way during a biologically significant time.
Parental Success and Behaviour In my experiments, there are differences between males which hatch and which do not hatch clutches. Males with small territories at the beginning of the reproductive cycle fail to hatch their clutches, but the loss of the eggs does not occur until just before hatching. Why this relationship holds is difficult to explain. Just before the eggs are to hatch, males whose eggs later hatch and those whose eggs fail to hatch have similar-sized territories. The data on activity indicate that males which do hatch clutches remain closer to the nest and spend more time at the nest than males which do not hatch clutches. Both types of males engage in the same proportion of aggressive encounters at similar distances from the nest. Perhaps something of the males' behaviour within their territories results in others attempting to raid the nest. Judged by my tests, males that hatch clutches are no more aggressive than those that do not. This seems inconsistent since the temporal pattern of changes in territory size of males hatching clutches is the same as the aggressive behaviour measures (partition interactions, initiations of interactions)(Figs lc and d), but the territory size measure does distinguish between males hatching clutches and those that fail (Fig. le). Either the behaviour tests are not sensitive to the difference between the elassi-
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3
fication of males, or there really may be no difference in these behaviours. Also, territory size must be a result of many attributes of the male, not only its aggressive behaviour. From my observations and from descriptions from the literature on sticklebacks, the following scheme seems plausible as an explanation of the observations on parental success. The males claim territories in suitable areas. Distances between nests are rather uniform (Black & Wootton 1970; van den Assem 1967), but there are differences in territory size dependent on structure of the habitat, order of settling, and many individual characteristics. Males interact continually. Each male learns the location of his neighbours' nests and assesses his neighbours' pugnacity. Males with the largest territories win the competition for gravid females (van den Assem 1967). Some males fail to obtain eggs. These males are attracted to nests of males which have clutches about to hatch. Males without clutches make successful raids on other nests, especially on nests of males which, during the first part of the reproductive cycle, have small territories. At the time of raiding, these territories have contracted to the same size as those of the other fish, but males susceptible to raids spend more time away from the nest.
Site Attachment and Hostility Components of Territoriality Site attachment. Both means of distances from the nest and means of distances from the nest squared were intended to give a measure of the area utilized by the fish. Means of distances from the nest squared are likely a better measure of area, since area is always proportional to the second rather than the first power of a dimension. Squaring in this ease gives greater weight to distances further from the nest. Thus, when occurrences at the nest are excluded, grouped males have larger means of distances from the nest squared. Van den Assem (1967) reports similar results. I interpret this in the following way: (a) solitary and grouped males are on the average at similar distances from their nests during the reproductive cycle; and (b) grouped males, however, tend more often to be at distances further from the nest. This seems reasonable because grouped males must engage in boundary conflicts with other males. This defence of a boundary would make them swim near the periphery of the territory more often than solitary males that do not defend a boundary because there are no conspeeifics.
BLACK: HATCHING SUCCESS AND BEHAVIOUR. OF STICKLEBACKS For solitary males, the comparison o f mean distance from the nest excluding occurrences at the nest with those including occurrences at the nest shows that much time is spent at the nest (where distance from the nest is 0) during the time just before the dutch hatches (Table I I I A and C). In effect, if the male moves from the nest, he is likely to go just as far away any time during the parental phase, but during the time just before hatching the male rarely leaves the nest. It seems, therefore, that stickleback males exhibit a high degree o f 'site attachment' whether or not conspecifics are present. When conspecifics are present, boundary defence puts males farther from the nest more often than if they are alone. Hostility. The consistently lower, although not significantly different, aggressive behaviour scores by grouped males over solitary ones (Fig. la) seems to be one result of the presence of conspecifics. During the 5-min tests, solitary males usually devoted the entire time to attacking the tester-male; grouped males were distraeted by other males, but only rarely engaged in encounters with males other than the tester. This may have caused the lower scores by grouped males, but it is possible that the sudden introduction of a male into the territory of a solitary male was a stronger stimulus to attack than for a grouped male accustomed to intruding males. The experiment with tester-males at different distances supports this latter idea because the first of a series often was abnormally high compared to tests at the same distance but later in the series on the previous or subsequent day. The aggression test scores for solitary and grouped males at 20 cm from the nest (Fig. la) are not strictly comparable to those made at the six distances (Figs Ib and 3). However, of the tests made at six distances, only those at 20 era were relatively constant during the parental phase; all the others showed U-shaped trends. This indicates that the tester-male at 20 cm is a maximal stimulus. An intruder that close to the nest is always attacked vigorously. The total number of bites per 5 min is a rather crude measure. Some of the other measures in Fig. 3 indicate other things besides the overall frequency of bites change. Bites per second oriented to tester-male remains relatively constant during the first part of the reproductive cycle. It is lowest the day before hatching and then increases drastically after the eggs hatch.
