Lucy M. I. Webster

Physiological stress links parasites to carotenoid-based colour signals

Vertebrates commonly use carotenoid‐based traits as social signals. These can reliably advertise current nutritional status and health because carotenoids must be acquired through the diet and their allocation to ornaments is traded‐off against other self‐maintenance needs. We propose that the coloration more generally reveals an individual’s ability to cope with stressful conditions. We tested this idea by manipulating the nematode parasite infection in free‐living red grouse (Lagopus lagopus scoticus) and examining the effects on body mass, carotenoid‐based coloration of a main social signal and the amount of corticosterone deposited in feathers grown during the experiment. We show that parasites increase stress and reduce carotenoid‐based coloration, and that the impact of parasites on coloration was associated with changes in corticosterone, more than changes in body mass. Carotenoid‐based coloration appears linked to physiological stress and could therefore reveal an individual’s ability to cope with stressors.

Oxidative stress and the effect of parasites on a carotenoid-based ornament.

SUMMARY Oxidative stress, the physiological condition whereby the production of reactive oxygen and nitrogen species overwhelms the capacity of antioxidant defences, causes damage to key bio-molecules. It has been implicated in many diseases, and is proposed as a reliable currency in the trade-off between individual health and ornamentation. Whether oxidative stress mediates the expression of carotenoid-based signals, which are among the commonest signals of many birds, fish and reptiles, remains controversial. In the present study, we explored interactions between parasites, oxidative stress and the carotenoid-based ornamentation of red grouse Lagopus lagopus scoticus. We tested whether removing nematode parasites influenced both oxidative balance (levels of oxidative damage and circulating antioxidant defences) and carotenoid-based ornamentation. At the treatment group level, parasite purging enhanced the size and colouration of ornaments but did not significantly affect circulating carotenoids, antioxidant defences or oxidative damage. However, relative changes in these traits among individuals indicated that males with a greater number of parasites prior to treatment (parasite purging) showed a greater increase in the levels of circulating carotenoids and antioxidants, and a greater decrease in oxidative damage, than those with initially fewer parasites. At the individual level, a greater increase in carotenoid pigmentation was associated with a greater reduction in oxidative damage. Therefore, an individuals ability to express a carotenoid-based ornament appeared to be linked to its current oxidative balance and susceptibility to oxidative stress. Our experimental results suggest that oxidative stress can mediate the impact of parasites on carotenoid-based signals, and we discuss possible mechanisms linking carotenoid-based ornaments to oxidative stress.

Testing the interactive effects of testosterone and parasites on carotenoid-based ornamentation in a wild bird

Abstract Testosterone underlies the expression of most secondary sexual traits, playing a key role in sexual selection. However, high levels might be associated with physiological costs, such as immunosuppression. Immunostimulant carotenoids underpin the expression of many red‐yellow ornaments, but are regulated by testosterone and constrained by parasites. We manipulated testosterone and nematode burdens in red grouse (Lagopus lagopus scoticus) in two populations to tease apart their effects on carotenoid levels, ornament size and colouration in three time‐step periods. We found no evidence for interactive effects of testosterone and parasites on ornament size and colouration. We showed that ornament colouration was testosterone‐driven. However, parasites decreased comb size with a time delay and testosterone increased carotenoid levels in one of the populations. This suggests that environmental context plays a key role in determining how individuals resolve the trade‐off between allocating carotenoids for ornamental coloration or for self‐maintenance needs. Our study advocates that adequately testing the mechanisms behind the production or maintenance of secondary sexual characters has to take into account the dynamics of sexual trait expression and their environmental context.

Characterising functionally important and ecologically meaningful genetic diversity using a candidate gene approach.

