physiological and life-history traits (see Roulin 2004 for a review). For example, dark .... light in a photo light tent cube. ..... investigate long term effects of body mass loss on survival probability, especially in the wild. In fact .... 87 â 88. MACARTHUR R.H., WILSON E.O. (1967), The Theory of Island Biogeography, Princeton,.
MELANIN-BASED COLORATION AS A SIGNAL OF ALTERNATIVE LIFE-HISTORY STRATEGIES IN FERAL PIGEON
COLUMBA LIVIA
March – July 2010
Feral pigeons in Paris - Steve Greaves 2008
Charlotte Récapet, École Normale Supérieure, Paris. Supervisors: Julien Gasparini and Lisa Jacquin, Evolutionnary Ecology, UMR 7625 CNRS-UPMC, Paris 6 University.
2
METHODS
5
1) 2) 3) 4)
5 6 7 8
EXPERIMENTAL SET-UP MEASUREMENT OF EUMELANIC COLORATION EVALUATION OF INVESTMENT IN IMMUNITY, MAINTENANCE AND REPRODUCTION STATISTICAL ANALYSES
RESULTS
10
1) IMMUNE RESPONSE 2) MAINTENANCE 3) REPRODUCTION 4) TRADE-OFFS MAINTENANCE AND IMMUNITY IMMUNITY AND REPRODUCTION MAINTENANCE AND REPRODUCTION
10 11 13 14 14 14 14
DISCUSSION
16
TRADE-OFF BETWEEN MAINTENANCE AND IMMUNE RESPONSE TRADE-OFF BETWEEN IMMUNE RESPONSE AND REPRODUCTION TRADE-OFF BETWEEN MAINTENANCE AND REPRODUCTION
16 16 17
CONCLUSION
19
ACKNOWLEDGMENTS
19
REFERENCES
20
3
Introduction Fluctuations of environmental conditions and selective pressures over time or space have been hypothesised to drive genetic diversification and genetic diversity maintenance in natural populations (Gillespie & Turelli 1989, Byers 2005). However, empirical evidences of this disruptive selection are still rare (Hedrick 2006). Mechanisms explaining the maintenance of polymorphism in the wild remain therefore to be tested. A species is said to be polymorphic when individuals from the same age- and sex-class can exhibit variations in a trait (at least two phenotypes must have a frequency higher than 0.5 %) that are genetically determined, independently of environment and body condition (Roulin 2004). Variations in melanin-based coloration in birds often follow Mendelian laws (see Roulin 2004 for a review) and have been shown to be highly heritable in some species (feral rock pigeon Columba livia, Johnston & Janiga 1995; barn owl Tyto alba, Roulin et al. 1998; tawny owl Strix aluco, Gasparini et al. 2009a; guillemot Uria aalge, Jefferies & Parslow 1976). As they can be easily measured, variations in melanin-based coloration constitute a good trait to study selective pressures potentially responsible for genetic polymorphism maintenance. Variations in melanin-based coloration has been shown to be related to variations in physiological and life-history traits (see Roulin 2004 for a review). For example, dark reddish females tawny owls Strix aluco maintain a higher level of antibodies for a longer period of time compared to pale reddish females when injected with a vaccine (Gasparini et al. 2009a). Moreover, tawny owls chicks from differently coloured genetic mothers have different abilities to convert food into body mass. Chicks from paler females were less affected by food restriction combined with an immune challenge (Piault et al. 2009). Differently coloured individuals may thus differ in the resolution of physiological and life-history trade-offs, which open the interesting possibility that melanin-based coloration could be a signal of genetic strategies facing variable environments (Roulin 2004). Feral rock pigeon Columba livia constitute an excellent model to study the maintenance of melanin-based coloration polymorphism. In fact, a large variety of melaninbased coloration patterns, called morphs, has been artificially selected during domestication for aesthetic purposes. Some of this coloration diversity has been maintained when pigeons escaped from captivity and colonized the urban environment, but the factors involved in the maintenance of coloration polymorphism still remain poorly known (see Johnston and Janiga 1995 for a review). A few studies have tried to investigate this phenomenon. For example, Obukhova (2001) showed that the proportion of strongly melanic pigeons is higher in urbanized area, suggesting different selective pressures along an urbanization gradient. A
