Testosterone upregulates lipoprotein status to control sexual attractiveness in a colorful songbird

A salient feature of many secondary sexual characteristics in animals is that their expression is controlled by sex-steroid hormones. However, for only a few types of ornaments do we know the precise molecular mechanism by which androgens like testosterone (T) enhance trait production. We studied the red carotenoid-based beak of male zebra finches (Taeniopygia guttata), which serves as a sexually selected trait and is thought to be T dependent. In previous research, we demonstrated that the extent to which male finches produce lipoproteins in the bloodstream, which bind carotenoid pigments acquired from the diet and transport them to peripheral tissues, regulates the accumulation of carotenoids in the body and beak pigmentation. Here we show that T acts to upregulate lipoprotein production and allows male zebra finches to display flashy, sexually attractive coloration. Levels of circulating T in blood positively and significantly predicted lipoprotein profile (as measured by cholesterol levels), blood carotenoid concentration, and beak color. Exogenous T administration elevated cholesterol and carotenoid status as well as beak redness. Last, experimental inhibition of T (using the anti-androgen flutamide) downregulated lipoprotein production and carotenoid circulation and faded the beak. This androgen- and lipoprotein-mediated system represents one of the more detailed physiological mechanisms underlying the development of a sex-steroid-dependent trait in animals.     

Short- and long-term consequences of prenatal testosterone for immune function: an experimental study in the zebra finch

Hormone-mediated maternal effects play an important role in the formation of a differentiated phenotype. They have been shown to influence a wide array of offspring traits, both early in life and in adulthood. One important offspring trait that is under the influence of maternal androgens is the immune system. In birds, a growing number of studies show that yolk androgens modulate immune function during the chick stage. However, there is a lack of knowledge regarding long-term effects of prenatal androgens on offspring immunity. In this study, we therefore investigated the influence of prenatal testosterone (T) on several measures of immunity in fledgling and adult zebra finches (Taeniopygia guttata). Cell-mediated immune response (towards phytohaemagglutinin, PHA) of fledglings hatching from control eggs was negatively related to brood size, whereas there was no such association for fledglings hatching from eggs with experimentally elevated T levels (T fledglings). Male control fledglings showed reduced mass gain compared to female control fledglings within 24 h after the PHA injection. This pattern was reversed in T fledglings. Total antibody levels in fledglings were not affected by egg treatment. Neither cell-mediated immunity nor total antibody levels in sexually mature zebra finches were influenced by egg treatment. However, there was an immuno-enhancing effect of elevated egg T on both primary and secondary humoral immune responses toward diphtheria and tetanus antigens in ca 5 and 7 month old zebra finches. In addition, the covariation between different immune components differed between T and control offspring, suggesting that egg treatment may have altered the potential trade-offs between different parts of the immune system. Our results suggest that prenatal androgens could be an important factor contributing to individual variation in immune function even in adulthood.     

A quantitative comparison of the commonly used methods for extracting carotenoids from avian plasma

Interest in animal carotenoids, especially in birds, has exploded in recent years, and so too have the methods employed to investigate the nature and function of these pigments. Perhaps the most easily and commonly performed procedure in this work has been the determination of carotenoid concentration from avian plasma. Over the past 20 years of research on avian carotenoids, numerous methods have been used to extract carotenoids from bird plasma, all of which have differed in several important parameters (e.g., number and types of solvents used, degree of mixing/centrifugation). However, to date, no study has systematically compared these methods to determine if any of them are more effective than others for recovering any or all types of carotenoids present. We undertook such an investigation on plasma samples from two bird species (house finch, Carpodacus mexicanus, and mallard, Anas platyrhynchos) using five of the most commonly employed methods for extracting carotenoids from avian plasma: (1) acetone-only, (2) methanol-only, (3) ethanol-only, (4) ethanol + hexane, and (5) ethanol + tert butyl methyl ether. We also manipulated the amount of time that extracts were centrifuged, which has varied tremendously in previous studies, to evaluate its importance on carotenoid recovery. We found that all methods equally recovered the polar xanthophylls (lutein and zeaxanthin), but that the methanol-only procedure poorly recovered non-polar carotenoids (less β-carotene in both species and less β-cryptoxanthin in house finches) compared to the other methods. These results suggest that the data accumulated to date on xanthophyll plasma carotenoids in birds should be comparable across studies and species despite the different chemical extraction methods used. However, care should be taken to use relatively strong organic solvents for fully recovering non-polar carotenoids. We also found no effect of centrifugation duration (1 vs. 10 min at 10,000 rpm) on carotenoid recoveries, demonstrating that researchers can save considerable time by centrifuging for a much shorter time period than is typically used.     

