Michal Vinkler

Carotenoid maintenance handicap and the physiology of carotenoid-based signalisation of health

Despite a reasonable scientific interest in sexual selection, the general principles of health signalisation via ornamental traits remain still unresolved in many aspects. This is also true for the mechanism preserving honesty of carotenoid-based signals. Although it is widely accepted that this type of ornamentation reflects an allocation trade-off between the physiological utilisation of carotenoids (mainly in antioxidative processes) and their deposition in ornaments, some recent evidence suggests more complex interactions. Here, we further develop the models currently proposed to explain the honesty of carotenoid-based signalisation of heath status by adding the handicap principle concept regulated by testosterone. We propose that under certain circumstances carotenoids may be dangerous for the organism because they easily transform into toxic cleavage products. When reserves of other protective antioxidants are insufficient, physiological trade-offs may exist between maintenance of carotenoids for ornament expression and their removal from the body. Furthermore, we suggest that testosterone which enhances ornamentation by increasing carotenoid bioavailability may also promote oxidative stress and hence lower antioxidant reserves. The presence of high levels of carotenoids required for high-quality ornament expression may therefore represent a handicap and only individuals in prime health could afford to produce elaborate colourful ornaments. Although further testing is needed, this ‘carotenoid maintenance handicap’ hypothesis may offer a new insight into the physiological aspects of the relationship between carotenoid function, immunity and ornamentation.

Contrasted evolutionary histories of two Toll-like receptors (Tlr4 and Tlr7) in wild rodents (MURINAE)

BackgroundIn vertebrates, it has been repeatedly demonstrated that genes encoding proteins involved in pathogen-recognition by adaptive immunity (e.g. MHC) are subject to intensive diversifying selection. On the other hand, the role and the type of selection processes shaping the evolution of innate-immunity genes are currently far less clear. In this study we analysed the natural variation and the evolutionary processes acting on two genes involved in the innate-immunity recognition of Microbe-Associated Molecular Patterns (MAMPs).ResultsWe sequenced genes encoding Toll-like receptor 4 (Tlr4) and 7 (Tlr7), two of the key bacterial- and viral-sensing receptors of innate immunity, across 23 species within the subfamily Murinae. Although we have shown that the phylogeny of both Tlr genes is largely congruent with the phylogeny of rodents based on a comparably sized non-immune sequence dataset, we also identified several potentially important discrepancies. The sequence analyses revealed that major parts of both Tlrs are evolving under strong purifying selection, likely due to functional constraints. Yet, also several signatures of positive selection have been found in both genes, with more intense signal in the bacterial-sensing Tlr4 than in the viral-sensing Tlr7. 92% and 100% of sites evolving under positive selection in Tlr4 and Tlr7, respectively, were located in the extracellular domain. Directly in the Ligand-Binding Region (LBR) of TLR4 we identified two rapidly evolving amino acid residues and one site under positive selection, all three likely involved in species-specific recognition of lipopolysaccharide of gram-negative bacteria. In contrast, all putative sites of LBRTLR7 involved in the detection of viral nucleic acids were highly conserved across rodents. Interspecific differences in the predicted 3D-structure of the LBR of both Tlrs were not related to phylogenetic history, while analyses of protein charges clearly discriminated Rattini and Murini clades.ConclusionsIn consequence of the constraints given by the receptor protein function purifying selection has been a dominant force in evolution of Tlrs. Nevertheless, our results show that episodic diversifying parasite-mediated selection has shaped the present species-specific variability in rodent Tlrs. The intensity of diversifying selection was higher in Tlr4 than in Tlr7, presumably due to structural properties of their ligands.

Phytohaemagglutinin skin-swelling test in scarlet rosefinch males: low-quality birds respond more strongly

The phytohaemagglutinin (PHA) skin-swelling test is one of the most widely used methods for cell-mediated immunity measurement in immunoecology. Although several studies have investigated the condition-dependent traits associated with the magnitude of cutaneous inflammatory response to PHA, the results concerning signalling of the responsiveness through ornamental traits are still controversial. This is especially true for carotenoid-based feather ornamentation in birds. We therefore examined the linkage between several condition-dependent traits, including the red ornamental coloration of the plumage, and the magnitude of the PHA-induced immune response in scarlet rosefinch, Carpodacus erythrinus, males. Our results show two important aspects of the PHA-induced inflammation in this species. First, histological analysis showed that the swelling response was dependent on basophil activity. Second, the magnitude of the response (increase in patagium thickness) was associated with individual size, carotenoid-based ornamental coloration and a ptilochronological marker of feather growth at the time of moulting (mean growth bar width), thus mirroring the long-term quality of the individual. The positive linkage between the individual size or mean growth bar width and the PHA response suggests an association between the magnitude of the response and individual metabolic rate. However, as the magnitude of the response was also related negatively to ornament saturation and positively to ornament lightness, our results indicate stronger responsiveness in inferior males. Highly ornamented, healthier individuals recruited fewer basophils into the inflamed tissue causing less intense swelling. To our knowledge, this study is the first to show a negative association between carotenoid-based plumage coloration and the magnitude of the PHA-induced immune response.

