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First published online February 27, 2009
Journal of Experimental Biology 212, 815-822 (2009)
Published by The Company of Biologists 2009
doi: 10.1242/jeb.022111
Yolk androgens and the development of avian immunity: an experiment in jackdaws (Corvus monedula)
Department of Animal Ecology, Lund University, SE-223 62, Sweden
* Author for correspondence (e-mail: maria.sandell{at}zooekol.lu.se)
Accepted 10 December 2008
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Summary |
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 TOP Summary INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Key words: development, immunocompetence, life-history, maternal effects, androgens
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INTRODUCTION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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).
Maternal effects are the direct influences on offspring fitness that is a
result of the phenotype or environment of its mother
(Falconer and Mackay,
1996).
In egg-laying vertebrates, maternal effects may act through differential
transfer of nutrients, maternal antibodies and hormones
(Schreck et al., 1991;
Williams, 1994). Maternal
hormones influence the development of offspring in a wide range of vertebrate
species (Schreck et al., 1991;
Schwabl, 1993;
Adkins-Regan et al., 1995;
Schwabl, 1996;
Conley et al., 1997). In
birds, it has been argued that females may enhance their reproductive success
by mediating sibling competition and growth of offspring by means of
differential hormone transfer to the egg yolk
(Schwabl, 1993;
McNabb and Wilson, 1997;
Eising et al., 2001). For
example, differential transfer of steroids to eggs within the same clutch may
mitigate or increase the effect of hatching asynchrony as yolk steroids
enhance nestling growth and competition
(Sockman and Schwabl, 2000;
Eising et al., 2001;
Groothuis et al., 2006)
Although several studies have shown how nestlings benefit from increased
yolk androgens, e.g. in terms of increased growth and survival (for a review,
see Groothuis and Schwabl,
2008), high levels of yolk androgens also involve costs. Exposure
to elevated androgen levels in developing chicks can increase metabolism
(Tobler et al., 2007) (but see
Eising et al., 2003) and may
possibly increase susceptibility to oxidative stress
(Royle et al., 2001;
Groothuis et al., 2006).
Several studies have also shown that yolk androgens can influence nestling
immune capacity (Andersson et al.,
2004; Groothuis et al.,
2005a; Mueller et al.,
2005; Navara et al.,
2005; Navara et al.,
2006) (but see Tschirren et
al., 2005). However, the majority of these studies involve a
single immunological test, namely the test of cell-mediated immunity by
injection with phytohaemagglutinin (PHA) (e.g.
Lochmiller and Deerenberg,
2000). Only one study has also investigated the effect of yolk
androgens on humoral immunity in combination with cell-mediated immunity
(Mueller et al., 2005). In
this study on black-headed gulls (Larus ridibundus), however, immune
tests were performed at a very early stage of the chick phase (seven days of
age), i.e. when the immune system of the young is immature and chicks rely on
maternally transferred antibodies for protection against antigens. Hence, it
is not known whether the effect of yolk androgens on immunity extends beyond
the first days of life and whether it involves other parts of the immune
system than PHA-induced cell-mediated immunity. There is, therefore, a need
for studies that investigate the effect of yolk androgens on several lines of
immunity during an extended part of the chick period when the chick is reliant
on its own immune defence. An integrative approach investigating several lines
of immunity is essential for understanding the trade-offs between
immunocompetence and other life history traits (e.g.
Norris and Evans, 2000;
Adamo, 2004).
During the first phase of life, young birds have a less effective defence
against parasites and infections than adult birds. Birds are born with an
incomplete immune system and young chicks have to rely on maternal antibodies
and the innate immune defence system to fight off pathogens
(Apanius, 1998). It has been
shown that activating or maintaining immune functions can be metabolically
costly and resource demanding (Saino et
al., 1997; Lochmiller and
Deerenberg, 2000;
Alonso-Alvarez and Tella, 2001;
Råberg et al., 2003)
(but see Svensson et al.,
1998), which indicates a trade-off between immune function and
life-history traits (Sheldon and Verhulst,
1996; Lochmiller and
Deerenberg, 2000). Similar trade-offs may also exist between
different parts of the immune system, e.g. if investment in cell-mediated
immune function impairs concomitant investment in humoral immune function
(Salvante, 2006) and these
trade-offs may be different during different life stages. Maternal steroids
may prime the development and expression of various components of the immune
system such as cell-mediated and humoral immune function differently.