How many color metrics do we need? Evaluating how different color-scoring procedures explain carotenoid pigment content in avian bare-part and plumage ornaments

For a variety of technical and conceptual reasons, biologists have come to use several different methods to quantify the colors of animals. However, the relative abilities of these different color-scoring procedures to capture variation in the actual color-generating mechanisms—pigment or structural composition of the integument—have never been tested systematically. Here, we examined which commonly employed color metrics predict carotenoid content of ornaments in three avian species (house finch Carpodacus mexicanus, mallard duck Anas platyrhynchos, and zebra finch Taeniopygia guttata). We used spectrophotometry to measure reflectance spectra from beak and feather tissue, calculated numerous color metrics (e.g., hue, chroma, brightness, principal components, and tetrahedral color space position) from these spectra, and determined carotenoid content at the site of color measurement with high-performance liquid chromatography. We found that several principal component, tristimulus, and avian visual model metrics significantly correlated with carotenoid content of house finch feathers and duck beaks. Carotenoid content of mallard beaks was most closely correlated with brightness and saturation metrics, whereas in house finch feathers, carotenoid concentration was best captured by hue and saturation metrics. According to tristimulus scores and visual models, we found that the ultraviolet portion of the spectrum was not an essential predictor of variation in carotenoid content. Also, visual model chromatic contrasts generally were not significant predictors of carotenoid content, although some achromatic contrasts and tetrahedral color space vector parameters were. Our results indicate that numerous methods, especially tristimulus scores, are suitable for capturing pigment-based color variation in two carotenoid-containing ornaments, and we discuss the merits and shortcomings of these different approaches. In contrast, there were no significant relationships between any color metrics and the carotenoid content of zebra finch beaks, suggesting that other color-generating mechanisms besides carotenoids may contribute to color variability in this species.     

Role of secondary metabolites and pigments in the epidermal tissues, ripe ovaries, viscera, gut contents and diet of the sea cucumber Holothuria atra

The epidermal tissues, ovaries, viscera, gut contents, and the diet of Holothuria atra (Jaeger), collected from the Great Barrier Reef (GBR), Australia, just prior to spawning, contained carotenoid pigments, mycosporine-like amino acids (MAAs) and three other metabolites in varying proportions. Mycosporine-glycine (mycosporine-gly), palythine, asterina-330, shinorine, mycosporine-2-gly, porphyra-334, and palythinol were present in the epidermal tissues. Mycosporine-gly was the major MAA in the epidermal tissues, gut contents and the diet, and the only MAA present in the ripe ovaries and the viscera. Gadusol, a metabolite with antioxidant properties, which co-occurs with MAAs, and in certain instances with trace amounts of 6-deoxygadusol in unfertilised and fertilised eggs and developing larvae of some marine invertebrates and vertebrates, was absent from H. atra. However, 6-deoxygadusol, with similar physical and chemical properties to gadusol, and the proposed biosynthetic precursor of MAAs, was present, but only in the ovaries. This is first report of the presence of this proposed biogenetic precursor of MAAs in significant amounts in a marine invertebrate. Approximately 90% of the total carotenoids of the epidermal tissues, ovaries and viscera of H. atra are highly oxidised, the main component being astaxanthin followed by canthaxanthin. These were the major carotenoids present in the ovaries and the total carotenoid content was highest in the ovaries. β-carotene, a common egg carotenoid, was present in trace quantities in the ovaries but, along with the xanthophylls lutein and zeaxanthin, it occurred in significant amounts in the gut contents and the diet of H. atra. Carotenoid patterns in the epidermal tissues and viscera were strikingly similar, containing β-carotene, canthaxanthin, astaxanthin, zeaxanthin, lutein, phoenicoxanthin and echinenone. The alkaloids homarine and isomeric trigonelline, (structurally unrelated to MAAs) and 6-deoxygadusol were present only in the ovaries and the viscera. It is suggested that these metabolites and pigments are either involved with photoprotection or reproduction, or associated with both processes. The origin, biogenetic relationships and the roles of these metabolites and pigments in H. atra are discussed. Received: 21 November 1997 / Accepted: 14 September 1998     