4 previous study in Paris showed differential parasite resistance between differently coloured free-living pigeons, with darker individuals being more resistant to blood parasites than paler ones (Jacquin et al. in press). Parasitism seems thus to be an important environmental factor involved in the maintenance of melanin-based coloration polymorphism in urban areas. As mounting an immune response is energetically costly (Lochmiller & Deerenberg 2000), birds showing a higher resistance to parasites are expected to invest less in other traits such as maintenance and reproduction. Such a trade-off might therefore have different effects depending on food resources in the environment. As parasite abundance and food availability probably fluctuate over time and space, the beneficiary morph will change over time and space, promoting polymorphism maintenance at a global scale. Thus, spatio-temporal variations in parasite diversity and food abundance within the urban environment could promote the maintenance of alternative genetic colour morphs as each morph could be better adapted to different parasite exposure levels and/or to alternative food availability levels (Roulin 2004). In this study, we aimed at comparing life-history traits of differently coloured captive feral pigeons under various parasitic pressures and food availability conditions. We wanted to determine whether melanin-based coloration was linked to different resolutions of trade-offs between maintenance, immunity and reproduction. Individuals were or not submitted to a non-pathogenic immune challenge and/or to food limitation. Investment in maintenance was evaluated by measures of body mass. Investment in immunity was measured as the intensity of specific immune response (specific IgG antibodies levels). Allocation to reproduction was evaluated both as the reproductive activity and the total reproductive output over the whole experiment. Trade-offs between these life-history traits are expected to vary between colorations depending on environmental conditions, as differently coloured individuals may be differently adapted to alternative environments. Previous studies showed that darker pigeons were more resistant to parasites (Jacquin et al. in press) and that darker barn owls Tyto alba lost more weight when immune challenged (Gasparini et al. 2009a). We thus expected darker pigeons to invest more in immunity at the expense of maintenance and/or reproduction than paler ones when exposed to an immune challenge, and to be more sensitive to food restriction (Piault et al. 2009).
5
Methods 1) Experimental set-up The experiment was conducted from March to July 2010. 120 adult rock pigeons Columba livia were captured in 3 locations of the Parisian suburbs and kept in 10 outdoor aviaries. Each aviary contained six males and six females. Birds were submitted to two treatments: a “food treatment” and an “immune treatment”. The food treatment was initiated two weeks before the immune treatment, on day -14 (see Table 1). Food was manipulated both in quantity and quality. 60 pigeons (five aviaries) were food limited (“limited food group”). They were fed with 30 grams wheat (low protein and lipid diet) per day and animal, which correspond to a basal food quantity to maintain nonbreeding pigeons (Hawkins et al. 2001). When chicks hatched, no food was added during their first week of life, then 15 g per chick and per day during their second week and 30 g per chick and per day afterward. 60 other pigeons (5 aviaries) were fed ad libitum (“non-limited food group”) with mixed corn, wheat and peas (high protein and lipid diet) (see Table 2). All pigeons were provided with mineral grit and water supplemented with vitamins. The immune treatment started on day 0. On day 0 and 14 (see Table 1), 60 birds (three pairs chosen randomly in each aviary, called the “immunised group”) were injected subcutaneously with 100 µL of a solution containing 0.5 mg.mL-1 KLH (Keyhole Limpet Hemocyanin). KLH is an artificial protein that birds do not encounter in their natural environment, therefore used to stimulate immune responses to a novel antigen in birds (Hasselquist et al. 2001, Duffy et al. 2002). A first injection at day 0 is expected to cause a primary immune response, whereas a second injection at day 14 should cause a secondary stronger and longer immune response. 60 other birds (“non-immunised group”) were injected with a neutral physiological saline (PBS) (see Table 2). Day
-14
0
14
Start of food
First injection
Second
Opening of the
injection
nests (start of
treatment
28
35
reproduction)
Table 1: Experimental schedule.
