Male song quality affects circulating but not yolk steroid concentrations in female canaries (Serinus canaria)
1 School of Biological Sciences, Royal Holloway, University of London,
Egham, Surrey, TW20 OEX, UK
2 Max Planck Research Centre for Ornithology, Vogelwarte Radolfzell, 78315
Radolfzell, Germany
3 Center for Reproductive Biology, School of Biological Sciences, Washington
State University, Pullman, WA 99164-4236, USA
* Author for correspondence at present address: Zoology Department, University of Gothenburg, Box 463, SE 40530 Gothenburg, Sweden (e-mail: rupert.marshall{at}zool.gu.se)
Accepted 25 October 2005
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Summary |
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Key words: song, faecal steroid, HVC, female choice, mate quality, yolk hormone
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Introduction |
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Comparisons of playback treatments containing either conspecific song,
heterospecific song or no song have demonstrated that song has a stimulatory
effect upon the female reproductive axis in canaries (Serinus canaria
L.), song sparrows (Melospiza melodia;
Bentley et al., 2000) and ring
doves (Streptopelia decaocto risoria;
Cheng, 1986
;
Cheng et al., 1998
). Previous
studies showed that song quality affects reproductive development in canaries
(Kroodsma, 1976
) and that song
affects female faecal oestrogen levels in zebra finches (Taeniopygia
guttata; Tschernichovski et al., 1998). But, to our knowledge, no studies
have investigated the effect of song quality on the secretion of reproductive
hormones. The brain regions involved in avian song perception are hormone
sensitive (Brenowitz and Arnold,
1992
) although the mechanism by which the song stimulus results in
an endocrine response in female birds remains unclear. Recently, two pathways
have been suggested, the first involving an indirect neural connection from
the higher vocal centre (HVC), the area of the brain involved in female
perception of song (Brenowitz,
1991
; Leitner and Catchpole,
2002
), to the thalamus (Foster
et al., 1997
), and the second involving control of gonadotrophin
releasing hormone in the HVC itself
(Bentley et al., 2004
).
Female canaries respond to specific attractive (`sexy'; A-phrase) syllables
(Vallet et al., 1998) with
copulation solicitation displays. Syllables that do not elicit such a response
may be artificially transformed into the attractive form by shortening the
period of silence between the two elements comprising a syllable or broadening
the frequencies (Draganoiu et al.,
2002
). Although such sexy syllables induced female copulation
solicitation displays, they had no stimulatory effects on egg laying
(Leboucher et al., 1998
). We
investigated whether sexy syllables influence female hormone secretion.
Female hormone levels depend on a wide range of factors, from time of day
to the stage of the reproductive cycle
(Sockman and Schwabl, 1999).
The eggs of female canaries contain varying amounts of testosterone
(Schwabl, 1993
), higher
concentrations leading to an increase in nestling growth and begging as well
as correlating with dominance and social rank in later life
(Schwabl, 1996b
). This has led
to the hypothesis that females may be able to influence the quality of their
offspring beyond their initial choice of a mate by actively varying the amount
of specific resources within each egg in accordance with their perception of a
partner's quality (e.g. Gil et al.,
1999
; Cunningham and Russell,
2000
; Ellegren et al.,
1996
). Canary eggs contained higher concentrations of testosterone
when the female was exposed to attractive syllables during yolk formation than
when she was exposed to songs without such syllables
(Gil et al., 2004
). Similar
experiments in zebra finches (using manipulation of coloured leg-rings) have
produced mixed results, one study showing that eggs of females mated to more
attractive males contain higher levels of testosterone
(Gil et al., 1999
), while a
recent study found no such effect
(Rutstein et al., 2004a
),
although in the latter study the authors investigated only the second egg from
each brood.
We tested the following two hypotheses: (1) male song quality has an acute affect upon circulating levels of testosterone and oestradiol in female canaries, as measured by their concentrations in faeces, and (2) the concentrations of maternal steroids in the egg yolks change in response to the presence or absence of attractive syllables.