Haematological health assessment in a passerine with extremely high proportion of basophils in peripheral blood

Haematological methods are widely utilised among avian ecologists as a means for individual health assessment. However, the technical simplicity of some of the tests may easily lead to oversimplification of the evaluation. Here, we show in the Scarlet Rosefinch (Carpodacus erythrinus) that haematological parameters other than the widely used heterophil/lymphocyte (H/L) ratio may be important to investigate. We give the full description of seven basic haematological traits (leukocyte differential count, immature erythrocyte count, haematocrit, mean cell volume, total red and white blood cell count and blood parasite occurrence). Most remarkably, the examination of 178 adults and 155 nestlings has revealed that this species has an extraordinarily high proportion of basophils among the peripheral blood leukocytes (on average about 42 and 56%, respectively). Although the high basophil count is a general trait even in healthy individuals of this species, the proportion of these cells is condition-dependent and is further increased by Haemoproteus infection. Our results also suggest that the immature erythrocyte count in the peripheral blood is a good predictor of the nestlings’ growth rate. We conclude that the Rosefinch haematology differs strikingly from other avian species with known values of basic haematological parameters. We therefore emphasise the importance of a general haematological examination, based on material obtained by an appropriate method (e.g. for smear preparation, we recommend using differential staining and avoiding prior methanol fixation).

Extra‐pair fertilizations contribute to selection on secondary male ornamentation in a socially monogamous passerine

Despite considerable research effort, it remains unclear whether extra‐pair fertilizations (EPF) drive the evolution of male secondary ornamentation in socially monogamous systems. In this study, we test the hypothesis that EPF contribute to the evolution or maintenance of male feather ornamentation in a sexually dichromatic passerine, the Scarlet Rosefinch, Carpodacus erythrinus. We show that the colouration of ornamental breast feathers is a good predictor of basic sources of variation in male annual reproductive output in rosefinches and that the annual realized reproductive success of males is positively associated with measures of ornamental colouration only when gains and losses because of EPF are considered. The results indicate that EPF in rosefinches may rely on absolute (good genes) rather than self referential (genetic complementarity) criteria of mate choice. Our study corroborates the potentially important role of EPF in the evolution and/or maintenance of elaborate male ornaments in socially monogamous taxa.

Opposing effects of oxidative challenge and carotenoids on antioxidant status and condition-dependent sexual signalling.

Several recent hypotheses consider oxidative stress to be a primary constraint ensuring honesty of condition-dependent carotenoid-based signalling. The key testable difference between these hypotheses is the assumed importance of carotenoids for redox homeostasis, with carotenoids being either antioxidant, pro-oxidant or unimportant. We tested the role of carotenoids in redox balance and sexual signalling by exposing adult male zebra finches (Taeniopygia guttata) to oxidative challenge (diquat dibromide) and manipulating carotenoid intake. As the current controversy over the importance of carotenoids as antioxidants could stem from the hydrophilic basis of commonly-used antioxidant assays, we used the novel measure of in vivo lipophilic antioxidant capacity. Oxidative challenge reduced beak pigmentation but elicited an increase in antioxidant capacity suggesting resource reallocation from signalling to redox homeostasis. Carotenoids counteracted the effect of oxidative challenge on lipophilic (but not hydrophilic) antioxidant capacity, thereby supporting carotenoid antioxidant function in vivo. This is inconsistent with hypotheses proposing that signalling honesty is maintained through either ROS-induced carotenoid degradation or the pro-oxidant effect of high levels of carotenoid-cleavage products acting as a physiological handicap. Our data further suggest that assessment of lipophilic antioxidant capacity is necessary to fully understand the role of redox processes in ecology and evolution.