In this study, we used an integrative approach to examine the influence of
yolk androgens on the immune function of jackdaw (Corvus monedula L.)
nestlings. This species shows strong hatching asynchrony with brood reduction
of up to 50% (Gibbons, 1987;
Heeb, 1994). Within-clutch
levels of yolk androgens (testosterone and androstendione) decrease with
laying sequence (M.I.S., unpublished data) and may, hence, promote size
differences in hatching asynchrony. Moreover, jackdaws have a relatively long
nestling period of over 30 days and are, therefore, well suited to study the
development of the immune system. This species is also a suitable contrast
with studies on yolk hormones and immunity in the black-headed gull [Larus
ridubundus (Muller et al., 2005)], which also has a long nestling period,
hatching asynchrony but an increase of yolk androgens with laying order.
Specifically, whether humoral immunity may be affected by yolk androgens even
long after hatching, at a time when the offspring is reliant on its own immune
system for protection against pathogens. We assessed humoral immune function
of chicks hatched from either androgen-treated or control eggs towards
different immune system activators; the mitogen lipopolysaccharide (LPS) when
chicks were 1–2 weeks old and two types of antigens (diphtheria toxoid
and tetanus toxoid) when chicks were 3–4 weeks old. In three-week-old
chicks, we also assessed cell-mediated immune response by injection with PHA.
Repeated exposure to different antigens and a mitogen allowed us to test
whether and how the influence of yolk steroids on adaptive immune function
changes over the developmental phase.
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MATERIALS AND METHODS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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When the fourth egg in a clutch was laid (all clutches contained at least four eggs), we manipulated yolk androgen concentration in the collected eggs. We assigned eggs to two groups depending on laying order – early (the two first eggs laid) and late group (egg number three and four in the laying sequence). One egg in each group was randomly injected with 70 ng testosterone and 300 ng androstendione suspended in 25 µl sesame oil (androgen chicks). The remaining eggs received an injection of 25 µl sesame oil only (control chicks). The holes were sealed with OpSite (Smith & Nephew, Mölndal, Sweden), a transparent adhesive dressing. The dose of injected androgens corresponds to approximately one standard deviation of the mean yolk androgen levels found naturally in our population (M.I.S., unpublished data) and is, therefore, assumed to be within the physiological range of the species.
After injection, eggs were returned to the nests in the colonies. Eggs from two nests were combined in a dyad so that one nest received only early laid eggs (one control egg and one androgen-injected egg from own nest and one control egg and one androgen-injected egg from another nest) or only late-laid eggs (one control egg and one androgen-injected egg from own nest and one control egg and one androgen-injected egg from another nest). This enabled us to analyse the effect of androgen manipulation and, at the same time, separate genetic effects (genetic nest) and environmental effects during the nestling rearing period (foster nest) as well as analyse potential laying order effects (early or late eggs). In 2003, all eggs laid after the fourth egg were collected and frozen. Due to the low hatching success and subsequent small clutches in 2003, we changed the experimental setup during 2004 and we returned any eggs laid after the fourth egg to the nest. The fifth and/or sixth egg were treated as control eggs (N=14) or received yolk androgen manipulation (N=9) and they were added to the nest one day later than the first four eggs. Due to brood reduction during the first week, only four chicks from eggs five or six were alive during the immune challenge. These chicks were not included in analyses of nestling growth or immune response but the `extra' eggs were accounted for in analyses by including actual brood size at different periods of the nestling cycle in the models (see below).
Hatching success, fledgling success and nestling growth were monitored during nest visits with 1–4 day intervals. Body mass was measured eight times; on day 0, 2, 6, 10, 14, 20, 24 and 28, tarsus length on day 10, 14, 20, 24 and 28 and wing length on day 14, 20, 24 and 28. Brood size was measured at hatching and when the oldest chick was 6, 14 and 28 days old.
Testing humoral immunity
We tested humoral immunocompetence using one mitogen and two types of
antigens during two stages of the nestling period. During the early
developmental period of the nestlings, we injected six-day-old chicks with
LPS. LPS is derived from cell walls of Gram-negative bacteria, Escherichia
coli, and mimics a natural infection with bacteria (e.g.
Kuby, 1998;
Bonneaud et al., 2003). Immune
response towards LPS involves both the innate defence (such as behavioural
responses, fever, inflammatory response and production of acute phase
proteins) and the adaptive defence
(Janeway and Travers, 1996).