A simple and inexpensive chemical test for behavioral ecologists to determine the presence of carotenoid pigments in animal tissues

Animals use several different types of pigments to acquire their colorful ornaments. Knowing the types of pigments that generate animal colors often provides valuable information about the costs of developing bright coloration as well as the benefits of using these signals in social or sexual contexts. It is often assumed that red, orange, and yellow colors in animals are derived from carotenoid pigments, when in fact there are other pigments that confer similar colors on animals. These include the pteridine pigments in a wide range of organisms, hemoglobin in blood-filled sinuses, the psittacofulvins of parrot feathers, and the phaeomelanin pigments in rufous or yellow feathers and fur. In this paper, we describe a quick and easy, two-step chemical method for field biologists to determine if their study species uses carotenoid pigments as integumentary colorants. This laboratory procedure first employs a thermochemical extraction technique, in which acidified pyridine is used under high temperature to free carotenoid pigments from tissue to produce a colorful, pigmented solution. Red, orange, or yellow tissues containing pteridines, hemoglobin, or eumelanins do not release colored pigments into heated pyridine. However, psittacofulvins, and occasionally phaeomelanins, will also solubilize using this method. Thus, a follow-up test is needed, using solvent transfer, to confirm the presence of carotenoids in animal tissues. The use of absorbance spectrophotometry on the colorful solution may also provide information about the predominant carotenoids that bestow color on your study animal.     

Carotenoids indicate differences in diet of the hydrothermal vent crabBythograea thermydron (Brachyura)

Carotenoid pigments were used as markers to investigate the sources of energy to two deep-sea hydrothermal vent communities. Specimens of the hydrothermal-vent brachyuran crabBythograea thermydron were collected at 2 500 m depth from the Rose Garden vent site in the Galápagos Rift Valley in February, November and December 1979, and 2 600 m depth on the East Pacific Rise at 21°N in May 1982. Four carotenoids (astaxanthin, canthaxanthin, echinenone and beta-carotene) have been identified as the pigments responsible for the red color of the eggs of the crabs from the Galápagos Rift site. Consistent with the fact that animals are unable to synthesize carotenoidsde novo, precursors were not present in the crabs' tissues, affirming that these pigments are of dietary origin. The number of ovigerous female crabs and the concentrations of carotenoids in the eggs suggest a readily available source of these pigments in the Galápagos vent environment. In contrast, the developing eggs ofB. thermydron from the 21°N vent site were cream-colored, with only trace quantites of carotenoids and fewer types of carotenoids. Analysis of carotenoid distribution in both females and males in these two populations indicates a very low level of carotenoids in the diet of the 21°N vent crabs, and reflects differences in trophic interactions and primary production at the two vent sites. The few types and low concentration of carotenoids inB. thermydron indicate a diet that is different from non-vent, deep-sea crustaceans. We hypothesize that the source of carotenoids are bacteria within the vent community, and not ultimately from photosynthetic production.     