Over the past two decades the fields of molecular ecology and population genetics have been dominated by the use of putatively neutral DNA markers, primarily to resolve spatio-temporal patterns of genetic variation to inform our understanding of population structure, gene flow and pedigree. Recent emphasis in comparative functional genomics, however, has fuelled a resurgence of interest in functionally important genetic variation that underpins phenotypic traits of adaptive or ecological significance. It may prove a major challenge to transfer genomics information from classical model species to examine functional diversity in non-model species in natural populations, but already multiple gene-targeted candidate loci with major effect on phenotype and fitness have been identified. Here we briefly describe some of the research strategies used for isolating and characterising functional genetic diversity at candidate gene-targeted loci, and illustrate the efficacy of some of these approaches using our own studies on red grouse (Lagopus lagopus scoticus). We then review how candidate gene markers have been used to: (1) quantify genetic diversity among populations to identify those depauperate in genetic diversity and requiring specific management action; (2) identify the strength and mode of selection operating on individuals within natural populations; and (3) understand direct mechanistic links between allelic variation at single genes and variance in individual fitness.

Transcriptomic response of red grouse to gastro-intestinal nematode parasites and testosterone: implications for population dynamics

A central issue in ecology is in understanding the relative influences of intrinsic and extrinsic effects on population regulation. Previous studies on the cyclic population dynamics of red grouse (Lagopus lagopus scoticus) have emphasized the destabilizing effects of either nematode parasites or territorial behaviour and aggression. The potential interacting effects of these processes, mediated through density‐dependent, environmentally induced alterations of host immunocompetence influencing susceptibility to parasites have not been considered. Male red grouse at high density are more aggressive, associated with increased testosterone, which potentially could lead to reduced immunocompetence at a stage when parasites are most prevalent. This could depress individual condition, breeding performance and survival and thus drive or contribute to overall reductions in population size. Here, we characterize the transcriptomic response of grouse to nematode parasite infection and investigate how this is subsequently affected by testosterone, using a microarray approach contrasting red grouse with high and low parasite load at both high and low testosterone titre. A suite of 52 transcripts showed a significant level of up‐regulation to either chronic parasite load or experimental parasite infection. Of these, 51 (98%) showed a reduced level of expression under conditions of high parasite load and high testosterone. The genes up‐regulated by parasites and then down‐regulated at high testosterone titre were not necessarily associated with immune response, as might be intuitively expected. The results are discussed in relation to the fitness and condition of individual red grouse and factors influencing the regulation of abundance in natural populations.

Identification of genes responding to nematode infection in red grouse.

The identification of genes involved in a host’s response to parasite infection provides both a means for understanding the pathways involved in immune defence and a target for examining host–parasite co‐evolution. Most studies rely on a candidate gene approach derived from model systems to identify gene targets of interest, and there have been a dearth of studies geared towards providing a holistic overview of immune response from natural populations. We carried out an experiment in a natural population of red grouse (Lagopus lagopus scoticus) to manipulate levels of Trichostrongylus tenuis parasite infection. The transcriptomic response of individuals was examined from standard cDNA and suppressive subtractive hybridization (SSH) libraries produced from gut, liver and spleen, enriching for genes expressed in response to T. tenuis infection. A total of 2209 and 3716 unique transcript sequences were identified from the cDNA and SSH libraries, respectively. Forty‐five of these had Gene Ontology annotation associated with immune response. Some of these genes have previously been reported from laboratory‐based studies of model species as important in immune response to gastrointestinal parasite infection; however, multiple novel genes were also identified. These may reveal novel pathways involved in the host response of grouse to T. tenuis and provide a resource that can be utilized as candidate genes in other species.