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Materials and methods |
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Song playback
Song was recorded from 12 males using a Marantz (Longford, Middlesex, UK)
CP430 cassette recorder with a Sennheiser (Wedemark, Lower Saxony, Germany)
MD211U microphone. Song was edited using Avisoft-SASLab Pro (Avisoft
Bioacoustics, Berlin, Germany). A maximum of two attractive syllables from
each of 12 males was selected to compile the playback treatment, selected to
maximise variation between syllables. Syllables were computer-edited to
increase their attractiveness to females by reducing the gap between the
elements of individual syllables
(Draganoiu et al., 2002).
Playback procedure was similar to that described by Gil et al.
(2004
). Each song lasted 30 s,
followed by 90 s of silence. Song order was randomised using the Random Order
Continuous Repeat function of the Sony Minidisc system (Sony MZ R700) and
included one `silent' song lasting 15 min to avoid habituation to the
stimulus. The control song contained five different syllables, which were
repeated for 30 s, none of which was attractive. 21 attractive syllables were
used in constructing the attractive playback. Syllables were used in a
randomised order, each 30-s segment of attractive song occurring only three
times in each playback session. Playbacks lasted for 3 h in the morning
(immediately after feeding/cleaning) and 3 h in the evening, finishing 1 h
before the lights were turned off.
Experimental design
Yolk hormone experiment
22 individually housed females were randomly divided into two groups, one
in each of two experimental rooms, where they were in auditory but not visual
contact with members of the same group. Each group received playback treatment
in a different order (attractivecontrol or controlattractive).
Nesting material was provided from the commencement of playback. Eggs were
removed and replaced with dummy eggs on the day that they were laid, and yolks
were frozen for later analysis. Nests were removed 3 days after the last egg
was laid, and the female was moved to the other room, where she was exposed to
playback of the alternate treatment immediately. Nest cups were returned after
3 days in the new room. 13 of the 22 females laid one clutch in each
treatment, and the analysis of yolk hormones presented here is limited to
these clutches. Since no males were present, the eggs were unfertilized.
Acute faecal hormone experiment
After completing two broods for yolk analysis, females were moved to a room
without playback for one week. The same 22 females that were used for analyses
of yolk steroids were then assigned to one of two experimental rooms where
they were in auditory but not visual contact with members of the same group.
Each group received its playback treatment in a different order
(attractivecontrol or controlattractive). During the 3 h of
playback, faeces were collected on paper placed on the cage floor. At the end
of each playback period, the collection sheet was removed and the faeces
scraped from it. There was a 1-h period of silence between treatments during
which faeces were not collected. Thereafter, the treatment was reversed and
faeces collected again. We used non-invasive measurements of steroids in
faeces to avoid handling effects. Faecal steroid concentrations correlate with
circulating levels in canaries, injected levels of radiolabelled hormone
correlating with the faecal profile
(Sockman and Schwabl, 1999).
Our method of faecal steroid measurement has been validated for the canary,
the concentrations of immunoreactive steroid in faecal buffer solution
correlating strongly with the figures obtained after steroids were separated
from faeces by hydrolysis, organic extraction and chromatographic separation
from other metabolites (Schwabl,
1996a
; Sockman and Schwabl,
1999
).
Hormone analysis
Collection and storage of faeces was standardised, and all faeces were
collected at the same time and allowed to dry at room temperature for 24 h
before being frozen until analysis
(Cockrem and Rounce, 1994;
Goymann, 2005
;
Sockman and Schwabl, 1999
).
Analyses of faecal androgens and oestrogens were conducted as described
elsewhere (Schwabl, 1993
;
Sockman and Schwabl, 1999
).
The limited quantity of faeces led us to restrict analyses to androgen
[testosterone (T) and 5
-dihydrotestosterone (5
-DHT)] and
oestrogen (oestradiol) only. Briefly, faeces were homogenized with
phosphate-buffered saline with glucose (PBSg) and assays were performed
without any further extraction or purification. These assays have been
validated for the canary (Schwabl,
1996a
; Sockman and Schwabl,
1999
). All samples were assayed for androgens (A) and oestrogens
(E) in two single assays. The antibody (Wien Laboratories, Succasunna, NJ,
USA) used to measure faecal A cross-reacts with T and 5
-DHT. The E
antibody (Arnel Products Co., New York, NY, USA) cross-reacts with
17ß-oestradiol and to a low extent with oestrone and oestriol. Yolk
steroids were measured according to Schwabl
(1993
). Briefly, whole yolks
were homogenised with dH2O, and steroids were extracted from the
homogenate and partially purified and separated using mini-chromatography
columns before radioimmunoassay for androstenedione (A4),
5
-DHT, T and 17ß-oestradiol were performed. All eggs were assayed
for A4, 5
-DHT, T and 17ß-oestradiol in a single assay.