Androgen-dependent maternal effects on offspring fitness in zebra finches

There is accumulating evidence that maternal hormones may play a role in offspring sex adjustment, but little is known about the costs of such hormone-mediated mechanisms. Recent studies have reported sex-specific effects of hormones on offspring viability. Specifically, we previously found that elevating the plasma androgen level in mothers results in a male-biased offspring primary sex ratio, but it affects the viability of sons negatively and daughters positively in zebra finches (Taeniopygia guttata; Rutkowska and Cichoń, Anim Behav, 71:1283–1288, 2006). In this study, we studied further fitness consequences of exposure to elevated yolk androgen levels in zebra finches. We measured growth rate and cellular immune response of nestlings that hatched from eggs laid by females injected with testosterone during egg laying and nestlings of unaffected control females. We found that sons of testosterone-treated females grew slower in comparison to sons of control females. The significant interaction between experimental group and offspring sex indicates that sons of testosterone-treated mothers suffered impaired immune responsiveness while daughters seemed to benefit from elevated androgen level in terms of enhanced immune responsiveness. We found no effects of androgens on offspring performance at adulthood—neither fecundity of females nor attractiveness of males was affected. We conclude that the benefits of biasing sex ratio towards males by increasing androgen level in the yolk may be limited due to negative effects on male offspring performance early in life.     

Developmental stress impairs song complexity but not learning accuracy in non-domesticated zebra finches (<Emphasis Type="Italic">Taeniopygia guttata</Emphasis>)

Food restrictions early in life can have adverse effects on the development of adult avian song structure. Nutritional deficiencies during brain development are thought to impair the growth of neural circuits responsible for learning and production of song in adulthood. Thus, the quality of song may reflect the quality of the singer due to the costs associated with neural development early in life. Recent investigations have focused on domesticated laboratory strains of zebra finches where early dietary deficiencies have significantly reduced the complexity of song and its sexual attractiveness. Domesticated zebra finches may be more sensitive to the early effects of moderate under-nutrition on song complexity than their non-domesticated counterparts. In an aviary experiment with non-domesticated zebra finch stock, we found that song complexity when measured by a linear combination of six variables was reduced in food-restricted birds, with syllable rate and maximum syllable frequency as the principal variables affected. The restriction had no effect on learning accuracy when song phrases of experimental birds were compared to those of their fathers.This result demonstrates that early nutrition may differentially affect the development of neural processes that influence learning accuracy and song complexity. While the finding of negative effects of dietary restriction on song complexity is robust for zebra finches and is not an artefact of domestication, it does not explain why some nutritionally stressed populations of wild zebra finches have more complex songs than those from other regions of Australia characterised by greater food availability.     

Carotenoids in the egg yolks of collared flycatchers (<Emphasis Type="Italic">Ficedula albicollis</Emphasis>) in relation to parental quality,environmental factors and laying order

Birds may influence the fitness of their offspring by transmission of different amounts of carotenoids to their eggs. Carotenoids play crucial roles in antioxidant protection and immune defence mechanisms, but they may be available to females in limiting amounts. Therefore, their allocation to the eggs may be influenced by the female’s condition, age and environmental circumstances. Furthermore, the quality of the male parent, which affects the reproductive value of the offspring, may also influence this investment. In this correlational study, we investigated proximate and ultimate factors that may lead to variation in yolk lutein, zeaxanthin and β-carotene concentrations among and within clutches of a wild passerine, the collared flycatcher (Ficedula albicollis). We found that carotenoid concentration was positively associated with caterpillar supply at the time of egg formation, which suggests a proximate constraint of carotenoid availability on yolk composition. Neither female condition, body size, age, nor male plumage ornamentation, age and body size correlated with carotenoid deposition. Yolk β-carotene concentration was found to be positively linked to yolk testosterone concentration. We suggest that females allocated more β-carotene to their eggs to mitigate the potentially detrimental effects of elevated steroid concentration. We found that concentration of β-carotene increased with laying order. The possible function of this pattern may be to enhance the resistance to oxidative stress and pathogens of the disadvantaged last-hatching nestling, suggesting that collared flycatchers pursue a compensatory, “brood survival” strategy.     