We injected nestlings with 7.5 µg LPS suspended in 100 µl PBS (0.1 mg of
LPS per kg of body mass) (Grindstaff et
al., 2006). We avoided measuring aspects of the acute phase
response to avoid disturbance at the nest when jackdaw nestlings were less
than 10-days-old. Prior to injections, we took a blood sample (50 µl) from
the jugular vein for analyses of pre-injection concentrations of antibodies
against LPS. We expected these six-day-old nestlings to have anti-LPS
antibodies as a consequence of maternal antibody transfer (most probably
including anti-LPS antibodies because exposure to Gram-negative bacteria
should be common among adult birds) but possibly also because these nestlings
already had functional B-cells. Our rationale for the LPS injection of
six-day-old chicks was to ensure that all chicks had a similar (moderate)
exposure to LPS at a time when their own B-cells are beginning to mature and,
hence, to be able to measure the T-cell-independent activation of their
endogenous B-cells based on a (as far as practically possible) similar level
and timing of LPS challenge. Because B-cells need to be activated to produce
antibodies against LPS and antibodies have a half-life of 5–7 days in
newly hatched chicks (Davison et al.,
2008), we decided to measure anti-LPS antibody titres eight days
after LPS injection. Hence, when nestlings were 14-days-old we took a second
blood sample for analyses of the nestlings' anti-LPS antibody response (100
µl). This response measures the T-cell-independent activation of B-cells to
produce IgM antibodies (Janeway and
Travers, 1996) and should reflect the ability of 14-day-old
nestlings to produce a first-line antibody defence to an infection with
Gram-negative bacteria. Maternally transferred antibodies (via egg
yolk) should be very low or non-existent in the circulation in 14-day-old
nestlings (Hasselquist and Nilsson,
2009). Moreover, the response in 14-day-old nestlings should be
relatively independent of difference in exposure to Gram-negative bacteria
before LPS injection at day six, because LPS is a T-cell-independent antigen
that does not induce formation of memory B-cells and, thus, does not induce
secondary antibody responses (Janeway and
Travers, 1996).
When nestlings were 14-days-old, we challenged other aspects of their
humoral immunity using a diphtheria/tetanus vaccine (DTV) containing two
antigens. Diphtheria toxoid and tetanus toxoid are potent antigens that
activate antigen-specific B-cells resulting in a T-cell-dependent primary
antibody response (i.e. first production of IgM followed by IgG antibodies
peaking ca. 14 days post-injection as well as the formation of memory
B-cells) (Janeway and Travers,
1996). Individuals were vaccinated with 100 µl human DTV (2 Lf
diphtheria toxoid, 5 Lf tetanus toxoid adsorbed in aluminium phosphate;
Aventis Pasteur, Toronto, Canada) in the pectoral muscle. Prior to the
injection, a blood sample of 100 µl was taken from the jugular vein. The
sample was used to analyse post-injection analyses of LPS treatment (see
above) as well as measurement of background values of antibodies to
diphtheria/tetanus. Primary antibody responses towards diphtheria/tetanus peak
around 9–15 days after vaccination in small to medium sized passerines
(Hasselquist et al., 1999;
Owen-Ashley et al., 2004) and
ca. 14 days after vaccination in, e.g. pheasants
(Ohlsson et al., 2002). We,
therefore, collected a blood sample after 14 days, when the young were
28-days-old (100 µl). All blood samples were centrifuged for 10 min at 1000
g and the separated plasma was stored at –50°C until
analysis. The fact that jackdaw nestlings stay for about a month in the nest
box, which is a much longer period than nestlings of other nest box breeding
passerine birds, which leave the nest when ca. two weeks old (e.g.
Grindstaff et al., 2006),
allowed us to measure primary antibody responses of nestlings
Antibody titres were determined using enzyme-linked immunosorbant assays
(ELISA). Protocols followed those previously developed for passerines [LPS
(Grindstaff et al., 2006);
diphtheria/tetanus (Owen-Ashley et al.,
2004)]. An individual's pre- and post-vaccination plasma samples
in duplicate were placed on the same ELISA plate. Pre- and post-injection
plasma samples were diluted 1:100 for LPS antibodies, 1:300 for the diphtheria
antibodies and 1:900 for the tetanus antibodies. The strength of the humoral
immune response was estimated as the difference between post- and
pre-immunisation antibody titres. For comparison of samples between plates, a
serially diluted standard (pooled serum from jackdaw chicks immunised with the
antigens) was run on all plates. However, in diphtheria/tetanus assays, we
used two different standards in 2003 and 2004 and, therefore, we cannot
distinguish between-year assay variation from other between-year variation due
to, e.g. environmental factors. We, therefore, controlled for year in all
statistical analyses of antibody titres against diphtheria and tetanus
toxoids.
