Negative effects of early developmental stress on yolk testosterone levels in a passerine bird
1 Departamento de Ecología Evolutiva, Museo Nacional de Ciencias
Naturales (CSIC), José Gutiérrez Abascal 2, 28006 Madrid,
Spain
2 Department of Animal Behaviour, Universität Bielefeld, PO Box 100
131, 33501 Bielefeld, Germany
3 Departamento de Fisiología (Fisiología Animal II), Facultad
de Biología, Universidad Complutense de Madrid, 28040 Madrid,
Spain
* Author for correspondence (e-mail: dgil{at}mncn.csic.es)
Accepted 5 April 2004
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Summary |
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Key words: yolk testosterone, developmental stress, androgens, maternal effects, reproductive investment, differential allocation, zebra finch, Taeniopygia guttata
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Introduction |
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Several studies in birds have experimentally manipulated the amount of
resources available to altricial nestlings by brood-size manipulations. This
approach has allowed researchers to detect early condition-dependent patterns
in a number of variables, such as clutch size
(Haywood and Perrins, 1992),
adult size and mortality (De Kogel,
1997b
) or the development of sexual ornaments
(De Kogel and Prijs, 1996
). It
has recently been shown that the amount of androgen deposited in the egg yolk
is an important component of reproductive investment in birds
(Schwabl, 1993
). Egg yolk
androgen has positive effects on the embryo and on the development of
nestlings, such as increased begging, growth and muscular development
(Eising et al., 2001
;
Lipar and Ketterson, 2000
;
Schwabl, 1993
), although a
study has also identified survival costs of high yolk androgen levels
(Sockman and Schwabl, 2000
).
Females have been shown to vary their allocation of yolk androgen according to
the attractiveness of their male, thus suggesting that there are costs
associated with this investment (Gil et al.,
1999
,
2004
; D. Gil, P. Ninni, A.
Lacroix, F. De Lope, C. Tirard, A. Marzal and A. P. Møller, manuscript
submitted for publication). There is some evidence that this may be the case,
such as a positive covariance between yolk-testosterone and arrival date in
the barn swallow Hirundo rustica (D. Gil, P. Ninni, A. Lacroix, F. De
Lope, C. Tirard, A. Marzal and A. P. Møller, manuscript submitted for
publication), or with female age in the starling Sturnus vulgaris
(Pilz et al., 2003
). However,
a recent study has failed to detect increased egg androgen concentrations in
the eggs of food-supplemented black-backed gulls Larus fuscus
(Verboven et al., 2003
).
In this study, we manipulated early development in zebra finches
Taeniopygia guttata by cross-fostering nestlings to small, medium and
large brood sizes. This manipulation typically influences early developmental
stress, resulting in strong effects in nestling and adult morphology and
fitness (De Kogel, 1997b;
Naguib et al., 2004
).
Subsequently, we allowed these birds to sexually mature, and paired females
randomly with non-experimental males from the same population. The hypothesis
was that, if yolk testosterone allocation is costly, females should lay eggs
with lower concentrations of testosterone with increasing levels of
developmental stress experienced as nestlings.
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Materials and methods |
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Breeding of the second generation
When female offspring from the above experiment were 9 months old, we
paired them up with unrelated males that had been raised in non-manipulated
broods. Overall, we used 14 females coming from 10 small manipulated broods,
19 females from 10 medium broods, and 19 females from 8 large broods. Females
were significantly lighter and had significantly shorter wing lengths as
rearing brood size increased, while tarsus length also showed a
non-significant trend in the same direction
(Table 1). Males that had not
been in visual or direct contact with these females were randomly allocated to
females, and did not wear colour rings that could bias their attractiveness to
females (Burley, 1988).
|
Pairs were kept in individual cages (83 cmx30 cmx40 cm) and supplied daily with both dried and germinated senegal, plata and red millet seeds and fresh water (plus additional vitamins three times a week). Each cage was provided with a wooden nest box attached to the side (12.5 cmx12 cmx14 cm) and with coconut fibres on the floor, to be used as nesting material. Cages were distributed over three rooms and arranged on rows of shelves placed on walls so that birds had visual contact with birds on the opposite side of the room. Rooms had a temperature of 23°C and a L:D regime of 16 h:8 h, with lights on at 06:00 h. Nests were checked daily between 10:00 and 12:00 h and eggs were labelled with a felt pen on the day of laying, allowing us to keep track of egg laying order. Eggs were removed on day 2 after laying and frozen at 20°C. We kept females in the experiment until they had not laid an egg for 5 days, after which we transferred them back to a larger aviary.
Hormone assay
Yolks were dissected after allowing the eggs to thaw for 10 min. After this
time, egg white is defrosted while the yolk still remains compact and can be
removed with a needle. Yolks were homogenised by stirring using a yellow
pipette tip. A small sample (around 50 mg) was taken and weighed in a
pre-tared glass tube. We added 0.5 ml of distilled water and several glass
beads to the tube and vortexed it until the yolk was completely homogenised.