42
6 Body mass and coloration score were balanced between food treatments (Body mass: χ2 = 0.016, df = 1, P = 0.90; Coloration score: χ2 = 0.32, df = 1, P = 0.57), immune treatments (Body mass: χ2 = 0.13, df = 1, P = 0.72; Coloration score: χ2 = 0.31, df = 1, P = 0.58), and their interaction (Body mass: χ2 = 0.77, df = 1, P = 0.38; Coloration score: χ2 = 1.33, df = 1, P = 0.25). Immune treatment Immunised
Non-immunised
(injection of KLH)
(injection of PBS)
30
30
30
30
Limited Food
(-)
treatment
Non-limited (+)
Table 2: Number of individuals involved in each experimental group.
2) Measurement of eumelanic coloration Colour variation (black, red or brown) in vertebrates is due to the deposition of two different types of melanin pigments: yellow to red phaeomelanins and black eumelanins (Haase et al. 1992). In this study we focused on eumelanic black coloration because it is the most widespread coloration in feral pigeon populations. Indeed, feral pigeons display a continuous variation in this eumelanin-based coloration from white to black (Johnston & Janiga 1995) which can be divided by human eye in 5 main groups (Johnston & Janiga 1995; Johnson & Johnston 1989): (0) White or almost white individuals; (1) Blue bar (grey mantle with two dark wing bars); (2) Checker (a checked mantle with moderate dark spots); (3) Tpattern (a dark mantle with small grey marks); and (4) Spread (a completely melanic plumage) (Figure 1). These patterns are mainly genetically determined (Jonhston & Janiga 1995) and differ by the surface of dark area on the wings that corresponds to different melanin deposition in feathers (Haase et al. 1992).
7
Figure 1: Main eumelanin-based coloration morphs in feral pigeons However, intermediary morphs are frequent and melanin-based coloration may be considered as continuous. In this study we thus calculated a continuous score as the percentage of dark surface on the wing compared to grey surface. All birds were photographed with a digital camera (Cyber-shot DSC-HX1, Sony) under a standardised white light in a photo light tent cube. The percentage of dark surface was calculated as the number of black pixels/number of white pixels x 100 after binary transformation of the picture on the wings of birds using ImageJ software (U.S. National Institutes of Health, Bethesda, USA). Dark surface measurements were highly repeatable between photographs of the same individual (n = 30, 4 photographs per individual, F29,90 = 40.28, P < 0.001, r = 0.91).
3) Evaluation of investment in immunity, maintenance and reproduction The aim of this study was to compare the relative investment of each morph in immunity, maintenance and reproduction. To assess the quality of the immune response, we took blood samples from the wing vein of each bird every two weeks from day 0 in order to assay specific anti-KLH antibodies production. Blood samples were left at 5 °C for 24 hours before centrifugation (10000 r.p.m. for 10 min). Plasma was then extracted and stored at -20 °C. The ELISA technique was used to quantify anti-KLH antibodies in plasma. To assess the investment in maintenance, we measured body mass to the nearest 5g with a spring balance (Medio-Line 40600, Pesola, Switzerland) every 2 weeks from day -14. To assess the investment in reproduction, we recorded the reproductive state as the reproductive activity on a particular week (0: no eggs; 1: incubating eggs; 2: feeding chicks) as well as the total reproductive output, i.e. the total number of eggs laid at the end of the experiment (day 126).