541
This means that the exact response to testermale changes in intensity o f attack by biting, even if the frequency per 5-min is the same. Biting is frequent and most intense after the fry hatch. This may be necessary for protection of the fry because, once hatched, they disperse from the nest and so are not discretely located within the territory. The experiments providing these data were not designed to analyse causal relationships. It is particularly hard to separate attendance at the nest because it is the focus of the territory and because it is the centre for parental care. This is because they are not mutually exclusive. Subtracting amount of fanning from orientation to nest gives an index o f site attachment less dominated by parental activities. Up to day 9, this figure is roughly 15 to 23 s per 5 min, with one day having much more, one much less than that. This suggests that 'site attachment' may remain relatively constant, a feature also suggested by the rather constant means of distances from the nest squared (excluding occurrences at the nest). Territory size, however, does change, and what appears to happen is that peripheral parts o f the territory are abandoned, that is, not visited or defended; and other males take up the slack. Once the fry hatch, the male again has time to devote to defence and he reclaims part of what he lost. Acknowledgments This work was done while I was receiving support from National Research Council of Canada Scholarships. I wish to thank Dr D. Chitty for his encouragement and D r R. Liley for hi~ review of the manuscript. Patricia Bright did the tedious job of transcribing the Rustrak charts. REFERENCES Assem, J. van den (1967). Territory in the three-spined stickleback Gasterosteus aculeatus L. Behaviour, SuppL, 16, 1-164. Black, R. & Wootton, R. J. (1970). Dispersion in a natural population of three-spined sticklebacks. Can. J. ZooL, 48, 1133-1135. Iersel, J. J. A. van (1953). An analysis of the parental behavior of the male three-spined stickleback Gasterosteus aculeatus L. Behaviour, SuppL, 3,
1-159. Siegel, S. (1956). Nonparametric Statistics .for the Behavioral Sciences. Toronto: McGraw-Hill. Tinbergen, N. (1953). Social Behaviour in Animals. London: Methuen. Tinbergen, N. (1957). The functions of territory. Bird Study, 4, 14--27. (Received 12 May 1970; revised I November 1970; MS. number: AI000)
HATCHING SUCCESS IN THE THREE-SPINED STICKLEBACK (GASTEROSTEUS ACULEATUS) IN RELATION TO CHANGES IN BEHAVIOUR DURING THE PARENTAL PHASE BY ROBERT BLACK* Department of Zoology, University of British Columbia, Vancouver 8, British Columbia Abstract. In the laboratory, there are consistent differences between individual three-spined stickleback males in aggressiveness during the parental phase. Changes in aggressive behaviour and territory size have similar U-shaped temporal patterns which are common to all males. Aggression is lowest and territory size smallest just before the clutch hatches when the male spends most of his time fanning, Males without clutches sometimes attack the nests of other males. Interference by these males is often responsible for hatching failure. Individuals that hatch clutches seem no more aggressive than those that do not. However, males hatching clutches have larger territories during the first part of the parental phase. This paper describes an investigation of the freshwater three-spined stickleback, Gasterosteus aculeatus L. The purpose was to describe changes in behaviour during the parental phase of the male stickleback, to determine whether reproductively successful males behave differently than unsuccessful ones, and to describe aspects of territorial behaviour in terms of site attachment and hostility (Tinbergen 1957). The approach I used was to quantify, in several ways, aggressive and other territorial behaviour in situations of increasing social contact: each way emphasized (a) the reaction of a male fish to another male, or (b) the location of these activities within the territory. A more detailed experiment examined territoriality. Because the nest is the focal point of the male's territory, I investigated the spatial relationships of males with their nests. I examined hostility closely in solitary males to describe an 'aggression landscape' about the nest (van den Assem 1967). Reproductive success is usually assessed by contribution to the next generation. In this laboratory study, such an approach was impossible. Therefore, I defined parental success as the ability to hatch a clutch of eggs. I chose this because some males hatched clutches and others failed, and the number of fry hatched from each successful clutch was similar, although variable, for different treatments of males. I attempted to find differences between sueeessful and unsuccessful males in the behavioural
characteristics which were easily measurable and seemed to have ecological significance. This approach is strictly correlative but, if a relationship exists, it provides predictions which can be tested independently. The rationale behind this approach is that a detailed analysis of interactions among animals and behaviour of individuals might give insight into processes operating at the population level. Behaviour is one expression of the way in which animals cope with their environment. Since environment includes conspecifics, the interactions between animals as well as the presence of others are likely to be involved in processes affecting numbers. The three-spined stickleback is a popular experimental animal. Tinbergen (1953) and van den Assem (1967) give accounts of its behaviour. The breeding male is territorial, builds a nest, courts a female and then cares for the eggs and fry. Methods