Cell-mediated immunity: PHA challenge
The mitogen PHA is commonly used in passerine birds as a T-cell stimulant.
Injection of PHA (Cat. L-8754, Sigma Chemical Co., St Louis, MO, USA) in the
wing web produces a swelling approximately 24 h after injection. PHA activates
the cell-mediated immunity in a specific way but it also stimulates parts of
the innate immunity, e.g. macrophages and inflammatory responses (e.g.
Martin et al., 2006).
On day 19, chicks were injected with PHA dissolved in PBS in the wing web
on both wings. The injection volume differed between years although the
concentration was the same. In 2003, we used 0.5 mg PHA in 0.1 ml PBS and in
2004, 0.25 mg in 0.05 ml PSB. Because chicks were injected on both wings, the
total amount of PHA was 1 mg and 0.5 mg for 2003 and 2004, respectively.
Granbom and colleagues showed that the spatial repeatability of PHA swellings
between both wings is relatively low and to improve the accuracy of the test,
both wings should be injected with PHA
(Granbom et al., 2005). Smits
and colleagues showed that it is not required to use a control injection of
saline to accurately assess the response to PHA
(Smits et al., 1999). Prior to
injection, the injection point was marked and three measurements of the wing
web were taken with a digital micrometer to the nearest 0.001 mm. 24 h later
(±20 min), three new measurements were taken on each wing. Median
values were used in all analyses.
Statistics
Statistical analyses were conducted with SAS System for Windows 9.1 (SAS
Institute Inc., Cary, NC, USA). We used repeated mixed-model analysis of
variance (ANOVA) (PROC MIXED) (Littell et
al., 2004) with foster nest and genetic mother as random factors,
egg treatment, year and sex as fixed factors and hatching date, body mass and
brood size as covariates. Random effects were estimated with the likelihood
ratio test as described in Littell et al.
(Littell et al., 2004).
Non-significant (P>0.1) fixed factors and covariates as well as
interactions were sequentially backward eliminated from the models. The
Sattherthwaite approximation was used to calculate the denominator degrees of
freedom (Littell et al.,
2004). The significance level was set at P<0.05.
There is a sex-specific growth and mortality in jackdaw nestlings
(Table 1)
(Arnold and Griffiths, 2003).
82% of all nestlings were sexed with molecular methods
(Fridolfsson and Ellegren,
1999). We ran statistical tests both including sex as a factor in
all models of nestling growth, nestling size and immune response (i.e. with
reduced sample size) and with the complete dataset excluding sex. However,
models with and without sex produced qualitatively the same results and we,
therefore, present only the results from analyses with sex included.
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RESULTS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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2=1.00, d.f.=1, P=0.32) and there was no effect
of laying order on hatchability (P=0.45).
Clutch size and brood size
Clutch size declined from 4.83±0.13 chicks (means ± s.e.m.)
at hatching to 2.26±0.12 chicks at day 14 and 1.5±0.16 chicks at
day 28. There was a significant difference in clutch size at hatching between
years due to the difference in experimental design (2003, 2.75±0.14
chicks; 2004, 3.8±0.13 chicks; t-test,
t54=5.45, P<0.001) but this difference
disappeared during the nestling period and there was no significant difference
in brood size at day 14 (2003, 2.42±0.17; 2004, 2.17±0.19,
t52=0.85, P=0.40), indicating that the degree of
brood reduction was higher in 2004, presumably due to larger broods.
Mortality
Mortality rate during the nestling period was relatively high (37.5%) and
was due to either starvation or predation. Predation (18.4%), which was most
probably due to pine martens (Martes martes), did not differ between
egg treatments (chi-squared test, 2=2.10, d.f.=1,
P=0.14).
Mortality not related to predation is assumed to be due to
starvation/sickness and when excluding predated nestlings, mortality was still
not significantly related to egg treatment (2=0.34, d.f.=1,
P=0.56). The timing of nestling mortality due to starvation did not
differ between treatments (mean age: control chicks, 5.23±0.96 days;
chicks from androgen-treated eggs, 7.23±0.96 days; t-test,
t24=0.98, P=0.33).
