Role of sensitisation to testosterone in the early development of aggression in the blackheaded

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1 University of Groningen Role of sensitisation to testosterone in the early development of aggression in the blackheaded gull Ros, Albert Frank Huáscar IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 1997 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Ros, A. F. H. (1997). Role of sensitisation to testosterone in the early development of aggression in the black-headed gull s.n. Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Download date:

2 Chapter IV Sensitisation of social behaviour to testosterone in young black-headed gulls Albert F.H. Ros, Sandra ten Holder, and Ton G.G. Groothuis Abstract In previous studies we found that in gull chicks temporary treatment with testosterone resulted in a long-lasting increase in responsiveness of aggressive behaviour to an aggressive challenge. Since these challenges induced a short lasting elevation in testosterone levels in both hormonally treated chicks and control chicks, it was hypothesised that the treatment increased the sensitivity to the hormone. The effect disappeared around day of fledging. In this paper we tested: (1) this sensitivity hypothesis, (2) whether the long-lasting effect is confined to an early sensitive period, or can be induced in juvenile gulls too, and (3) whether the effect can be extended to several months. Juvenile fledged gulls were temporarily treated with testosterone or sham operated at the age of 3.5 and 10 months. After cessation of testosterone treatment the birds still performed aggressive behaviour in higher frequencies than untreated controls, especially during a social challenge. Birds treated with testosterone at 3.5 months of age showed a much faster increase in aggressive behaviour during subsequent treatment at 10 months of age than birds treated for the first time at 10 months of age. It is concluded that in a wide range of ages temporary exposure to testosterone can induce a long-lasting increase in sensitivity of aggressive behaviour to testosterone. It has been found in several animal species, including adult birds, that a social challenge outside the breeding season, when basal testosterone levels are low, is related to a short lasting elevation in levels of this hormone that mediates the aggressive response to the challenge. Our results open the possibility that the sensitivity of the aggressive response to the challenge is influenced by the elevated basal testosterone levels during the beginning of the breeding season. Submitted

3 46 Chapter IV Introduction In many animal species, including palearctic birds, basal levels of testosterone are elevated in spring, necessary for the high level of competition during territory establishment and mate selection at that time. During parental care or territorial defence outside the breeding phase, testosterone may only be elevated during an aggressive challenge. This enables the animal to respond with aggressive behaviour without incurring the costs of persistently high levels of the hormone. (For reviews see Wingfield et al. 1990, Ketterson et al. 1996). In this paper we test whether the sensitivity to this short lasting elevation in testosterone levels during a social challenge, is increased by earlier exposure to high basal levels of the hormone. The subject of this study is the black-headed gull. Birds of this species are monogamous and breed in dense colonies during late spring and summer. Males and females both produce testosterone, have the same nuptial plumage, and perform similar types and frequencies of aggressive behaviour (van Rhijn 1985). Black-headed gull chicks respond aggressively to an adult intruder on the territory. We showed experimentally that chicks that had been exposed to elevated levels of testosterone for some time respond much more aggressively to a standardised aggressive challenge than chicks not exposed to such levels of testosterone. Recently we showed that in both groups testosterone plasma levels are elevated for a short period just after the challenge. A possible explanation for the group difference in aggression despite similar hormone levels is that exposure to testosterone increases the sensitivity of aggressive behaviour to testosterone (Chapter II). One of the two aims of this paper is to test this idea experimentally. In chicks the long-lasting effect of testosterone treatment disappeared around fledging (Chapter II, Chapter III). Since long-lasting hormonal effects are often reported to result from hormonal exposure early in life, we questioned whether our previous results hold true 1) for older birds as well, and 2) for a longer time span than the few weeks between testosterone treatment and fledging in our earlier study. Therefore the second aim of the paper is to extend our earlier studies to older gulls over a longer time span. In the first experiment juvenile birds were treated with testosterone or sham operated. Social behaviour of both groups was compared for some time after termination of treatment. To test the influence of testosterone treatment on the later sensitivity to the hormone, both groups were subsequently treated with testosterone in next spring. In the second experiment the effect of social