The sticklebacks came from the upper Little Campbell River in south-west British Columbia; they were all form leiurus. A combination of periodic collection of fish and 10~ water temperature and natural photoperiod, which tends to retard maturation, or 20~ water temperature and L6-hr day length, which tends to speed maturation, provided reproductively mature fish at most times of the year. When I needed fish for an experiment I selected the most mature fish in the warm stock tank; thus the state of maturity was not constant. However, I assume that all fish building a nest were equally mature; my experiments deal only with fish which built
*Present address: Department of Biological Sciences, University of California, Santa Baxbara, California 93106, U.S.A. 532
BLACK: HATCHING SUCCESS AND BEHAVIOUR OF STICKLEBACKS nests. As nearly as possible, the fish used in experiments experienced the same environmental conditions: day length of 16 hr and water temperatures of 16~ in winter to 22~ in summer. Food for the fish was mainly frozen adult brine shrimp; occasionally Daphnia and tubifex worms supplemented the diet of females. Fry received newly hatched brine shrimp nauplii until they could eat the adult shrimp. I fed the fish once or twice at similar times each day. The aquaria used for experiments were: (a) 17.5-, 37.5- and 49.4-1itre glass aquaria, (b) 269-1itre wooden tanks 240 x 56 x 20 cm, with one Plexiglass side, used as a single container, or, with partitions, as eight separate ones, and (e) 190 x 127 x 20-cm vinyl paddling pools. Aquaria and tanks had a layer of sand on the bottom, and artificial plastic plants or Ceratopteris sp. and Chara sp. planted or floating. The paddling pools had both sand and live plants located in fifteen uniformly distributed dishes. All containers had an air supply, most had a filtering system. Experimental males were kept under one of the following conditions: (1) Solitary in glass aquaria or wooden tanks. These fish could not see other fish in adjacent tanks, but perhaps could see fish in the tanks across the room. (2) Isolated-with-visual-contact (hereafter referred to as IVC) in 33.6-1itre compartments in the wooden tanks. The partitions were partially Plexiglass so neighbouring males could see and interact with each other at the transparent section. (3) Grouped in wooden tanks or paddling pools. There were four males with nests in the wooden tanks and six males with nests in the paddling pools. These fish could see and fight each other all the time. All males were territorial, but were unsynchronized in their reproductive cycles. This meant that some males were caring for eggs when others had none. Grouped males competed for females. Males of all treatments had free choice of nest site.
Recording Procedures I made daily recordings for all my measures of behaviour, except for aggression tests, at 20 cm from the nest, of solitary, IVC and grouped males; these were made every 2 days. For this test, I placed a nuptially coloured male stickleback in a Plexiglass cylinder, 7 cm in diameter and 10.5-era high; the cylinder was usually 20 cm from the experimental male's
533
nest. During each test, I listed the behaviour patterns of the experimental male for 5 rain after he first approached the tester-male. The scores of the test were total frequencies of some of the types of behaviour: bites per 5 rain, bites and bumps per 5 min, and approaches, charges, bites, bumps, and fluttering per 5 rain. A bite was a contact by an open-mouthed male with the Plexiglass container or another fish. A bump was similar contact but the fish's mouth was closed. In an approach, the male swam slowly toward the tester-male; a charge was a rapid movement toward the other male. Fluttering was persistent swimming up and down and around the Plexiglass cylinder. In a separate experiment on solitary males in the wooden tanks, a modification of the aggression test involved a cylinder 5.5 cm in diameter filled with water to the level in the tank. Instead of making handwritten notes, I used a Rustrak four-channel event recorder to show: (a) how long the experimental male oriented to the tester-male, (b) how long the experimental male oriented to the nest and how long the male fanned, (c) the number of bumps, and (d) the number of bites. This method gave higher numbers of bites than the handwritten record because the fish could bite faster than I could write. This experiment served to describe the 'aggression landscape' during the reproductive cycle. Each day I presented the testermale at 20, 40, 60, 80, 100 and 120 cm from the nest, in a random order with the six 5-rain tests following each other immediately. IVC males attacked each other at the transparent partitions. I recorded all the behaviour patterns in each attack; but as an aggressive behaviour score, I used the number of aggressive interactions at the partitions during 15 min each day: each male could attack two neighbours. Grouped males interacted aggressively, with varying frequencies and intensities. High intensity interactions are charging, charging and biting, charging and chasing, and spine fighting. Low intensity interactions are approaching and hesitating. In chasing, one fish swam rapidly after another which tried to avoid the aggressor. Hesitating was short movements toward and then away from another male; orientation to the other male was maintained. A spine fight occurred when two fish attacked each other simultaneously. They circled around each other rapidly with raised ventral spines, each attempting and often succeeding in biting the other. For each interaction, I recorded the initiating and
534
ANIMAL
BEHAVIOUR,