8 Anti-KLH antibody assay High-binding plates (96 wells, flat bottom, Microlon® 600 ; cat. 655101, Greiner BioOne, Germany) were coated overnight at 5 °C with 100 µL KLH (40 µg.mL-1 in 50 mM carbonate/bicarbonate buffer, pH 9.6), then washed five times with phosphate buffered saline (0.1 M PBS, pH 7.4). Wells were blocked with 200 µL 3% milk powder (Régilait Bio) in PBS for two hours at room temperature with agitation. After five washings, two 100 µL dilutions in PBS with 0.5% milk powder were distributed for each sample (1:500 and 1:5000). A standard pool of pigeon serums with high concentrations of anti-KLH antibodies, diluted serially from 1:500 to 1:32000, was added to each plate, with a duplicate for each dilution. Plates were then incubated either one night at 5 °C or two hours at room temperature with agitation. After five washings, plates were incubated two hours at room temperature with 100 µL rabbit-anti-pigeon IgG conjugated to horseradish peroxidase (10 mg/ml in PBS, pH 7.2; cat. RAP/IgG(H+L)/PO, Nordic Immunology, Netherlands), diluted 1:5000 in PBS with 0.5% milk powder. Plates were washed five times, then 100 µL OPD (o-Phenylenediamine dihydrochloride, 0.4 mg.mL-1 with 0.4 mg.mL-1 urea hydrogen peroxide and 0.05 M phosphate-citrate in distillated water, pH 5.0 ; cat. P9187, Sigma-Aldrich, USA) was added to the wells. OPD is the substrate of peroxidase and the reaction produces a coloured compound with a maximal absorption at 450 nm. After 10 minutes in the dark at room temperature, the reaction was stopped by adding 50 µL HCl (1 M). Plates were then read at 490 nm in a microplate reader (Model 680, Bio-Rad Laboratories, UK) and data were computed using Microplate Manager (Bio-Rad Laboratories). A logistic curve was fitted to the standards to calculate the unknown concentrations of the samples. Only values within the standard concentration limits were kept. When the 1:5000 dilution gave a value above 40, a higher dilution was realised (1:50000). Log-transformed anti-KLH titres were highly repeatable within-plates (n = 59, 2 wells per sample, F46,46 = 13.94, P < 0.001, r = 0.93) as well as between-plates (n = 40, 2 wells per sample, F29,31 = 38.32, P < 0.001, r = 0.81), showing that this method is reliable to measure anti-KLH antibody levels.
4) Statistical analyses Immune response Antibody levels were log-transformed to obtain a normal distribution. We studied the dynamics of the immune response separately for both immune treatment groups, by including time as a linear effect in mixed-effects models and an individual random effect (on slope and intercept) to account for repeated measures on a same individual over time. Generalized linear models (GLM) were fitted to study both quantitative (coloration score) and qualitative effects
9 (food treatment) and their interactions on the intensity of the primary and secondary immune responses (day 14 and day 28) in the immunised group. Maintenance Body mass dynamics over the experiment were analysed using restricted maximumlikelihood fit (REML) of mixed-effects models with food, immune treatment and time as factors, coloration score as a covariate and all interactions. To account for repeated measures on a same individual over time, we added an individual random effect on the intercept. Reproduction The total number of eggs laid was analysed by fitting GLM with both treatments as factors and coloration score as a covariate. Trade-offs First, we examined the trade-offs between maintenance and immunity by modelling the relationship between antibody levels and body mass loss in a GLM. Then we investigated the trade-off between immunity and reproduction by testing the impact of reproduction on antibody production. As reproduction had not started during the first month (see Table 1), we studied the effect of reproductive state on the decrease in antibody levels from day 14 (primary response) to day 84 (stop of the decrease in antibody levels, see Figure 2). The food treatment was also included as a factor. Finally we tested the trade-off between maintenance and reproduction by modelling the link between body mass loss and antibody production between day 0 and day 14 or day 28 in a GLM. All final models were assessed by a step-wise model selection using the AIC criterion and an analysis of variance was used to estimate the significance of each effect of the selected model. Significance levels were set to 0.05 and tests were two-tailed. Each model was checked for homoscedasticity and normality of the residuals. Cook’s distances were inferior to one. All analysis were performed with R (R Foundation for Statistical Computing, Vienna).