The antibodies used to measure yolk concentration of T, 5
-DHT and
17ß-oestradiol were the same as those used for faecal assays; the
A4 antibody was purchased from Wien Laboratories.
Analysis
Brood size ranged from three to six eggs, most females laying four or five.
We used mean values of neighbouring eggs in the laying sequence in accordance
with the matrix in Table 1 in
order to produce a standardised brood size focussed upon the beginning, middle
and end of the laying cycle (Rutstein et
al., 2004b). Females differed significantly in the levels of all
four hormones in their eggs, in some cases by factors of 10 or more (data not
shown). All results were analysed on a within-female basis, with eggs as
repeated measures, each female thereby acting as her own control.
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Results |
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Although the main effect of song treatment on faecal E was not significant, Fig. 1 indicates that this was probably due to a carry-over effect from the previous treatment with attractive song in one of the two experimental groups (indicated by the highly significant interaction term). Comparing the two control playback regimens directly, levels of E were significantly lower when control playback was received before the attractive song treatment than when it was played after the attractive treatment (t=3.8, P=0.001, N=22). E levels were lower in faeces from the control treatment than from those during the subsequent attractive playback when control playback was the first treatment, although marginally non-significant (t=2.01, P=0.071, N=10).
|
Yolk hormone experiment
Mean yolk steroid levels calculated across treatments and all females were
as follows: A4=9.63±7.79 mg pg1 (mean
± S.D.), N=117;
5-DHT=13.53±6.83 mg pg1, N=119;
T=143.41± 79.47 mg pg1, N=119;
E2=8.81±5.65 mg pg1, N=119. All
yolk steroids, except for E2 and A4, were correlated
with each other: T vs A4, F=116.442,
P=0.0001; 5
-DHT vs A4, F=16.755,
P=0.0001; E2 vs A4, F=3.354,
P=0.696; 5
-DHT vs T, F=31.113,
P=0.0001; E2 vs T, F=16.011,
P=0.0001; E2 vs 5
-DHT, F=22.987,
P=0.0001.
|
Since the amount of yolk as well as the concentrations of yolk steroids may vary, we also analysed the ratio of yolk mass to whole egg mass. As with the steroid concentrations, we found a significant effect of lay order upon this ratio, yolks becoming smaller in relation to the size of the egg towards the end of a brood (F=28.42, P<0.0001). However, there was no effect of song treatment upon this ratio (F=0.3, P=0.59).
Correlation of yolk and faecal steroid concentrations
There were no significant correlations between treatment effects on levels
of steroids in yolks and faeces: yolk 5-DHT vs faecal
androgens, r=0.243, P=0.423; yolk T vs faecal
androgens, r=0.039, P=0.9; T+5
-DHT vs
faecal androgens, r=0.079, P=0.798; yolk E2
vs faecal oestrogens, r=0.13, P=0.672.
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Discussion |
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The link between song perception and an endocrine response remains unclear,
although a neural pathway between the HVC and the thalamus
(Foster et al., 1997) and the
release of gonadotrophin releasing hormone direct from the HVC itself
(Bentley et al., 2004
) have
been suggested. The HVC is the most important nucleus of the passerine neural
song system for song production in males
(Nottebohm et al., 1976
) and
is present as a smaller feature in female birds as well
(Brenowitz, 1991
;
Leitner and Catchpole, 2002
).
Female canaries that both respond stronger to male songs and discriminate more
accurately among songs of different quality have a larger HVC compared with
less sensitive and discriminative females
(Leitner and Catchpole, 2002
).