Intraspecific variation of photosynthesis,respiration and photoprotective carotenoids in<Emphasis Type="Italic"> Gracilaria birdiae</Emphasis> (Gracilariales: Rhodophyta)

In most rhodophytes, the quenchers zeaxanthin or lutein seem to be used for carotenoid photoprotection next to β-carotene. However, our study shows that the tropical intertidal alga Gracilaria birdiae possesses high concentrations of antheraxanthin, as well as lower concentrations of both zeaxanthin and violaxanthin. In higher plants and some algal groups, these three oxygenated carotenoids are known to protect photosystem II in a xanthophyll cycle. We were able to manipulate the carotenoid concentrations in G. birdiae by varying irradiance and temperature in laboratory experiments. Higher concentrations of violaxanthin were found in dark incubations and higher concentrations of zeaxanthin in light incubations, which is an indication that a xanthophyll cycle is active in G. birdiae. Our results also suggest a biosynthesis pathway from lycopene to β,β-carotene and β-cryptoxanthin in the light and synthesis of new lycopene in darkness. This would imply that the production of functional carotenoids is regulated by their need in photoprotection. Despite cultivation in the laboratory under the same conditions for at least 6 years, there were differences in photosynthetic properties and carotenoid dynamics between algae obtained from a population close to the Equator and another population close to the Tropic of Capricorn. These differences most probably have their origin in genetic differentiation mediated by ambient irradiance and temperature regimes. Four phycoerythrin-deficient mutant strains of G. birdiae were studied as well. Photosynthetic properties and carotenoid dynamics were basically the same in the mutants and the wild strains, but two green strains had remarkably low dark respiratory rates. This is an indication of low production of metabolites, which may be a competitive disadvantage.     

Primary sex ratios in zebra finches: no evidence for adaptive manipulation in wild and semi-domesticated populations.

When breeding diet is restricted, domesticated zebra finches,Taeniopygia guttata, produce male-biased primary and secondary sex ratios, but unexpectedly produce unbiased ratios when food is unrestricted. We investigated the primary sex ratios (at laying) of wild zebra finches in southeastern Australia in response to food supplementation and environmental factors predicted to enhance female breeding condition and to bias the primary sex ratio towards daughters. Molecular sexing of all nestlings in 54 complete broods where every egg hatched, failed to show any significant biases from random. Time of egg laying (month, season) and environmental conditions (rainfall, temperature) did not significantly predict variation in the primary sex ratio, but time of breeding did affect clutch size. Wild zebra finches at our colony did not bias their sex allocation as there were no differences in the primary sex ratio and no differences in the numbers and mass of sons and daughters at the end of parental care (day 35–40 post-hatch). Biases in primary sex ratio of our wild population are probably weak or non-existent possibly due to the unpredictable environment and/or multiple contrary selective forces acting on sex ratios. We also investigated the effects of photoperiod, biases in the adult sex ratio, and parental attractiveness on primary sex ratios of semi-domesticated, laboratory zebra finches. Molecular sexing of three-day old embryos from complete clutches, failed to reveal significant biases from random. In contrast to previous studies, sex of eggs did not correlate with laying order and egg mass declined with order, rather than increased. Domestication may be responsible for these differences.     

Pigments of meso- and bathypelagic chaetognaths

Pigments of the meso- and bathypelagic chaetognaths Sagitta macrocephala and Eukrohnia fowleri were studied by chromatographic analysis. Supplementary histological studies were also performed. Fat-soluble properties and absorption spectra of the chaetognath pigments indicated that all pigments were carotenoid, independent of chaetognath species or habitat. The major carotenoid in chaetognaths was very soluble in nonpolar solvents such as carotenes, although its absorption spectrum formed a single broad peak at around 460 nm. The characteristics of the carotenoids in the chaetognaths were different from those of the pigments in the plankton which formed their diet. It is therefore inferred that carotenoids in chaetognaths are not formed by the deposition of food pigments in the intestinal tissue, but are synthesized by the chaetognaths themselves.     