A transcriptomic investigation of handicap models in sexual selection

Handicap models link the evolution of secondary sexual ornaments to physiological costs and thus provide a mechanistic explanation for signal honesty in sexual selection. Two commonly invoked models, the immunocompetence handicap hypothesis (ICHH) and the oxidative stress handicap hypothesis (OSHH), propose suppression of immunocompetence or increase of oxidative stress by testosterone, but empirical evidence for both models is controversial and based on morphological and physiological assays. Here, we investigated these two models on the gene transcription level using microarrays to quantify the transcriptomic response of red grouse (Lagopus lagopus scoticus) caecal, spleen and liver tissues to experimental manipulation of testosterone levels. We used a geneontology framework to identify genes related to immune function and response to reactive oxygen species and examined how transcription levels changed under experimentally increased testosterone levels in birds with parasites present or absent. Contrary to our expectations, testosterone had virtually no effect on gene transcription in spleen and liver. A small number of genes were significantly differentially regulated in caecum, and while their functions and transcription changes are consistent with the ICHH, we found little support for the OSHH. More genes responded to testosterone in the presence rather than absence of parasites, suggesting that handicap mechanisms may be context dependent and more pronounced in the presence of adverse environmental conditions. These findings illustrate the utility of transcriptomics to investigating handicap models, suggest that classic models may not underlie the handicap mechanism, and indicate that novel emerging models involving different mediators and physiological systems should be examined.

Erratum to: Pronounced genetic structure and low genetic diversity in European red-billed chough (Pyrrhocorax pyrrhocorax) populations

In the original publication, Tables 3 and 6 were published with incorrect estimates of population heterozygosities. All other diversity statistics were correct as originally presented. Updated versions of Tables 3 and 6 with corrected heterozygosity estimates confirmed using Arlequin 3.5 (Excoffier and Lischer 2010) as in Davila et al. (2014) are provided in this erratum. Discrepancies were minor for populations on the British Isles. The correct estimates for Spain are slightly larger than those reported for La Palma by Davila et al. (2014), but this does not necessarily affect their interpretation that choughs on La Palma may have originated from multiple migration events. The original conclusion that chough populations on the British Isles have low genetic diversity compared to continental European populations remains and is now, in fact, strengthened.

Oxidative stress and the effect of parasites on a carotenoid-based ornament. Corrigendum.

It has been drawn to our attention that Fig. 1a,c,d in Mougeot et al. (2010a) appears to show the same data as Fig. 1a,d,e in a paper published by (some of) the same authors in Journal of Evolutionary Biology (Mougeot et al., 2010b). The authors wish to clarify the relationship between these two papers and rectify the lack of crossreferencing. Both papers focus on red grouse and are outputs from the same large-scale field manipulation undertaken in 2006. They represent two, very different, stand-alone studies addressing different conceptual issues. The central experimental manipulation of both papers is the drug treatment of birds. The authors felt it was necessary to include background data in each paper to give the reader confidence in the efficacy of the experimental treatment and to allow the reader to better understand and interpret the main results of the papers, which explore very different physiological mechanisms that influence ornamental colouration. For this reason, the treatment effects on parasite burden and comb colouration were presented in both papers, although a different index of comb redness was used [adjusted R-values (Fig. 1e in J. Evol. Biol.) vs. adjusted R ⁄(G + B) values (Fig. 1c in J. Exp. Biol.)]. Both indices are accurate reflections of comb redness, gave similar results and have been used in previous studies in different contexts to characterize comb colour. Both papers also include the same data on levels of circulating carotenoids [Fig. 1d in J. Exp. Biol.; Fig. 1d in J. Evol. Biol.]. The authors feel the inclusion of these data in both papers can be justified conceptually. In the J. Exp. Biol. paper, the trade-off between the use of carotenoids for colouring ornaments vs. dealing with oxidative stress is central to the key question. In the J. Evol. Biol. paper, these data reinforce the argument that carotenoid levels and ornamental colouration covary with corticosterone levels. The reiteration of background data on parasite removal and carotenoid levels does not compromise the uniqueness of each paper. The papers were submitted to, and published by, the respective journals in parallel, and the absence of cross-referencing was an oversight on the part of the authors, who acknowledge that the figure legends should have cited the corresponding paper and that the different indices used for comb redness should have been clarified. The paper by Mougeot et al. (2010a) should have been cited and referenced in the Journal of Evolutionary Biology article (Mougeot et al., 2010b), and we apologise for any inconvenience caused.