We propose that our results suggest an evolutionary feedback mechanism that
maintains the honesty of song as a signal under sexual selection; female
canaries solicit more copulations from males that sing more elaborate songs,
containing more attractive syllables
(Vallet et al., 1998
). If male
song is an honest signal of quality
(Draganoiu et al., 2002
) then
it is necessary that females, as receivers of the signal, are able to
discriminate amongst the variance in signal quality
(Guilford and Dawkins, 1991
),
a task in which the HVC is heavily implicated
(Leitner and Catchpole, 2002
).
Here, we have shown that high quality song promotes higher levels of
circulating (faecal) androgens and oestrogens in females.
Since testosterone and oestradiol both increase the recruitment and
survival of HVC neurons in female canaries
(Rasika et al., 1994;
Hidalgo et al., 1995
;
Harding, 2004
), the increased
levels of androgens and oestrogens in response to high-quality song may
enhance female abilities of discrimination and accuracy, allowing them to
select high-quality males as mates based on their song. Thus, by singing
attractive songs, males may be indirectly improving their selective advantage,
and/or that of their progeny, over males who sing less attractive songs. Such
a system would benefit both signaller and receiver, selecting for more
attractive songs whilst preserving signal honesty and enhancing the
discriminatory power of the receiver. The requirement to signal is maintained
as males must sing in order to elicit a copulatory solicitation display and
copulate.
Although our findings demonstrate an immediate effect of male song quality
upon female steroid secretion, we did not find an effect of song quality upon
the levels of these hormones in their eggs. While the first- and middle-laid
eggs of a clutch showed a trend towards higher levels of the three measured
androgens in the attractive song treatment, this was not the case for the
late-laid eggs (Fig. 2).
Overall androgen concentrations (A4, 5-DHT and T combined)
were not different in our study (data not shown). We also did not find
significant differences when using non-standardised brood size in the analyses
(data not shown). These results conflict with those of Gil et al.
(2004
), who showed higher yolk
T in all eggs when females were presented with attractive songs and implied
significance for 5
-DHT and A4 based on a correlation of
these androgens with T in a small sub-sample of their yolks. Although sexy
song affected female endocrine state, it affected neither overall nor
intra-clutch patterns of yolk steroids in our study, which used a similar
design. Such a lack of repeatability seems also to be the case in another song
bird, the zebra finch. While Gil et al.
(1999
) found increased yolk
androgens when females were mated to red-banded as opposed to green-banded
males, a similar study by Rutstein et al.
(2004a
) was unable to find
such an effect, although females did lay larger eggs for more attractive males
in the first breeding round (Rutstein et
al., 2004a
). Thus, it is premature to accept the generality of the
hypothesis that females adjust yolk steroid concentrations in response to male
quality.
An emerging feature of experimental investigations of differential
allocation are carry-over effects of previous experience into subsequent
reproductive bouts. In our study, this was evident as higher yolk steroid
concentrations in the second exposure to song regardless of its quality. Such
carry-over was also present in the zebra finch studies of Gil et al.
(1999) and Rutstein et al.
(2004a
). Future tests of the
differential allocation hypothesis need to take such physiological inertness
into account. Despite claims that female birds regulate yolk steroid
concentrations, the extent to which females exert control over yolk steroid
sequestration remains unclear (Birkhead et
al., 2000
; Gil,
2003
). It has previously been shown that yolk androgen levels
reflect the hormonal state of the female canaries
(Schwabl, 1996a
) during egg
formation. Although we cannot directly address the association of female
circulating and yolk steroid concentrations in our study, the comparison of
faecal and yolk steroids in the two experiments revealed no correlation. Thus,
the possibility for regulation of circulating and yolk androgens remains an
unanswered question. We can conclude, however, that among-female variation in
circulating steroids does not necessarily predict yolk steroid concentrations
and vice versa.
In summary, findings from an increasing number of studies present a mixed picture as to whether females can `manipulate' the hormone levels in their eggs. We found no support for the hypothesis that female canaries allocate reproductive resources in relation to the perceived attractiveness of their mate. Facultative manipulation of yolk hormone levels by females, if it exists, may not be linked to male quality, as has previously been suggested. Nonetheless, we find that variations in song quality of canaries can have a significant and immediate effect upon the female endocrine state and suggest a feedback mechanism for maintaining signal honesty and improving discrimination amongst signals by the receiver.
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Acknowledgments |
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