Mind the gap: the ratio of yolk androgens and antioxidants varies between sons and daughters dependent on paternal attractiveness

Females are expected to partition resources between offspring in a context-dependent way to maximise total fitness returns from a reproductive attempt. Female zebra finches (Taeniopygia guttata) vary the allocation of yolk androgens and antioxidants among offspring. Importantly, the balance between androgens and antioxidants in yolks may be more important than their independent absolute amounts in terms of fitness consequences for developing young. Therefore, we tested whether the relative allocation of these two resources in yolks varies according to either the Trivers–Willard, positive or compensatory maternal investment hypothesis. We manipulated male attractiveness using coloured leg bands (red-banded males appear attractive; green-banded males, unattractive) and measured yolk androgens and antioxidants in each egg, egg sex, clutch sex ratio and female condition. While female zebra finches manipulated the balance of androgens and antioxidants within and between clutches in response to mate attractiveness, offspring sex and their own condition, they did not do so in a way that consistently followed any of the hypotheses. Mothers paired with unattractive males allocated a larger antioxidant/androgen ratio to daughters than sons. This pattern was reversed when paired to an attractive male; sons received a larger antioxidant/androgen ratio than daughters. We also found offspring sex ratio decreased with increasing female condition for unattractive males, but not for attractive males. However, without knowing the fitness consequences of the balance of different egg constituents, it is difficult to interpret the patterns consistently in terms of the Trivers–Willard, compensatory and positive investment hypotheses.     

Investigations of carotenoids in some fauna of the Adriatic Sea. III. Leander (Palaemon) serratus and Nephrops norvegicus (Crustacea: Decapoda)

Investigations have been carried out on the carotenoids in Leander (Palaemon) serratus (Pennant) and Nephrops norvegicus (L.) (Crustacea: Decapoda) from the Adriatic Sea. The presence of the carotenoids was determined by means of columnar and thin-layer chromatography. The following carotenoids were found: Leander serratus: -carotene, echinenone, isocryptoxanthin, cryptoxanthin, canthaxanthin, lutein, isozeaxanthin, zeaxanthin, astaxanthin, and astacene; Nephrops norvegicus: -carotene, lutein, zeaxanthin, astaxanthin, and astacene.     

Are yolk androgens and carotenoids in barn swallow eggs related to parental quality?

Recently, evidence is mounting that females can adaptively engineer the quality of their offspring via the deposition of yolk compounds, including carotenoids and androgens. In this study, we simultaneously consider how both carotenoids and androgens in egg yolk relate to parental quality in barn swallows (Hirundo rustica erythrogaster). First, we found no relationship between concentrations or amounts of yolk androgens and carotenoids. Yolk carotenoids decreased with laying order, whereas we found no relationship between yolk androgens and laying order. Second, we tested the Investment Hypothesis, which predicts that high-quality females or females paired to high quality mates, allocate differentially more of these yolk compounds to their offspring. For carotenoids, we mostly found evidence to counter predictions of the Investment hypothesis: (1) Carotenoid concentrations varied among females, (2) heavier eggs contained lower carotenoid concentrations, although heavier yolks contained greater amounts of carotenoids, (3) eggs of earlier-laying females had lower concentrations in their eggs, and (4) yolk carotenoids were not correlated with clutch size or male plumage ornamentation. For androgens, we found weak support for the Investment Hypothesis: (1) Yolk androgens varied among females, (2) heavier eggs and yolks contained greater amounts, although not concentrations of androgens, (3) females paired to more colorful males laid eggs with greater concentrations of androgens, and (4) no effects of laying date or morphological correlates of female quality on androgen concentrations in egg yolks. Overall, these findings suggest that each yolk compound may have different functions and therefore may be regulated by different mechanisms.     