Nestling growth
Hatching mass was not influenced by yolk androgen treatment
(F1,40.7=1.45, P=0.23 with egg mass as a
covariate). Nestling growth rate from hatching until day 28 was significantly
different between years with a faster growth rate in 2003 but overall size did
not differ significantly between years
(Table 1). There was a
significant effect of sex on body mass (males were heavier than females) but
there was no significant sex difference in tarsus length or wing length
(Table 1). However, there was
no effect of egg androgen treatment on nestling growth measured as increase in
body mass, tarsus length or wing length
(Table 1). Final body mass at
day 28 was also unrelated to egg treatment (body mass,
F1,36.5=0.001, P=0.96 with effect of sex,
F1,44.9=11.24, P=0.0016 and year,
F1,35=12.14, P=0.0013).
Nestlings that hatched early during the breeding season grew faster than those hatched late resulting in a significant effect of hatching date on body mass and wing length and a near significant effect on tarsus length (Table 1). However, the effect of hatching date was independent of androgen treatment. Rearing environment was important as indicated by the significant effect of foster nest (Table 1).
LPS challenge
Jackdaw chicks had plasma antibodies against LPS already prior to LPS
challenge at day six [mean 3.15±0.35 (log: 1+mOD
min–1)]. There was a significant difference in antibody
concentrations between control and androgen chicks in the pre-injection
samples; control chicks had higher antibody titres than androgen chicks
[control chicks, 3.48±0.48 (log: 1+mOD min–1);
androgen chicks, 2.82±0.14 (log: 1+mOD min–1);
F1,52.2=6.16, P=0.016]
(Fig. 1). There was also a
significant interaction between egg treatment and hatching date
(F1,46.7=5.71, P=0.021) but no overall effect of
hatching date (P=0.45). The interaction was due to a trend towards a
seasonal decline in LPS antibody titres among control chicks
(F1,38.9=3.54, P=0.06) compared with no seasonal
effect among androgen chicks (P=0.29). There was no effect of brood
size or body mass on antibody concentration prior to injection. There was a
significant effect of foster nest (2=9.8, d.f.=1,
P=0.0017) but not of the genetic parents (2=2.8,
d.f.=1, P=0.091) on pre-injection values.
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Diphtheria/tetanus challenge
Pre-injection concentrations against diphtheria and tetanus were low
[diphtheria toxoid, 0.85±0.08 (log: 1+ODmin–1);
tetanus toxoid, 0.71±0.78 (log: 1+mOD min–1)] and
significantly correlated (r70=0.79, P<0.0001).
There was no significant effect of egg treatment, body mass, hatching date or
brood size on pre-injection values (P>0.2 in all cases). The
diphtheria/tetanus challenge produced significantly higher antibody titres
than pre-injection values (diphtheria, t69=4.42,
P<0.0001; tetanus, t69=6.62,
P<0.00019). The antibody titres on average showed an increase that
was 24-fold for tetanus toxiod and 5-fold for diphtheria between pre-injection
and post-injection samples (14 days later at the presumed peak of the primary
antibody response), showing that nestlings 2–4 weeks old can already
produce a normal T-cell-dependent antibody response. A high correlation
between the antibody production towards diphtheria toxoid and tetanus toxoid
(r70=0.54, P<0.0001) showed that these two
responses were congruent and we combined these two responses into one humoral
immune score with principal component analysis. Androgen egg treatment had a
significant effect on humoral immune responses. Control chicks had
significantly higher antibody titres against diphtheria/tetanus than androgen
chicks (Table 2;
Fig. 2). There was also a trend
towards stronger immune responses early in the breeding season
(Table 2) but no effect of sex,
body mass or brood size (day 14).
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PHA challenge
T-cell-mediated immune response differed significantly between years
probably due to variation in injection volume (see Materials and methods)
(2003, 1.03±0.45 mm; 2004, 0.45±0.30 mm;
F1,37.1=51.16, P<0.001). However, after
controlling for year, there was a significant difference in response to PHA
injection between treatment groups. Nestlings from androgen-injected eggs had
a significantly lower cell-mediated immune response than control chicks
(Table 2;
Fig. 3). There was a marginally
significant effect of foster nest on the intensity of response
(2=3.9, d.f.=1, P=0.047) but there was only a weak
tendency for a genetic effect (2=2.9, d.f.=1,
P=0.09). None of the covariates (hatching date, body mass or brood
size) was correlated with T-cell-mediated immune response (all
P-values>0.25).