4 Long lasting effects of testosterone in gulls 47 challenges on social behaviour after termination of treatment was analysed in more detail. Experiment I Method Rearing conditions and treatment Twenty-four black-headed gulls of 2 weeks of age were collected from the field and housed in groups of three or four peers. The groups were kept in cages of 1 1 m. At 1 month of age the groups were randomly divided in two groups of 12 birds that were housed in outdoor aviaries of m each. Food and water were available ad libitum. The birds were fed with dry food pellets for trout (Trouvit, Trouw, Gent, Belgium). To facilitate individual recognition by the observer the birds were individually marked on head, tail, wing, breast, or back with rhodamine or picrine (ICN Biochemicals, Cleveland, Ohio, USA; chemicals were dissolved in acetone), and received a unique colour ring combination on the leg. Testosterone was administered using crystalline pellets of 25 mg testosterone propionate. These were implanted subcutaneously in the neck region under local anaesthesia with lidocaine (Xylocaine, Astra, Rijswijk, The Netherlands). The incision was closed with stitches. For measuring levels of testosterone blood was drawn from the brachial wing vein with a heparin-rinsed needle and syringe within 5 minutes after capture of the bird. After centrifugation, plasma was stored at 30 C until analysis. Experimental design At 3.5 months of age (day 0 of the T1 period) in each aviary seven birds were randomly chosen and implanted with a testosterone pellet. The other 10 birds were sham operated. At day 23 after implantation (the end of the T1 period and the beginning of the T-out period) the testosterone pellets were removed and the controls were sham operated again. In spring, at 10 months of age, in each aviary three birds of the T group and three birds of the C group were implanted with a testosterone pellet for 8 days (T2 period). All analyses presented in this paper concern only those birds that were present at the end of the T2 period: T group n = 6, C group n = 6 (in the course of the year one control and one testosterone treated bird died and three control and seven testosterone treated birds were used for other experiments).

5 48 Chapter IV Behavioural observations were carried out almost daily between day 1 and day 23 after the first implantation when 3.5 months of age, during the first 2 weeks after termination of this treatment, and on day 1, 2, 3, 4, 5, and 8 after implantation when 10 months of age. Two aviaries were simultaneously observed by two observers during sessions of 1 to 1.5 h spread over the day. Observations were carried out from a hide at a distance of 4 m and with the help of a cassette recorder. Classification of behaviour The following behaviours were analysed (for extensive descriptions see Groothuis 1989): 1) Overt Aggression: all (in)complete aggressive pecks; 2) Displays used both in aggressive and sexual interactions: Oblique: an erect posture accompanied by a loud call which is often used over long distances; Forward: in which the head is held in front of the body and the bill is held horizontally or upwards. This display is often used when birds are approaching each other. Choking: a posture accompanied by a repetitive call and in which the bill is pointing downwards. It is used when a bird claims a particular spot; Head-flagging: an erect posture (Upright) without call in which the birds faces away from another bird; 4) Sexual behaviour: the sum of: Luring (used to attract the partner), Sexual-begging, and Copulation. Sex of the birds Sex of the birds was determined by measuring the length of head and bill. This length is greater for males than females (breakpoint at 8.1 cm) and this method has a reliability of 95% (Coulson et al. 1983, Koopman 1990). Radioimmunoassay Testosterone concentrations were measured by radioimmunoassay according to the methods described in Pratt et al Assays were carried out in plasma without extraction. Interassay coefficient of variation was 6 7% and the intraassay coefficient of variation was 4 5%. The lower detection level was 0.1 ng/ml. Statistical treatment The data of levels of testosterone and of frequencies of behaviour showed skewed distributions. In order to apply parametrical statistical tests, including ANOVA and t-tests, the data were logarithmically transformed in case of levels of testosterone, and Poisson transformed in case of behavioural frequencies (see Zar 1984). After transformation the data showed normal distributions. Two-sided p values were used with alpha set to Results of repeated measurement ANOVAs were corrected for violation of the sphericity