Parental effects on carotenoid-based plumage coloration in nestling great tits, Parus major

Carotenoid pigments have attracted much interest in behavioural and evolutionary ecology because of their dual function in immune physiology and as color signals. In vertebrates, carotenoids must ultimately be obtained from the diet, and the mechanisms and magnitude of this environmental dependence are central for understanding carotenoid signal functions and evolution. In the present cross-fostering experiment with great tits Parus major, we investigate pre- and postnatal parental effects (egg yolk carotenoids, parental coloration) on nestling size and carotenoid coloration, using HPLC analysis of egg yolk carotenoids, and a reflectance-based measure of ‘chroma’ that reflects the plumage pigment concentration. Both rearing environment and origin influenced offspring size and plumage chroma. Maternal allocation of carotenoids to eggs had a weak positive effect on nestling plumage chroma, whereas we found no prenatal maternal effects (egg size or yolk carotenoid concentration) on size. Nestling plumage chroma was also significantly predicted by the chroma of the rearing father, but not by the color of the rearing mother or either of the original (genetical) parents. Thus, both prenatal maternal effects and postnatal paternal effects influence the carotenoid-based plumage coloration of nestling great tits. Future studies will reveal if parental effects have long-term consequences for plumage development and associated fitness components.     

Song amplitude and body size in birds

Bird song is a sexually selected multidimensional signal. A fundamental question regarding the evolution of sexually selected signals is what information they convey and how their honesty is maintained. Song amplitude is a performance-related signal trait that varies considerably between individuals, but this signal dimension has been neglected in past studies. I found that median song amplitude in male nightingales (Luscinia megarhynchos) and zebra finches (Taeniopygia guttata) did not vary significantly with body size or residual body mass. In contrast, I found a significant negative correlation between body size (and also residual mass) and the maximum song amplitude during interactive singing in nightingales. However, the function of these more subtle differences in song amplitude remains to be investigated. By and large, the results of this study suggest that mean song amplitude is unlikely to indicate a bird’s body size or current condition (measured as residual mass).     

Female mate choice in the zebra finch — the effect of male beak colour and male song

Studies of female mate preference in zebra finches (Taenopygia guttata) have shown that male beak colour and song rate are important. However, the two characters are correlated. Here the effect of beak colour and song rate on female choice are examined independently. In mate choice tests involving two males, beak colour was manipulated artificially using nail varnish. The results showed that females showed a significant preference for males with a high song rate, but not with a red beak. Females did not prefer males with a red beak if song rate was low and females preferred males with orange beaks who expressed a high song rate. Female preference for males with red beaks was not found when beak and song characters were no longer correlated.     

Singing activity stimulates partner reproductive investment rather than increasing paternity success in zebra finches

Song is used as a signal in sexual selection in a wide range of taxa. In birds, males of many species continue to sing after pair formation. It has been suggested that a high song output after pair formation might serve to attract extra-pair females and to minimise their own partner’s interest in extra-pair copulations. A non-exclusive alternative function that has received only scant attention is that the amount of song might stimulate the own female’s investment into eggs in a quantitative way. We address these hypotheses in a captive population of zebra finches, Taeniopygia guttata, by relating male undirected song output (i.e. non-courtship song) to male egg siring success and female reproductive investment in two different set-ups. When allowed to breed in aviaries, males with the highest song output were no more attractive than others to females in an analysis of 4,294 extra-pair courtships involving 164 different males, and they also did not sire more offspring (both trends were against the expectation). When breeding in cages with two different partners subsequently, females produced larger eggs with more orange yolks when paired to a male with a high song output. These findings suggest that singing activity in paired zebra finch males might primarily function to stimulate the partner and not to attract extra-pair females.