10
Results 1) Immune response In the immunised group, anti-KLH antibody level increased significantly after the first injection (Figure 2; paired t-test between day 0 and day 14: t = 17.50, df = 59, P < 0.001). Antibody level then decreased continuously throughout the experiment (mixed model, linear effect of time from day 14 to day 98: F1,353 = 196.99, P < 0.001). There was no increase in antibody levels between the primary response on day 14, and the secondary response on day 28, when birds were challenged for a second time with the same antigen (paired t-test between day 14 and day 28: t = 0.21, df = 59, P = 0.84). Antibody level of non-immunised individuals showed no variation over time (mixed model; linear effect of time from day 0 to day 98: F1,411 = 1.06, P = 0.30).
Mean antibody level
○ non-immunised ● immunised
injection 1 injection 2
Figure 2: Mean antibody level as a function of time, for immunised birds (solid line) and nonimmunised birds (dotted line). Immunised individuals from the non-limited food group had a higher secondary response than individuals from the limited food group (Figure 3; GLM for the immunised group at day 28; effect of food treatment: F1,56 = 8.95, P = 0.004). There was a quadratic relationship between coloration score and the strength of the secondary response (F2,56 = 6.19, P = 0.004), but no interaction with the food treatment (F2,54 = 0.29, P = 0.75). Immune response is lower for intermediate melanic individuals in both food treatments (Figure 3). A similar relation to coloration score was found for the primary response (F2,57 = 3.72, P = 0.03) but with no effect of the food treatment (F1,58 = 0.96, P = 0.33).
11
Log-transformed antibody level
○ non-limited ● limited
Figure 3: Log-transformed antibodies levels of immunised individuals at day 28 (secondary response) as a function of the coloration score, for the non-limited food group (white dots, dotted line) and the limited food group (black dots, solid line).
2) Maintenance Food treatment had a significant effect on body mass dynamics (Figure 4; food × time: F10,1132 = 2.79, P = 0.002). There was a significant difference in mass between the two treatment groups on day 42 (Table 3), one week after the starting of egg-laying and from day 70 (beginning of chick rearing) to the end of the experiment (Table 3). Both treatment groups lost mass during chicks rearing (paired t-test between day 56 and day 126, in the unlimited food group : t = -2.95, df = 57, P = 0.005 ; in the limited food group : t = -6.43, df = 57, P < 0.001), but this effect was more pronounced in the food limited group (body mass loss between day 56 and day 126 in the limited group: -26.8 ± 4.2; in the non-limited food group: 15.9 ± 4.4 g). There was no effect of the immune treatment or its interaction with the food treatment on body mass dynamics (KLH × time: F10,1132 = 0.31, P = 0.98; food × KLH × time: F10,1132 = 0.54, P = 0.86). Day
-14
0
14
28
42
56
70
84
98
126
t
-0.67
-0.56
-0.90
-1.06
-2.86
-0.87
-5.79
-2.66
-1.81
-2.76
df
114
115
116
117
118
116
117
117
116
114
P
0.51
0.58
0.37
0.29
0.005
0.39
< 0.001
0.009
0.07
0.007
Table 3: Comparison of mean body mass between the two food-treatment groups by Welch two-sample t-test
12 ○ non-limited ● limited
Start of food treatment
* *
Injection 1
Injection 2
* First egg
First chick
*
Figure 4: Variation in mean body mass for the two food-treatment groups. Stars indicate significant differences between the two treatment groups (Table 3). Analyses show a significant interaction of food treatment and coloration on body mass dynamics (food × coloration score × time: F10,1132 = 2.19, P = 0.016), whereas coloration alone had no effect (coloration score × time: F10,1132 = 0.93, P = 0.50). Darker individuals lost more weight than paler ones in the food limited group but lost less weight than paler ones in the non-limited food treatment (Figure 5).
○ non-limited ● limited
Figure 5: Mass loss over the whole experiment (from day -14 to day 126) as a function of the coloration score, for the non-limited food group (white dots, dotted line) and the limited food group (black dots, solid line).