6 Long lasting effects of testosterone in gulls 49 Fig. 7: Levels of testosterone (mean ± sem) resulting from 25 g crystalline testosterone propionate pellets. At day 0 the T group was implanted and the C group sham operated. At day 23 the pellets were removed. Values next to the data points refer to n values. assumption by means of the Huynh-Feldt epsilon correction for small sample sizes (HF). To correct for the increased chance of type I statistical errors due to multiple testing, p values were adjusted for comparison with alpha on the base of the number of tests, Ntest, as follows: the most significant value was multiplied with Ntest, the second most with Ntest-1, etc. (our modification of the Bonferroni method of Rice 1989). Results Levels of testosterone During the T1 period plasma levels of testosterone were much higher in the T group than in the C group (Fig. 7) (two sample t-tests: C group day 0 versus T group day 2, t = 9.24, df = 7, p < 0.001; C group day 0 versus T group day 4, t = 5.99, df = 6, p < 0.001; C group day 23 versus T group day 23, t = 10.4, df = 9, p < ). The levels in the T group were within the range of adult males during the breeding season (Groothuis and Meeuwissen 1992). After termination of testosterone treatment levels of testosterone decreased significantly within 2 days (two sample t-test, day 23 versus day 25, independent samples, t = 6.18, df = 4, p = 0.003; paired t-test, day 23 versus day 28, t = 4.64, df = 4, p = 0.01). During the T-out period levels of testosterone in the T group were slightly lower than in the C group but this difference was not significant (C group day 23 versus T group day 28, t = 1.22, df = 8, p = 0.26).

7 50 Chapter IV Fig. 8: Frequencies (mean ± sem) per hour observation of different types of social behaviour in testosterone treated birds (T group), and sham operated birds (C group). Activating effects of testosterone To assess whether testosterone treatment affected social behaviour, the average of the total frequency for each behaviour pattern was calculated over the T1 period for the C group and T group separately. Birds of the T group displayed higher frequencies of all behaviour patterns than control birds (Fig. 8). An ANOVA was carried out over these data with the frequencies of the different social behaviours as repeated measurement (6 levels: Overt Aggression, Sexual Behaviour, Oblique, Forward, Choking, and Headflagging), and treatment (2 levels: T group and C group) and sex (2 levels: male and female) as independent factors. The effect of treatment was highly significant (f = 64.92, df = 1/8, p < 0.001). Sex of the birds did not have any significant effect (sex, f = 0.61, df = 1/8, p = 0.46; all interaction effects with sex, p > 0.4). Testosterone treatment did not affect all social behaviours in a similar way (treatment type of behaviour interaction, f = 21.76, df = 5/40, p < 0.001, HF = 1.0). This is probably due to the lower effect of testosterone on Overt Aggression (see Fig. 8). The effect of testosterone was significant for all analysed behaviours (Two-sample t-tests, corrected for Bonferroni, Ntest = 6, Oblique, Choking, Forward, Head-flagging, and Sexual behaviour, p < 0.01; Overt Aggression, p < 0.05). Long lasting effect of testosterone After termination of testosterone treatment the frequency of all testosterone induced behaviours strongly decreased. Because of the resulting low frequencies and because all behaviours showed the same trend, the sum of the frequencies of the different types of social behaviour was used for further