receiving participants and the intensity of the encounter. I tabulated these raw data to show which males initiated and which received interactions, during the 15-min recording each day of the parental phase. After each recording session, I mapped the territory of each male. By 'territory', I mean a topographical area in which a male spends most of his time, and from which he excludes conspecifics (van den Assem 1967). The exact boundaries of the territories were clear because the fish engaged in boundary disputes, even though they occasionally went outside their territories. I made two measurements of location of activity of fish in the wooden tanks. For grouped males, I recorded the behaviour of individuals in the groups of males. Each 30-cm 2 area of the bottom had a name to describe its location. For 30 rain each day, I wrote down the behaviour pattern and location of each fish every 20 s (ninety observations per fish). For solitary males in the wooden tanks, I recorded the location of the fish every 5 s for 15 rain (180 observations). I converted these data into distances from the nest of the male stickleback. For each day of the parental phase, I calculated the mean of distances from the nest and the mean of distances from the nest squared, in two ways" I excluded the instances of the fish at the nest so the means reflect only the males' activity away from the nest, and I included the instances at the nest so the means reflect the proportion of activity at and away from the nest. I made all these behaviour recordings during the parental phase of the male stickleback. This is the period during which the male builds a nest, fertilizes a clutch of eggs, and cares for the eggs and fry. I recorded on the day of nest-building (NB), on the day of fertilization (day 0), and on each subsequent day up to about day 10. Time between NB and 0 varied from 1 day to several. Eggs hatched from day 6 to day 9, dependent on water temperature. Because males were extremely aggressive to females, I introduced the females only when they were ripe, and removed them as soon as they had spawned.
Results Changes in Behaviour I measured behaviour during the parental phase by six methods. The aggression test at 20 cm from the nest, the mean aggression test from those at six distances from the nest, partition
19,
3
interactions, and initiations of interactions provide indices of aggressive behaviour. Territory size was a direct measure of territory size; mean distance from the nest provides an index of utilized area. The daily changes in these scores during the parental phase have a similar U-shaped temporal pattern for five of them (Fig. lb to f). Analysis of variance indicates that among days the scores are significantly different. The aggression test scores at 20 cm from the nest have a temporal pattern which is constant with time rather than U-shaped. Figure l a shows that the scores are different between treatments of males, but each treatment has scores constant during the entire experiment. The comparison of measures of aggressive behaviour and territory size is complicated because the days of hatching are different. This is due to differences in water temperature and seasons of the year. However, if the time scale is adjusted so that biologically significant events, day of fertilization and day of hatching, occur in phase and the intervening time is scaled accordingly, the correspondence improves greatly. Figure 1 shows only the mean levels of the measured behaviour. Table I gives the analysis of the individual's scores answering two questions: (a) are an individual's scores consistently higher or lower than scores of other males and (b) do the changes in frequency of response of each individual follow the same temporal pattern? The sample size for mean bites per 5 min and mean of distances from the nest were too small for such an analysis to be worthwhile. Visual examination of plots of these for each individual suggest that patterns were similar and levels were different. For the other measures, the analysis of Table I shows this to be generally statistically true. Scores of individuals are consistently different from the others. A temporal pattern common to all males is present for only paritition interactions, initiations of interactions and territory size of males hatching clutches. I examined the relation of scores made by different methods on the same day on the same fish by correlation analysis. There was no significant correlation between aggression test scores and initiations of interactions of eleven grouped males. Partition interactions and aggression test scores were significantly correlated in only a few eases (six of twenty-eight).
BLACK: HATCHING SUCCESS AND BEHAVIOUR. OF STICKLEBACKS
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Fig. 1. Measures of aggressive behaviour and territory size during the parental phase. NB is day of nest-building and 0 is day of fertilization. (a) Aggression test (handwritten recording) scores (bites plus bumps per 5 rain) with tester-male 20 cm from the nest, for solitary (S), isolated-with-visual-contact (IVC), and grouped (G) males. N=ten fish for each treatment of males. (b) Mean aggressiontest fish scores (Rustrak recording, bite per 5 rain) for six distances from the nest for solitary males in the wooden tanks. Mean day of hatching is 7.0. N=three fish. (c) Partition interactions of IVC males. Mean day of hatching is 6.0. N=twelve fish. (d) Initiations of interactions of grouped males. Mean day of hatching is 8.5. N=nine fish. (e) Territory size in m2 for grouped males which do hatch ((3) and which do not hatch (Q) clutches. Mean day of hatching is 8.0. N= Four fish for both kinds of males. (f) Index of utilized area (mean distance from nest) for solitary males in the wooden tanks N--two fish. Spearman rank correlation between mean territory size and mean initiations of interactions per neighbour is 0.501 (P<0.05, N = 9 ) .