13
3) Reproduction Food limited birds laid significantly less eggs (n = 60, mean number of eggs laid per individual ± SE: 3.30 ± 0.35) than the non-limited food group (n = 60, 4.23 ± 0.45) (see Table 4). The number of eggs laid was also positively correlated with coloration score when all treatment groups were considered together (slope ± SE = 0.045 ± 0.017) (see Table 4). However this relationship differed between food and immune treatment groups (Table 4). Under food limitation or immune challenge, the number of eggs laid tended to be higher for darker individuals, whereas under mild environmental conditions (non-immunised or nonlimited food group) differently coloured individuals laid a similar number of eggs (Figure 6). F1,112
p
Food
6.79
0.010
Immune treatment
1.75
0.19
Coloration score
6.69
0.011
Food × Immune treatment
2.44
0.12
Food × Coloration score
6.10
0.015
*
Immune treatment × Coloration score
4.18
0.043
*
Food × Immune treatment × Coloration score
4.14
0.044
*
* *
Table 4: Type III-Ancova on the number of eggs laid over the whole experiment with food and immune treatment as factors and coloration score as covariate. The final model was assessed by a step-wise model selection using the AIC criterion. Adding initial body mass as a covariate and its interactions did not improve the model (AIC = 622 in both cases after model simplification). Sex and its interactions had no significant effect (P > 0.10). Immune treatment
○ non-immunised ● immunised
Food treatment
○ non-limited ● limited
Figure 6: Number of eggs laid as a function of coloration in immune treatment groups (left; immunised: black dots, solid line; non immunised: white dots, dotted line) and food treatment groups (right; limited: black dots, solid line; non limited: white dots, dotted line).
14
4) Trade-offs Maintenance and immunity There was no significant link between the increase in antibody levels and body mass changes for the primary response (F1,57 = 0.08, P = 0.78) nor the secondary response (F1,57 = 0.01, P = 0.91).
Immunity and reproduction The decrease in antibody levels from day 14 to day 84 in the immunised group was linked to the reproductive state (having eggs or chicks) on day 84 (GLM with food treatment as factor, reproductive state as covariate and their interactions; reproductive state: F1,55 = 8.86, P = 0.004). Birds with no eggs kept circulating antibodies for a longer time in their plasma than birds incubating eggs or raising chicks (Figure 7). This correlation was independent of
Decrease in antibody level
melanin-based coloration.
Figure 7: Mean decrease in log-transformed antibody level between day 84 and day 14 (primary response) as a function of reproductive state on day 84, in the immunised group.
Maintenance and reproduction Body mass loss over the whole experiment was not related to the number of eggs laid (GLM with food treatment and sex as factors and number of eggs as a covariate: F1, 109 = 0.24, P = 0.63). Nevertheless changes in body mass were negatively influenced by reproductive state (Table 5; Figure 8). Differently coloured birds were similarly affected by reproduction in
15 their body mass dynamics (reproductive state × coloration score × time: F1,1173 = 0.93, P = 0.33). Females were more negatively affected by reproduction than males (Table 5). F
df
p
Time
5064.84
1, 1184
< 0.001
Food
1.08
1, 115
0.30
Sex
13.72
1, 115
< 0.001
Coloration score
0.35
1, 115
0.55
Reproductive state
12.18
1, 1184
< 0.001
Time × Food
1.01
1, 1184
0.31
Time × Coloration score
5.03
1, 1184
0.025
*
Time × Reproductive state
14.20
1, 1184
< 0.001
***
Food × Sex
0.37
1, 115
0.54
Food × Reproductive state
22.87
1, 1184
< 0.001
***
Sex × Reproductive state
40.65
1, 1184
< 0.001
***
Time × Food × Reproductive state
3.52
1, 1184
0.061
*** *** ***
Table 5: Mixed-effects final model (AIC criterion selection) explaining body mass changes over time (individual as a random intercept effect). There were no differences in body mass dynamics between birds without eggs and birds incubating eggs (Figure 8; t = 1.20, df = 1179, P = 0.23). But birds that were feeding chicks exhibited significantly higher body mass losses than non reproductive birds (Figure 8; t = -2.10, df = 1179, P = 0.036).