8 Long lasting effects of testosterone in gulls 51 Fig. 9: Changes in the frequency (mean ± sem per hour observation) of testosterone dependent social behaviour (Overt Aggression + Oblique + Choking + Forward + Head-flagging) in testosterone treated birds (T group) and sham operated birds (C group). At day 23 the testosterone implants were removed. analyses. This score decreased quickly after termination of treatment and remained low during the T-out period (Fig. 9). The decrease in the T group was highly significant (paired t-test, T group day 22 versus day 25, t = 11.73, df = 5, p < 0.001). Interestingly testosterone treated birds stayed somewhat more active with testosterone dependent behaviour than control birds (ANOVA with factor treatment (2 levels) and repeated measurement factor day after termination of treatment (5 levels: days 25, 28, 32, 36, and 39), factor treatment: f = 7.45, df = 1/10, p = 0.021; no other significant effects). Increased sensitivity to testosterone After implantation of both the C and the T group in the T2 period in spring, the total frequency of social behaviour increased much more steeply in the T group than in the C group (Fig. 10). To test this increase statistically an ANOVA was carried out on the behavioural data with as repeated measurement factor the day after implantation in the T2 period (5 levels: day 1, 2, 3, 4, and 5), and as independent factor treatment during the T1 period (2 levels: C and T). This ANOVA showed a significant effect of treatment (f = 8.17, df = 1/10, p = 0.017) and a significant interaction effect between treatment and day after implantation (f = 3.08, df = 4/4, p = 0.043, HF = 0.75). At day 8 after implantation, normally the period in which the effect of testosterone is maximal, the frequency of social behaviour has reached similar levels in the C and the T group (two sample t-test, t = 0.18, df = 10, p = 0.86). A second way of testing increased sensitivity is to compare the increase in the social behaviour of birds of the T group in the T2 period with the increase

9 52 Chapter IV Fig. 10: Changes in the frequency (mean ± sem per hour observation) of Social Behaviour in temporarily testosterone treated birds treated with the hormone for a second time (T2 period T group) and in birds of the same age treated with testosterone for the first time (T2 period C group). Fig. 11: Changes in the frequency (mean ± sem per hour observation) of Social Behaviour in testosterone treated birds during a first treatment with testosterone (T1 period) and during a second treatment with testosterone (T2 period). of these birds in the T1 period. The frequency of social behaviour increased significantly more steeply in the in the T2 period than in the T1 period (Fig. 11). To test this statistically an ANOVA was carried out with as repeated measurement factors: a) the day after implantation (5 levels: day 1, 2, 3, 4, and 5), and b) the treatment period (2 levels: T1 and T2). The effect treatment period was significant (f = 10.34, df = 1/5, p = 0.024). Also the interaction effect between day after implantation and treatment period was significant (f = 5.81, df = 4/20, p = 0.003, HF = 1.0). Again the birds of the T group reached similar frequencies of social behaviour in both periods at day 8 after implantation (matched pairs t-test: t = 0.14, df = 5, p = 0.90).

10 Long lasting effects of testosterone in gulls 53 Conclusion Exposure of juveniles to testosterone increases the sensitivity of social behaviour to the hormone later in life. Experiment II Introduction In experiment I, social behaviour quickly approached control levels after termination of testosterone treatment. This seems contradictory to our earlier finding for chicks of the same species, namely that after termination of testosterone treatment aggressive behaviour is still performed in a much higher frequency than in control birds. However, these chicks had been challenged experimentally by confronting them in a standardised way with a model of an adult bird simulating an adult intruder on the territory, whereas in the experiments with the juveniles only spontaneous behaviour was recorded. This is because the juveniles do not respond anymore with aggressive behaviour to such standard stimulus tests. In this experiment we have used other ways to challenge the juveniles for assessing the long-lasting effect of testosterone treatment. The main aim was to determine whether birds of the T group are more responsive to these social challenges than birds of the C group after termination of treatment. Method Rearing conditions and experimental design Black-headed gulls were collected as in experiment I and housed in outdoor aviaries of m. Food and water was provided as in experiment I. One month before the experiment the birds were re-housed in outdoor aviaries of m. In spring, at 9 months of age, three groups were formed of three birds each which were implanted in a similar way as in experiment I with a 10 mm silicon tube (silicon tubes Medica BV, 's Hertogenbosch, The Netherlands: internal diameter 1 mm, external diameter 3 mm, length 12 mm sealed on both sides with 1 mm of silicon glue) filled with 8 mg of crystalline testosterone (Diosynth, Oss, The Netherlands). Several other experiments with these implants in similar aged birds resulted in plasma levels of testosterone of 2.5 ng/ml (Chapter VIII). We have chosen for this way of testosterone application instead of giving crystalline implants in order to reduce individual variation in levels of testosterone resulting from treatment. Two other groups were formed