Parental Success and Behaviour From general observations on solitary, IVC and grouped males it became apparent that, in my aquaria at least, the effect of other male sticklebacks on the fate of the clutch was great. Since in nature males are grouped, I include here a description of the influence of other males. Van den Assem (1967) reported similar effects. Each male in a grouped treatment engages in aggressive encounters in order to maintain his territory. Males repulse not only attacks on themselves, but also on their nests. Each male behaves as if he recognizes his neighbours, and knows the exact location of their nests.
Males raid each other's nests, not only when the owner is absent, but also when he is present. These attacks are of two kinds. One results in the theft of nesting material, the other in loss of well-developed eggs which are rarely adopted, but usually eaten. The attacking male takes little notice of the resident male; he does not flee, but persists in boring into the nest, keeping his body vertical and tail waving above the nest. At the same time, the resident male attacks the raider energetically. This activity seems to attract other males if it lasts long enough. I f other males come to the nest, the owner cannot keep them all away, and they destroy the nest and steal or eat the eggs. Loss of eggs usually occurs just before they hatch. I observed enough raids resulting in loss of clutches to feel assured that this was the usual cause for failure to hatch
536
ANIMAL
BEHAVIOUR,
19,
3
Table I. Coefficients of Concordance for tile Aggressive Behaviour and Territory Size Measurement
Treatment of males
Measurement
No. of males
Consistency of individual's level of response W
P
Common tempora pattern W
P
Aggression test
Solitary IVC Grouped
10 I0 10
0.334 0-310 0.372
0"02 0.02 0.01
0.089 0.111 0.067
0.50 0-40 0.60
Partition interactions
IVC
12
0-716
0.001
0-266
0-05
8
0-841
0"001
0.381
0-001
Grouped hatching clutch
4
0.550
0"001
0.493
0.05
Grouped not hatching clutch
4
0.861
0.001
0-355
Initiations of interactions Grouped Territory size
<0.20 >0-10
A significant coefficient indicates that temporal changes in the size of the measurements are common to all males or that the males are consistent in their scores relative to the other fish in the treatment. [See Siegel 1956 for the coefficient of concordance (W).] clutches. These attacks did not occur as frequently as van den Assem (1967) reports that sneaking and egg-robbing occurred in his experiments, although it is likely that we are referring to the same phenomena. A n analysis o f variance shows the trend o f the means o f the tests over successive days, the data being repeated observations on each subject each day. The only scores which vary from d a y to day are partition interactions, for twelve fish, initiations o f interactions, for nine fish, and territory size, for eight fish. The same analysis o f variance shows that only territory size is different for males hatching and not hatching clutches (Figure le, Table II). This difference occurs only during the first part o f Table II. Differences in Mean Territory Size of Males Hatching and Males Not Hatching Their Clutches
Mean territory size (m2) 4- sE Day
Hatch
Not hatch
t
NB
0.42 4- 0.095
0.33 -4- 0.071
0-758
0
0.62 -l- 0.095
0.32 4- 0.063
2.643
1
0.67 ::k 0.093
0-32 4- 0.069
3.026
2
0.68 ::[:0"092
0.40 4- 0"064
2.489
3
0.49 4- 0.101
0"46 • 0'054
0"261
t -- 0.05, 7 df = 2.365 Sample size for each class of fish is four.