Steroids were extracted by adding 3 ml of a 30:70 mixture of petroleum/diethyl
ethers and vortexing the mixture for 1 min. We separated the ether phase by
decanting the contents of the tube after having snap-frozen them in a bath of
alcohol and dry ice. The ether phase was then dried under a stream of nitrogen
and dry extracts reconstituted into 0.5 ml of a 0.01 mol l1
phosphate buffer with bovine serum albumin (1 g l1).
Although we have already shown the reliability of this extraction method
(Gil et al., 2004), we tested
it again for this species by re-extracting ten samples using the more thorough
method, which involves ethanol precipitation and hexane washing (for details,
see Schwabl, 1993
). Although
concentrations using the latter method were larger than those obtained using
the simplified method (16.9±7.37 vs. 10.45±4.12 pg
mg1, means ± S.D.), the regression between
the two extractions was highly significant (F1,10=70.9,
P<0.001, r2=0.87), showing that the simplified
extraction can provide an accurate assay of yolk androgens. We assayed the
concentration of testosterone using a commercially available radioimmunoassay
(DSL-4000, made by Diagnostic Systems Laboratories, Texas, USA), with high
specificity with testosterone (cross-reactivity with other androgens <6%).
Samples were assayed in duplicate, with an intra-assay coefficient of
variation of 3.13%. The between-assay coefficient of variation was 10.02%.
Statistical analysis
We analysed egg data with repeated-measures mixed lineal models using Proc
Mixed (SAS V. 8.1). Experimental brood size was declared as a fixed factor. In
order to control for random effects of females sharing a common environment as
nestlings or being genetically related, data were firstly cross-classified by
entering as random factors the original and experimental broods where the
females had been raised as nestlings. However, none of these factors explained
a significant part of the variance in any of the tests, and were removed from
the models. Individual egg data were cross-classified within females
(Littell et al., 1996), and
the effect in this case was always highly statistically significant (all
measures P<0.001). Therefore, in all models we conserved female as
a random factor. We used the Satterthwaite correction to approximate the
degrees of freedom (Littell et al.,
1996
). The statistical significance of the treatment (brood size
experienced by females) was adjusted following a two-tailed ordered
heterogeneity test, in the expectation that the groups would show progressive
responses to the manipulation (Rice and
Gaines, 1994
).
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Results |
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Brood size experienced by females as nestlings did not affect the time interval between pairing and laying (F2,45=0.11, P=0.89; small broods, 16.27±3.64 days; medium broods, 14.88±3.42 days; large broods, 17.18±3.54 days; means ± S.E.M.), clutch size (F2,45=0.37, P=0.69; small broods, 2.2±0.33 eggs; medium broods, 2.58±0.31 eggs; large broods, 2.44±0.32 eggs) or egg mass (F2,32.6=1.09, P=0.34; small broods, 809.94±37.37 mg; medium broods, 838.71±29.27 mg; large broods, 777.51±29.25 mg).
We obtained data for a sub-sample of females on nestling plasma
testosterone levels at 11 days of age
(Naguib et al., 2004), and
tested whether these levels would positively correlate with mean yolk
testosterone concentrations in adulthood, but found no significant
relationship (Spearman's r=0.104, N=19,
P=0.67).
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Discussion |
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This finding has important implications for the differential allocation of
resources in reproduction (Burley,
1988; Sheldon,
2000
). Life history theory predicts that reproductive investment
in iteroparous organisms should increase with increasing fitness value of a
given reproductive attempt (Stearns,
1992
; Trivers,
1972
). Male ornament size can provide females with an honest
signal of male quality (Møller and
Alatalo, 1999
), and females have been shown to vary their
reproductive investment according to male ornamentation in a wide array of
organisms (Sheldon, 2000
). We
have previously shown that the concentration of yolk androgens in three
different bird species increases with increasing size of manipulated male
ornamentation (Gil et al.,
1999
,
2004
; D. Gil, P. Ninni, A.
Lacroix, F. De Lope, C. Tirard, A. Marzal and A. P. Møller, manuscript
submitted for publication). As yolk androgens have been shown to positively
affect embryo and nestling development
(Eising et al., 2001
;
Lipar and Ketterson, 2000
;
Schwabl, 1993
; but see
Sockman and Schwabl, 2000
),
this investment may represent an example of differential allocation by
reproducing females. However, it needs to be shown that yolk androgen is
costly to females (Sheldon,
2000
). Our results are in line with this hypothesis, by
identifying a constraint in egg testosterone allocation set up in early
development.
Alternatively, we need to consider whether the differences in yolk
testosterone that we found are determined by other factors. For instance,
females fledging from enlarged broods may have attained lower social ranks,
and experienced different levels of aggression, which in turn may have
influenced yolk testosterone levels
(Schwabl, 1993;
Whittingham and Schwabl,
2002
). This seems to be a less likely possibility, because females
were allowed to breed in separate cages, without competition for mates or nest
sites. Moreover, de Kogel
(1997a
) did not find any
significant effect of brood size manipulation on later competitive behaviour.