11 54 Chapter IV of five birds which were sham operated and served as controls. After 11 days, at the end of the T period, the testosterone implants were removed (T-out period). Earlier experiments of this kind revealed that levels of testosterone decreased within a few days to control levels (Chapter III). To obtain comparable conditions between the C and T group during the T-out period, the birds were randomly divided over two groups of nine birds 2 days after termination of treatment. Behavioural challenges In addition to observations of spontaneous social interactions as conducted in experiment I, several manipulations were carried out to analyse the interaction between long-lasting effects of testosterone and social challenges: (1) Refraining the birds from swimming and bathing in the water basin for 1 day. The following day the behaviour was observed when the birds were given the opportunity to bath in a small water basin giving space for two birds only; (2) Placing a small tray in the cage with attractive food (a mash of eggs and fish); (3) Observing the birds during the time that a flock of wild black-headed gulls visited the area and performed high levels of social behaviour close to the aviaries (at 7 to 8 o'clock in the morning). Results The effect of testosterone during treatment and after termination of treatment on spontaneous behaviour showed comparable results as in experiment I: a clear increase (sum of Oblique, Choking, Forward, Head-flagging, and Overt Aggression) during treatment and a strong decrease to control levels after termination of treatment (Fig. 12). This interpretation was supported by the results of an ANOVA with period as repeated measurement factor (2 levels: T and T-out), and treatment (2 levels: T and C) and sex (2 levels: males and females) as the other factors. This revealed a significant effect of treatment (f = 35.31, df = 1/14, p < 0.001) and a significant interaction effect between period and treatment (f = 52.72, df = 1/14, p < 0.001). The factor sex did not show any statistical significant effects (f = 0.41, df = 1/14, p = 0.53; all interaction effects with sex: p > 0.40). After termination of treatment, testosterone treated birds did not differ from control birds in their frequency of social behaviour (two-sample t-test, t = 0.22, df = 16, p = 0.83). Interestingly, all three types of social challenge increased social behaviour in birds that had previously been treated with testosterone but not in control birds (Fig. 12). To test this statistically, an ANOVA was carried out on the behavioural data with type of challenge as repeated measurement factor (3

12 Long lasting effects of testosterone in gulls 55 Fig. 12: Frequencies (mean ± sem per hour observation) of Social Behaviours of testosterone treated birds (T group) and sham operated birds (C group) during experimental treatment: T1, after termination of treatment: T-out. After termination of treatment different manipulations were carried out in which the birds received extra stimulation: T-out bath: new bathing water, T-out food: attractive food, and T-out wild gulls: exposure to displaying wild gulls. levels: bath, food, and wild gulls), and treatment (2 levels: T and C) and sex (2 levels: males and females) as the other factors. The effect of treatment was highly significant (f = 18.92, df = 1/14, p < 0.001). The different types of challenge were equally effective (type of stimulation, f = 10.11, df = 3/42, p < 0.001, HF = 1.0). Sex did not show any significant effects (sex, f = 0.16, df = 1/14, p = 0.69; all interaction effects with sex, p > 0.25). Discussion The increased sensitivity to testosterone We hypothesised that exposure to testosterone causes an increase in sensitivity of social behaviour to testosterone. This study presents clear evidence for this: testosterone dependent social behaviour increased more quickly during subsequent testosterone treatment than during a first testosterone treatment. That exposure to gonadal hormones can influence later sensitivity to these hormones has been shown in castration experiments. These experiments show that the sensitivity of the male to the activating effects on behaviour of subsequent treatment with testosterone decreased when the time interval between castration and subsequent treatment becomes larger (Davidson 1972, Hutchison 1978) Several mechanisms may underlie the changes in sensitivity to testosterone, including testosterone or experience dependent changes in neurotransmitters, neuropeptides, receptor and enzyme systems and neuronal