the parental phase; at this time, those males which later fail to hatch clutches have smaller territories. The activity data on grouped males are for four fish only; two that hatched clutches, and two that did not. The conclusions drawn f r o m this experiment must be tentative. O n any 1 day, only one fish appeared in some o f the 30-cm 2 areas. I n that case, that fish was an 'exclusive occupant' o f that square. The n u m b e r o f squares which a male occupied exclusively is one measure o f territory size. A n o t h e r measure is the n u m b e r o f squares in which a male appeared at least 75 per cent o f the time that any fish was in that square. Analysis o f variance shows that for selected measures o f territory size and activity, males hatching clutches are similar to males not hatching clutches in the area o f exclusive occupancy, the mean distance f r o m the nest at which aggressive encounters occur, and the p r o p o r t i o n of activities which are aggressive. However, males not hatching clutches spend less time at the nest, have almost significantly greater m e a n distances f r o m the nest, and larger (but not significantly so) areas o f 75 per cent occupancy d u r i n g m o s t o f the reproductive cycle (Fig. 2). Site Attachment and Hostility Components o f Territoriality
The object o f this section is to examine h o w fish use the space a r o u n d their nest, and how this use and their aggressive behaviour at different
BLACK: H A T C H I N G SUCCESS A N D B E H A V I O U R O F STICKLEBACKS 75%
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distances from the nest change during the parental phase. Analysis of variance showed the following. The means of distances from the nest (excluding occurrences at the nest) during the reproductive cycle were no different for three solitary or four grouped males (Table I I I A ) . The means of distances from the nest squared, however were greater for the grouped males (Table III B). Neither set of means showed changes with time during the reproductive cycle, although there is an indication of this in the solitary males. The means of distances from the nest (including occurrences at the nest) for solitary males have a U-shape pattern during the reproductive cycle (Table III C). The results of the aggression tests at 20 cm from the nest for ten solitary and ten grouped males show that little change occurs during the reproductive cycle in the frequency of the response (Fig. la). Even though the 95 per cent
confidence intervals of the points for solitary and grouped males do overlap, the response of solitary males is consistently higher. A far more detailed analysis of this aggressive behaviour towards a tester-male was possible from the Rustrak recording for tests at six distances from the nest done on three solitary males. Figure 3 shows each of the measures described in the methods in two ways: (a) by distances, averaged over the 14 days for each distance, and (b) by days, averaged over the six distances for each day. There are two patterns of change with increasing distance from the nest. Bites, seconds oriented to tester, mean bout oriented to tester, and bites per second oriented to tester all decrease; seconds oriented to nest, mean bout oriented to nest, seconds of fanning, and mean bout length of fanning all increase. Of the last four measures, all but fanning tend to level off at the two greatest distances. There are three general patterns in the changes
538
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with time. Bites per 5 min has a definite U-shape with the trough at days 6 and 7 just before hatching of the clutch on day 8. If bites per 5 min are plotted for each distance separately, those at 20 cm from the nest are fairly constant during the entire cycle, while at all the other distances there is the U-shape pattern. Seconds oriented to the tester and the mean bout length oriented to the tester both decrease with time, while bites per second oriented to tester increases. All t h e measures related to the nest have an
inverted U-shape with highest values on day 6or7. The analyses of variance for these measures show,, in all cases, the variables are significantly different for distances and days. The interactions are not significant. So these behaviours measured by the aggression tests change with time and distance, and we can try to interpret the significance of these changes in relation to the biology of the fish. One relationship which appears is the inverse
539
BLACK: HATCHING SUCCESS AND BEHAVIOUR OF STICKLEBACKS Table HI. Area Utilized by Solitary and Grouped Males
Day of parental phase Treatment
1
2
3
4
5
6
7
47.0 41-0
54.5 35.1
38.8 34.8
44.7 28-7
44.1 27.4
63.4 27.7
45.8 22.5
43.3
B. Mean distances from the nest squared Grouped 4 Solitary 3
3598 2290
4346 1737
2727 1626
3390 1132
3546 1060
4263 1056
3778 590
2641 2528
C. Means of distances from the nest Solitary 2 24-8 7 7.5
22.6 9 18-7
24-8 10 19.8
16"3 11 31"5
10"6 12 61.0
9.5
9.1
A. Means of distances from the nest Grouped Solitary
N
NB
0
4 3
19"7 8 11.5
8
38.8
The raw data for each fish are distances from the nest. For each day of the parental phase for each fish the data analysed are the means of distances from the nest or means of distances from the nest squared. In A and B, the instances when the fish was at the nest are excluded from the calculation of the means. In C, means were calculated including distances at the nest. trends for biting, and nest orientation and fanning. The respective correlation coefficients are --0.44 and --0-29; both are significantly different from zero ( N = 2 5 2 - - t h r e e fish x six tests/day x 14 days). A decrease in biting is associated with an increase in nest orientation and fanning. A further analysis of the characteristics of the 'aggression landscape' was one in which the aggressive behaviour scores for the three fish are regressed on the six distances from the nest. The initial analysis of variance shows a difference in the level of aggressive behaviour scores between fish. The final analysis o f variance indicates that the linear regression accounts for a very large proportion of the variance, and even though there is some heterogeneity among the slopes of the regression lines for the individual fish (--0.65, --0.97 and --1.3), the intercepts for each fish are very different (141.3, 105.0 and 208.0 bites). The pooled regression equation is bites=151-5 -- 0.99 cm from nest. A plot of bites on distances from the nest for each fish separately shows that the curvature indicated in the analysis of variance comes from only one of the fish. This fish has the smallest mean level of bites per 5 rain and the smallest intercept. His aggressive behaviour at 80 cm and beyond is almost identical; that is, his response to a testermale at 80 cm and beyond is minimal. Discussion Changes in Bchaviour