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Correlations between different immune responses
To analyse correlations between immune responses we used residuals from
models including year as dependent variable because all three measurements of
immune function were significantly different between years (see
Table 1 and Materials and
methods for a potential explanation of between-year differences). There were
no significant correlations between any of the three tests of immunity (LPS
versus PHA, r50=0.06, P=0.69; LPS
versus diphtheria/tetanus, r60=0.07,
P=0.58; PHA versus diphtheria/tetanus,
r41=0.03, P=0.89). Within each experimental
category, the result was similar with no significant correlations
(P>0.1 in all cases).
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DISCUSSION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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;
Adamo, 2004;
Salvante, 2006), few studies
have analysed several lines of immunity simultaneously [but see Faivre et al.
(Faivre et al., 2003;
Forsman et al., 2008)].
Immunosuppression caused by exposure to elevated yolk steroid levels has been
found in studies of other bird species but the majority of these studies have
used a single immunological test, that is the test for (unspecific)
cell-mediated immunity by injection with PHA
(Andersson et al., 2004;
Groothuis et al., 2005a;
Navara et al., 2006). One of
the few studies that has assessed the effect of yolk androgens on avian
immunity with an integrative approach is the study by Mueller and colleagues
(Mueller et al., 2005) on
black-headed gulls (see also Navar et al., 2005). However, this study focused
only on the early stage of the chick phase, that is the first week of life
when the chicks endogenous immune system has just begun to develop and it is
not clear whether the effect of yolk androgens may extend beyond this first
period of life but whether they also affect the chick when its endogenous
immune system is further developed. Our experimental study on jackdaw chicks
shows that elevated yolk androgen levels result in a general immunosuppression
in nestlings and this conclusion was based on congruent results for three
different immunological tests of both humoral and cell-mediated immunity
conducted at 1–2 and 3–4 weeks of age.
The bird's immune system begins developing before hatching and is complete
by sexual maturity. Our understanding of the ontogeny of the avian immune
system originates almost exclusively from studies on poultry and is,
therefore, biased towards precoccial birds. Little is known about potential
differences between altricial and precoccial birds
(Apanius, 1998). One exception
is the American kestrel (Falco sparverius) where studies demonstrate
that chicks acquire antibody responsiveness to antigens when approximately
1–2 weeks old (Apanius,
1993). Because young jackdaws remain in the nest for almost five
weeks, it was possible for us to follow potential effects of elevated yolk
androgen exposure over a relatively long period. Androgen exposure during
embryonic development resulted in lower antibody titres during the early phase
(six-day-old chicks) when the chicks' own immune system just has begun to
develop. Moreover, we also found that in fully grown chicks (i.e. 3–4
weeks old chicks) both cell-mediated and humoral immune responsiveness,
measured as immune responses to injected non-pathogenic antigens, were
suppressed in chicks from androgen-treated as compared with control eggs.
These results clearly show that both arms of the adaptive immune defence were
negatively affected by yolk androgens. At the age of 3–4 weeks, the
immune system of the jackdaws is still under development and we can therefore
not conclude that the adaptive immune system has been permanently suppressed
by the yolk androgen treatment. Still, suppressed adaptive immune
responsiveness when ca. one month old, i.e. at an age when juvenile
jackdaws become fledged, should potentially have a huge impact on fitness. The
first period after fledgling is a stressful period for passerine birds, when
the young have to learn to feed and avoid predators and mortality is typically
high at this stage (Newton,
1998). A newly fledged bird will also be exposed to a broader
range of pathogens than when inside the nest; therefore, the need for a
well-developed, efficient immune system should be crucial for survival.
Antibody titres towards LPS differed between chicks from androgen-treated
eggs and control chicks already prior to the injection at day six. LPS
contains antigens that trigger a T-cell-independent B-cell response and at
high concentrations a non-specific antibody response
(Janeway et al., 2005). LPS
antibodies found in six-day-old nestlings could be of maternal origin or
produced by the nestlings. Maternal transfer of antibodies reflects the
pathogens that mothers have been exposed to and they may be present 1–2
weeks after hatching, although in decreasing concentrations
(Smith et al., 1994;
Lung et al., 1996;
Grindstaff et al., 2006).