13 56 Chapter IV Behavioural challenges in the field. structures. The amount of vasopressin in some brain areas of the rat is known to change in relation to levels of testosterone. Furthermore, after castration the depletion of vasopressin in testosterone sensitive brain areas in the rat takes 15 weeks. This time span coincides with the extinction of mounting and intromission after castration (de Vries et al. 1984). The possible role of experience, gained during the first treatment is the subject of further studies (Chapter V, VI). Another possible mechanisms is a shift in the conversion of testosterone to the different metabolites of testosterone. The types of social behaviour we measured are sensitive to estradiol and 5α-dihydrotestosterone (unpublished data). Testosterone has been shown to increase the aromatase activity in the brain (Hutchison and Steimer 1986, Balthazart et al. 1989). Consequently, our first testosterone treatment might have increased aromatase activity and thereby the proportion of testosterone that is converted to estradiol when the birds are subsequently exposed to testosterone. Age and long-lasting effects of testosterone In a previous experiment we found that chicks, temporarily treated with testosterone, continued to perform aggressive behaviour at a higher level than untreated birds, when challenged with an aggressive stimulus (Chapter III). This long-lasting effect of testosterone disappeared around 45 days of age. Around this age young gulls in the field fledge and become much less involved in territorial conflicts. This experiment shows that these long-lasting effects can still be induced long after fledging and are not restricted to a specific sensitive period early in life. Long lasting effects of testosterone on behaviour after puberty have been found in both mammal and bird species (for a review see Arnold and Breedlove 1985). The long-lasting effect of testosterone in the juveniles was much more pronounced when the birds were challenged than when spontaneous

14 Long lasting effects of testosterone in gulls 57 behaviour was observed. In chicks we found a short lasting elevation in plasma levels of testosterone directly after such a challenge (Chapter II). Although similar levels were found in chicks previously treated with testosterone and in untreated chicks, only the former showed substantial levels of aggression. It is likely that in juveniles too a social challenge leads to a short lasting elevation in testosterone levels. This may explain that the difference in behaviour between the testosterone treated and control group only showed up during a social challenge. Exposure to testosterone makes the birds behaviourally more sensitive to these short lasting elevated levels of the hormone, which is in line with the results of experiment I in which we found such an increased sensitivity. Consequences for the adult stage It has become increasingly clear that an aggressive response to a social challenge outside the season when basal levels of testosterone are not elevated can be explained by the occurrence of a short lasting elevation in testosterone levels induced by that challenge (Wingfield et al. 1990). Our results indicate that this may also be true for black-headed gulls. This is in line with the expectation that such a challenge mechanism is important for species in which the males have seasonal territoriality and engage in parental care, the latter being incompatible with high levels of testosterone (Wingfield et al. 1990) since the black-headed gull is a typical member of this group. It is a colonial breeder in which both sexes have to defend their territory heavily during the phase of parental care. More interestingly, our results indicate that exposure to elevated basal levels of testosterone increase the sensitivity of aggressive behaviour to short lasting elevations of the hormone released during challenges. This makes it a potentially interesting mechanism for the adult stage in which temporary exposure to high levels of testosterone occurs annually in spring. Increasing the sensitivity to testosterone by elevated levels of testosterone in spring may enhance the response to aggressive challenges by intruders on the territory later in the reproductive stage when levels of testosterone are low. Acknowledgements We thank the following persons: Jaap Kruijt and Jaap Koolhaas for their help throughout the study; the isotope laboratory of the University Hospital of Groningen for doing the hormonal assays; Martine Maan and Peter Korsten for helping with collecting the behavioural data; Sjoerd Veenstra and Roelie Wiegman for their assistance with rearing the birds. Sex-steroids were kindly provided by Diosynth, Oss, the Netherlands. This project was funded by grant NR SLW by SLW.

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