The essence of the results of this section is
that, if we think in terms of a biological time scale, rather than a daily one, most measures of aggressive behaviour and territory size indicate similar changes in intensity at corresponding biological times. One of the measures, the testermale score at 20 cm from the nest, does not conform to this temporal pattern. The explanation for non-conformity of one measure and the close correspondence of the others may be the same. A tester-male presented at 20 cm from the nest for 5 rain is a rather unnatural situation. Most natural intruders rarely get that close to the nest; and, if they do, the defending male chases them off or they flee almost immediately. It seems likely that my tests at 20 cm from the nest produced a maximum response from the solitary males. The decreased level of response in grouped and IVC males (Fig. la) was probably due to stimuli from other males, because IVC males attacked their neighbours at the partitions, and neighbours distract grouped males. However, for all three treatments of males the proximity to the nest left day to day levels unchanged during the reproductive cycle. All the other aggressive behaviour measures are related to the activity within the territory. Partition interactions occur at the artificial boundaries of the transparent partitions. Initiations of interactions are strictly boundary defence. The mean of aggression test scores for six distances from the nest is certainly influenced by the low intensity of attacks at distances far from the nest. The territory size measurement was based on the interacting system of a male and
540
ANIMAL
BEHAVIOUR,
his neighbours and was a resultant of the male's activity within his territory. Besides aggressive behaviour, the males must perform parental behaviour, i.e. fanning of the eggs in the nest. The amount of fanning reaches a peak just before the clutches hatch (van Iersel 1953). The mean distance from the nest (Fig. lf) is art index of parental care because a large number of occurrences at the nest (distance:0) lowers the mean. The changes in amount of parental care necessitate changes in the location of activities about the nest (mean distance from the nest). This is reflected in changes in the measured territory size (Fig. l e) and aggressive behaviour, especially that expressed far from the nest (Fig. 3, mean bites per 5 min); yet aggressive behaviour close to the nest remains great (Fig. 1a). These experiments are purely descriptive, and leave the question of the causal relationship between parental activities, aggressive behaviour and territory size unanswered. They have shown, however, that five measures of behaviour change in the same way during a biologically significant time.
Parental Success and Behaviour In my experiments, there are differences between males which hatch and which do not hatch clutches. Males with small territories at the beginning of the reproductive cycle fail to hatch their clutches, but the loss of the eggs does not occur until just before hatching. Why this relationship holds is difficult to explain. Just before the eggs are to hatch, males whose eggs later hatch and those whose eggs fail to hatch have similar-sized territories. The data on activity indicate that males which do hatch clutches remain closer to the nest and spend more time at the nest than males which do not hatch clutches. Both types of males engage in the same proportion of aggressive encounters at similar distances from the nest. Perhaps something of the males' behaviour within their territories results in others attempting to raid the nest. Judged by my tests, males that hatch clutches are no more aggressive than those that do not. This seems inconsistent since the temporal pattern of changes in territory size of males hatching clutches is the same as the aggressive behaviour measures (partition interactions, initiations of interactions)(Figs lc and d), but the territory size measure does distinguish between males hatching clutches and those that fail (Fig. le). Either the behaviour tests are not sensitive to the difference between the elassi-
19,
3
fication of males, or there really may be no difference in these behaviours. Also, territory size must be a result of many attributes of the male, not only its aggressive behaviour. From my observations and from descriptions from the literature on sticklebacks, the following scheme seems plausible as an explanation of the observations on parental success. The males claim territories in suitable areas. Distances between nests are rather uniform (Black & Wootton 1970; van den Assem 1967), but there are differences in territory size dependent on structure of the habitat, order of settling, and many individual characteristics. Males interact continually. Each male learns the location of his neighbours' nests and assesses his neighbours' pugnacity. Males with the largest territories win the competition for gravid females (van den Assem 1967). Some males fail to obtain eggs. These males are attracted to nests of males which have clutches about to hatch. Males without clutches make successful raids on other nests, especially on nests of males which, during the first part of the reproductive cycle, have small territories. At the time of raiding, these territories have contracted to the same size as those of the other fish, but males susceptible to raids spend more time away from the nest.