○ non-limited ● limited
Figure 8: Mean mass (± SE) loss between day 126 and day -14 (start of food treatment) as a function of reproductive state on day 126.
16
Discussion Trade-off between maintenance and immune response Birds injected with KLH had a higher immune response in the non-limited food group than in the limited food group. This could be due to a negative effect of food limitation on antibody production, as that process is known to be energetically demanding (Lochmiller & Deerenberg 2000). However it is impossible to draw such a conclusion in our experimental design since individuals in the non-limited food group had already been injected with KLH antigen in year 2009, whereas individuals in the limited food treatment were not. Therefore the higher antibody production in the non-limited food group could be due to a stronger reactivation of the immune system in 2010 when challenged with the same antigen. Moreover, body mass changes of the immunised individuals were not related to the intensity of the immune response. Therefore we provide no evidence of a direct trade-off between maintenance and immune response. Moreover, differently coloured individuals exposed or not to an immune challenge lost similar weight. This is surprising as Gasparini et al. (2009a) found a significant effect of immune challenge on body mass loss in dark barn owls but not in pale ones. However, our study takes place in artificial aviaries where predation and climate pressure may be milder than in the wild, making the expression of a trade-off between immunity and maintenance difficult to bring to light. It is also possible that the immune challenge was not strong enough to cause significant differences in weight losses between different treatment groups and differently coloured individuals.
Trade-off between immune response and reproduction Birds with no eggs maintained higher antibody levels in their plasma for a longer time than birds incubating eggs or raising chicks, suggesting a trade-off between reproduction and immunity. This correlation was independent of coloration. Two mechanisms are possible: investment in reproduction impairs antibodies production, or the cost of the immune response decreases investment in reproduction. As there was no clear effect of the immune treatment alone or in interaction with the food treatment on the number of eggs laid, the first proposal seems more likely. This is consistent with studies on pied flycatchers Ficedula hypoleuca that found no effect of maternal immunisation with LPS on the size of replacement clutch after eggs removal (Grindstaff et al. 2006). However, further studies evaluating hatching and fledging success could show an impact of immune challenge on reproductive output. In fact, Ilmonen et al. (2000) found that female pied flycatchers injected with a vaccine had lower
17 feeding rates and fledging success (fledglings / hatched eggs) than females injected with a saline. Another interesting result is that intermediate morphs had a lower immune response than lighter and darker individuals. This could be an advantage for extreme morphs and result in disruptive selection under high exposure to parasites. However a higher antibody production would have a higher cost and may result in a lower fitness when exposition to parasites and/or parasites virulence are low. Moreover, other components of the immune system might be differently related to melanism. In fact, when feral pigeons were injected with phytohaemagglutinin PHA (an antigen eliciting a cellular response), lighter birds had a lower response than darker birds (Jacquin et al. in press). Therefore, different components of the immune system seem to be linked to melanin-based coloration in a complex way (Gasparini et al. 2009b). Moreover, our study failed to show any significant link between antibody production and body mass loss or reproductive output. The impact of these differences in humoral response on fitness thus remains to be tested, for example by testing directly parasite resistance.
Trade-off between maintenance and reproduction After the start of reproduction, individuals from both food treatment groups decreased in mass, but body mass dynamics were different between groups. Individuals from nonlimited food group exhibited a slow and continuous mass loss, whereas individuals from the limited food group showed rapid and discontinuous weight losses at the peaks of chicks feeding. Effect of food limitation was visible only in combination with reproduction. Food limitation also had a negative impact on investment in reproduction, measured as the number of eggs laid over the whole experiment. On the other side, analyses suggest a negative impact of chicks feeding on body mass changes, but no impact of incubating eggs. Our results therefore suggest a trade-off between reproduction and maintenance. Furthermore, differently coloured individuals reacted differently to the food treatment. Darker individuals lost less mass than paler ones when food was unlimited, but lost more mass than paler ones when food was restrained. Darker individuals seem thus to better convert food into body mass in good food conditions, whereas paler ones would do better in harsh food condition. This is consistent with previous studies conducted by Piault et al. (2009) and Roulin et al. (2008), who demonstrated that chicks from darker barn owls and tawny owls seemed to grow better in good food conditions than paler ones, but to lose more weight in bad food conditions. This could be due to a higher metabolic rate of darker eumelanic individuals caused by pleiotropic effects of melanocortin ligands on energy homeostasis control centres (Ducrest et al. 2008).