Maternal transfer of antibodies against LPS was not manipulated in this study
and, hence, unrelated to yolk androgens. If antibody levels in six-day-old
chicks were mainly of maternal origin, this would indicate that maternal
antibodies are catabolised or used in immune defence at different rates in
control and androgen chicks. However, the chicks may have started to produce
endogenous antibodies already before they were six-days-old and the antibody
titres we measured would then been a mixture of maternal and endogenous
antibodies. If the latter explanation is true, then our results may also
reflect a difference in the timing of the onset of endogenous antibody
production caused by the yolk androgen treatment. This would then indicate
that the elevated yolk androgen could delay the development of neonatal immune
function so that androgen chicks started their endogenous production later
than control chicks. This is supported by the observation that
androgen-treated chicks increase their LPS-specific antibody titres between
day six and 14, resulting in no difference between treatment groups at day 14.
This could also be explained if both treatment groups had reached their limit
of production of LPS-specific antibodies at day 14. In the experiment with
black-headed gulls it was found that embryonic androgen exposure had negative
effects on antibody production towards LPS
(Mueller et al., 2005). In the
latter study, chicks were challenged at day seven and antibody response was
measured after 48 h. The pre-injection antibody titres were not significantly
different but post-injection titres were lower in androgen chicks. These
results were also interpreted as a delayed response in androgen chicks because
production of LPS antibodies was still in its early phase at this time. Our
result on jackdaw chicks are in contrast with these results since we found
that elevated yolk androgens in jackdaws had a negative effect already prior
to the LPS challenge.
In the present study, the cell-mediated response to PHA was tested during a
period when the nestlings were mounting a humoral immune response. PHA
response is thought to be a trade-off against other life-history components
and varies with individual condition
(Martin et al., 2006).
Although both PHA response and diphtheria/tetanus antibody titres were lower
in androgen chicks compared with control chicks, there was no direct
correlation between the two responses. Similarly, there was no correlation
between responses to LPS and diphtheria/tetanus. Exposure to elevated androgen
levels during embryonic development seems to have an overall negative effect
on the development of the adaptive immune system in jackdaw chicks. However,
other environmental factors, such as food availability and rearing conditions,
also influenced the immune responsiveness of jackdaw chicks. If these effects
vary within the nestling period, this could explain the lack of clear
correlations between the different tests.
The mechanisms by which yolk steroids influence immune function are not yet
known. One of the important stages of the development of the avian immune
system occurs during the first weeks of the chick's life when the
diversification of antibody repertoire through gene conversion takes place in
the bursa of Fabricius (Glick and Sadler,
1961). Studies have shown that high doses of testosterone
administrated in ovo early in embryonic development caused regression
of the bursa in chickens resulting in a reduction in IgG production
(Erickson and Pincus, 1966;
Lerner et al., 1971).
Experiments with mibolerone, an androgen analogue of testosterone, showed
negative dose-dependent effects on the embryonic development of the bursa and
the maturation and differentiation of B lymphocytes
(Bhanushali et al., 1985).
Hence, androgens transferred from the mother to the egg may have negative
consequences on nestling immunity by acting on the development of or the
antibody repertoire diversification in the bursa of Fabricius. We suggest that
such an effect could be long-lasting influencing immunocompetence even in
adulthood. There could also be other potential explanations for why yolk
androgen-treatment resulted in suppressed immune responsiveness. It has been
suggested that differences in prenatal exposure to androgens between males and
females can explain sex-specific differences in immune function in mammals, in
which females generally have greater humoral and cell-mediated immunity than
males (Martin, 2000). These
prenatal effects in mammals are the result of differential steroid production
by the embryos, not by exposure to maternal hormones. The link between yolk
androgens and circulating endogenous androgens in nestlings is not yet clear
but if embryonic exposure to yolk androgens influences circulating levels of
testosterone in bird nestlings, the negative effects of yolk treatment could
be explained by direct or indirect negative effects of circulating
testosterone. Immunosuppression as a result of high circulating levels of
testosterone has been shown in adult birds (e.g.
Duffy et al., 2000;
Owens-Ashley et al., 2004)
(but see Hasselquist et al.,
1999).
In conclusion, our study demonstrates that there are immunological costs
associated with increased levels of yolk androgens that are detectable even
after several weeks of chick development. The potential for long-lasting
effects on offspring fitness is large and may be mediated both through
organisational effects on immunocompetence and indirect ways through
trade-offs with other life-history traits
(Carere and Balthazart,
2007).
|
Footnotes |
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References |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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