Site Attachment and Hostility Components of Territoriality Site attachment. Both means of distances from the nest and means of distances from the nest squared were intended to give a measure of the area utilized by the fish. Means of distances from the nest squared are likely a better measure of area, since area is always proportional to the second rather than the first power of a dimension. Squaring in this ease gives greater weight to distances further from the nest. Thus, when occurrences at the nest are excluded, grouped males have larger means of distances from the nest squared. Van den Assem (1967) reports similar results. I interpret this in the following way: (a) solitary and grouped males are on the average at similar distances from their nests during the reproductive cycle; and (b) grouped males, however, tend more often to be at distances further from the nest. This seems reasonable because grouped males must engage in boundary conflicts with other males. This defence of a boundary would make them swim near the periphery of the territory more often than solitary males that do not defend a boundary because there are no conspeeifics.
BLACK: HATCHING SUCCESS AND BEHAVIOUR. OF STICKLEBACKS For solitary males, the comparison o f mean distance from the nest excluding occurrences at the nest with those including occurrences at the nest shows that much time is spent at the nest (where distance from the nest is 0) during the time just before the dutch hatches (Table I I I A and C). In effect, if the male moves from the nest, he is likely to go just as far away any time during the parental phase, but during the time just before hatching the male rarely leaves the nest. It seems, therefore, that stickleback males exhibit a high degree o f 'site attachment' whether or not conspecifics are present. When conspecifics are present, boundary defence puts males farther from the nest more often than if they are alone. Hostility. The consistently lower, although not significantly different, aggressive behaviour scores by grouped males over solitary ones (Fig. la) seems to be one result of the presence of conspecifics. During the 5-min tests, solitary males usually devoted the entire time to attacking the tester-male; grouped males were distraeted by other males, but only rarely engaged in encounters with males other than the tester. This may have caused the lower scores by grouped males, but it is possible that the sudden introduction of a male into the territory of a solitary male was a stronger stimulus to attack than for a grouped male accustomed to intruding males. The experiment with tester-males at different distances supports this latter idea because the first of a series often was abnormally high compared to tests at the same distance but later in the series on the previous or subsequent day. The aggression test scores for solitary and grouped males at 20 cm from the nest (Fig. la) are not strictly comparable to those made at the six distances (Figs Ib and 3). However, of the tests made at six distances, only those at 20 era were relatively constant during the parental phase; all the others showed U-shaped trends. This indicates that the tester-male at 20 cm is a maximal stimulus. An intruder that close to the nest is always attacked vigorously. The total number of bites per 5 min is a rather crude measure. Some of the other measures in Fig. 3 indicate other things besides the overall frequency of bites change. Bites per second oriented to tester-male remains relatively constant during the first part of the reproductive cycle. It is lowest the day before hatching and then increases drastically after the eggs hatch.
541
This means that the exact response to testermale changes in intensity o f attack by biting, even if the frequency per 5-min is the same. Biting is frequent and most intense after the fry hatch. This may be necessary for protection of the fry because, once hatched, they disperse from the nest and so are not discretely located within the territory. The experiments providing these data were not designed to analyse causal relationships. It is particularly hard to separate attendance at the nest because it is the focus of the territory and because it is the centre for parental care. This is because they are not mutually exclusive. Subtracting amount of fanning from orientation to nest gives an index o f site attachment less dominated by parental activities. Up to day 9, this figure is roughly 15 to 23 s per 5 min, with one day having much more, one much less than that. This suggests that 'site attachment' may remain relatively constant, a feature also suggested by the rather constant means of distances from the nest squared (excluding occurrences at the nest). Territory size, however, does change, and what appears to happen is that peripheral parts o f the territory are abandoned, that is, not visited or defended; and other males take up the slack. Once the fry hatch, the male again has time to devote to defence and he reclaims part of what he lost. Acknowledgments This work was done while I was receiving support from National Research Council of Canada Scholarships. I wish to thank Dr D. Chitty for his encouragement and D r R. Liley for hi~ review of the manuscript. Patricia Bright did the tedious job of transcribing the Rustrak charts. REFERENCES Assem, J. van den (1967). Territory in the three-spined stickleback Gasterosteus aculeatus L. Behaviour, SuppL, 16, 1-164. Black, R. & Wootton, R. J. (1970). Dispersion in a natural population of three-spined sticklebacks. Can. J. ZooL, 48, 1133-1135. Iersel, J. J. A. van (1953). An analysis of the parental behavior of the male three-spined stickleback Gasterosteus aculeatus L. Behaviour, SuppL, 3,
1-159. Siegel, S. (1956). Nonparametric Statistics .for the Behavioral Sciences. Toronto: McGraw-Hill. Tinbergen, N. (1953). Social Behaviour in Animals. London: Methuen. Tinbergen, N. (1957). The functions of territory. Bird Study, 4, 14--27. (Received 12 May 1970; revised I November 1970; MS. number: AI000)