Alternatively, as female condition may bias sex ratio
(Nager et al., 1999
), it could
be argued that differences in yolk testosterone between treatments could be
due to differences in sex ratio between treatments, and females investing
higher concentrations of testosterone in male than in female-bearing eggs
(Petrie et al., 2001
). Even
though we cannot rule out this possibility, the evidence for sex-specific
differential allocation of testosterone still remains controversial. Recent
research has shown that reported differences in yolk androgen levels between
male and female eggs in the peacock Pavo cristatus
(Petrie et al., 2001
) may have
been an experimental artefact, possibly because of sexual differences in
embryo steroid production and uptake
(Eising et al., 2003
;
Müller et al., 2002
).
A recent study has used food supplementation to test the cost of androgen
deposition in eggs in the black-backed gull
(Verboven et al., 2003). The
results showed that food-supplemented females had higher androgen levels than
controls, but laid eggs containing lower androgen concentrations. This
evidence does not support a cost of yolk androgen to the female, and suggests
that investment in egg testosterone is not a linear function of maternal
condition. The discrepancy between the results of the present study with those
of the gull experiment (Verboven et al.,
2003
), may suggest that limitations of androgen deposition are
more dependent on variation in early development than in the adult
condition.
Although our results suggest a constraint in the production of egg
androgens, they do not identify where the cost may lie. Several possibilities
exist (Gil, 2003), although so
far no research has tackled this problem directly. One possibility is an
inhibitory effect of circulating androgens on immune defence
(Duffy et al., 2000
;
Folstad and Karter, 1992
;
Hasselquist et al., 1999
),
possibly through increases in oxidative stress
(Chainy et al., 1997
;
von Schantz et al., 1999
).
Immunocompetence constraints could act in either the female or the offspring.
In the case of the female, it would need to be shown that higher androgen
biosynthesis results in higher circulating levels. Recent evidence challenges
this seemingly straightforward assumption
(Mazuc et al., 2003
;
Verboven et al., 2003
).
Immunocompetence may, on the other hand, constrain egg androgen allocation in
the offspring, even at the embryo stage. Although there is some positive
evidence along these lines from studies using pharmacological levels of
androgens (Al Afaleq and Homeida,
1998
; Henry and Burke,
1999
), it is unclear whether this effect would still be present
within the natural range of androgen yolk levels.
Another possibility is that the cost of androgen allocation may be brought
about by nestling behaviour. Levels of yolk androgen have been shown to
determine begging intensity in offspring
(Eising and Groothuis, 2003;
Schwabl, 1993
). This suggests
that that females may benefit by using yolk androgens to manipulate offspring
demands as a function of their own condition.
In a previous paper (Naguib et al.,
2004) we showed that testosterone levels in nestlings at day 11
increased with manipulated brood size, both for males and females. We
interpreted this pattern as suggesting a functional link between testosterone
and offspring competition. The results of the present paper, showing a pattern
in the opposite direction in the case of yolk testosterone, and no correlation
between nestling and adult yolk testosterone, suggest that the hormonal
milieus of the nestling and adult stage are functionally independent of one
another.
We failed to find any effects of early development on other indicators of
reproductive investment, such as clutch size or egg mass, as reported in
previous studies (Haywood and Perrins,
1992; Schluter and Gustafsson,
1993
). One possible explanation for the lack of effect of early
condition on clutch size in our study may be that we used the first clutch
laid by the females, whereas Haywood and Perrins used later clutches, and were
thus more likely to obtain larger variation among females. Additionally, the
large heritability of clutch size
(Christians, 2002
) may provide
more limited scope for variation than plastic allocation of egg components,
and thus may require larger sample sizes to detect any influence of maternal
effects.
Yolk-testosterone concentration decreased with laying order, confirming
previous results reported in this species
(Gil et al., 1999). This
pattern strongly contrasts with those of a considerable number of studies that
have reported increasing androgen concentrations with increasing laying order
in other passerine species (Schwabl,
1999
). Decreasing levels of testosterone with laying order suggest
that zebra finches hatching from the last eggs in a clutch will suffer from a
double handicap in sibling competition: to hatch later than their siblings
(Zann, 1996
), and to develop
with a lower testosterone concentration. Future research should address
whether these within-brood differences in testosterone levels affect the
outcome of sibling competition within a brood, and are related to patterns of
brood reduction.
To summarise, our experiment has detected negative effects of early developmental stress in yolk-testosterone concentration in adulthood. This suggests that egg androgens are directly or indirectly costly for the female, and that differences in yolk-androgen between broods with respect to male attractiveness represent patterns of adaptive differential allocation.
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Acknowledgments |
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