18 However, investment in reproduction showed an opposite pattern: when food was limited, darker individuals laid more eggs than paler ones. This suggests that pale individuals kept a relative good body condition in harsh conditions maybe by investing less in reproduction, whereas darker individuals produced more eggs, but at the expense of a greater body mass loss. It thus seems that dark and pale individuals adopt different strategies facing variable environments (Figure 9). One possible scenario is that when energy is limited, darker individuals adopt a “r” or “quick” strategy, with higher reproductive rate and lower survival rate, whereas lighter individuals would adopt a “K” or “slow” strategy favouring survival to reproduction (Wilson & Mac Arthur 1967; Promislow & Harvey 1990). Consistently, behavioural studies conducted on the same birds showed that darker females invest more time in reproductive activities but less in grooming necessary to feathers maintenance (Bouche et al., unpublished data). Reproduction
Maintenance
DARK
DARK light
light
Parasitic pressure Food limitation
(Parasitic pressure ?) Food limitation
Figure 9: Hypothetic pattern of investment in reproduction and maintenance in variable environments for dark and light coloured individuals as suggested by this study. It can be argued that the number of eggs laid do not necessarily reflect the reproductive success as some eggs could be sterile or chicks mortality be high. As eggs were cross-exchanged between nests for further experiments on parental effects, I did not evaluate the proportion of eggs hatched and the number of youngs fledged. Murton et al. (1974) showed that males of different morphs varied in their ability to rear chicks rather than the number of eggs laid in a year. It would thus be interesting to further investigate the components of breeding success under various environmental conditions and compare the number of fledglings between differently coloured individuals. Evaluating the possible impact of both strategies on fitness would also require to investigate long term effects of body mass loss on survival probability, especially in the wild. In fact, birds in captivity do not have to invest energy in moving between foraging sites and
19 they are not subject to predation that could decrease survival rate in a non-linear way, since birds in poor condition are more prone to be captured. Ongoing demographic studies in wild populations of the Parisian area will permit to compare survival rates between differently coloured individuals and to evaluate the effects of temporal variations in the environment.
Conclusion We found evidences of trade-offs between reproduction and maintenance, as well as between reproduction and immunity. These correlations are often found in natural systems and has been shown to result from structural and energetic limitations (e.g. Norris & Evans 2000). In agreement with this, the relationships found in our study were similar between experimental conditions and between colorations, suggesting that they are physiologically constrained. However, differently coloured individuals seemed to adjust their life-history traits to experimental conditions in different ways. Darker individuals maintained a higher reproductive output whatever the food limitation and immune challenge, with a negative impact on body mass. On the contrary, lighter individuals seemed to adapt their reproductive output to environmental conditions, and showed lower body mass losses in harsh food conditions (Figure 9). The link between melanin-based coloration and resistance to parasites is more complex and seem to depend on the component of the immune system considered. Because of such adjustment in energy allocation, differently coloured individuals may attain similar fitness facing the same parasite and food conditions by adopting different strategies. Further studies on the field are now called for to test whether such strategies could lead to different population dynamics in variable environments and how this could explain coloration polymorphism maintenance in urban environment.
Acknowledgments I would like to thank L. Jacquin and J. Gasparini for associating me in their research project and helping me to complete the lab work as well as the writing of this report; C. Haussy for introducing me to ELISA procedures; and S. Perret, L. Blottière, P. Bouche and S. de la Bardonnie as well as the other students at the Foljuif research station who helped me during manipulations on the birds.
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