Protein evolution of Toll-like receptors 4, 5 and 7 within Galloanserae birds

BackgroundToll-like receptors (TLR) are essential activators of the innate part of the vertebrate immune system. In this study, we analysed the interspecific variability of three TLR (bacterial-sensing TLR4 and TLR5 and viral-sensing TLR7) within the Galloanserae bird clade, investigated their phylogeny, assessed their structural conservation and estimated site-specific selection pressures.ResultsPhysiochemical properties varied according to the TLR analysed, mainly with regards to the surface electrostatic potential distribution. The predicted ligand-binding features (mainly in TLR4 and TLR5) differed between the avian proteins and their fish and mammalian counterparts, but also varied within the Galloanserae birds. We identified 20 positively selected sites in the three TLR, among which several are topologically close to ligand-binding sites reported for mammalian and fish TLR. We described 26, 28 and 25 evolutionarily non-conservative sites in TLR4, TLR5 and TLR7, respectively. Thirteen of these sites in TLR4, and ten in TLR5 were located in functionally relevant regions. The variability appears to be functionally more conserved for viral-sensing TLR7 than for the bacterial-sensing TLR. Amino-acid positions 268, 270, 343, 383, 444 and 471 in TLR4 and 180, 183, 209, 216, 264, 342 and 379 in TLR5 are key candidates for further functional research.ConclusionsHost-pathogen co-evolution has a major effect on the features of host immune receptors. Our results suggest that avian and mammalian TLR may be differentially adapted to pathogen-derived ligand recognition. We have detected signatures of positive selection even within the Galloanserae lineage. To our knowledge, this is the first study to depict evolutionary pressures on Galloanserae TLR and to estimate the validity of current knowledge on TLR function (based on mammalian and chicken models) for non-model species of this clade.

The health signalling of ornamental traits in the Grey Partridge (Perdix perdix)

AbstractBirds express various secondary ornaments that can indicate individual condition and health. Amongst these, red-coloured carotenoid-based ornaments are supposed to be particularly valuable predictors of individual quality, due to their sensitivity to oxidative stress. Nevertheless, melanin-pigmented traits might also signal health and immune functions. Both types of ornaments may be either skin-based or feather-based, each differing in their dynamics. In the present study, we compared the health- and stress-indicating capacity of melanin-based feather ornamentation and putatively carotenoid-based skin ornamentation in a single species—the Grey Partridge (Perdix perdix), a vulnerable avian species of the European agricultural landscape. In captive males, we firstly verified the carotenoid content of the red-coloured skin tissue behind the eye by chromatography (HPLC). Secondly, we assessed the individual health status of all males by examining differential leukocyte count, the frequency of immature erythrocytes, malaria prevalence and proinflammatory immune responsiveness to phytohaemagglutinin (PHA). Both the size of the melanin-based ornament and red chroma of the carotenoid-based ornament were related to the heterophil:lymphocyte (H/L) ratio. Hence, in the Grey Partridge, both redness of the skin ornament and area of the feather ornament may serve as honest indicators of individual health and long-term stress. However, the two ornamental components were unrelated to each other, and the directions of their association to the H/L ratio were opposite. We therefore propose that, in this species, larger melanin-based feather ornamentation size is linked to higher levels of stress (possibly caused by more intensive social interactions with other males), while the level of expression of the carotenoid-based skin ornamentation more reliably signals actual individual health status. Our results are potentially valuable from the perspective of Grey Partridge conservation efforts, as they indicate a simple method for assessing individual quality in this species.ZusammenfassungOrnamentmerkmale als Signale für den Gesundheitszustand beim Rebhuhn (Perdix perdix) Vögel schmücken diverse sekundäre Merkmale, die die Kondition und den Gesundheitszustand eines Individuums anzeigen können. Aufgrund ihrer Empfindlichkeit gegenüber oxidativem Stress gelten unter diesen rote, auf Karotinen basierende Schmuckmerkmale als besonders wichtig. Dennoch können auch auf Melaninpigment basierte Merkmale Gesundheitszustand und Immunfunktionen signalisieren. Beide Ornamenttypen treten sowohl in der Haut als auch in Federn auf, wo sie sich hinsichtlich der Dynamik unterscheiden. In der vorliegenden Arbeit verglichen wir die Kapazität von melaninbasierten Gefiedermerkmalen und die vermutlich karotinbasierte Hautfärbung hinsichtlich ihrer Gesundheitszustands- und Stressindikation beim Rebhuhn (Perdix perdix), einer gefährdeten Vogelart der europäischen Agrarlandschaft. Von Rebhuhnmännchen aus Gefangenschaft bestimmten wir zunächst mittels Chromatographie (HPLC) den Karotingehalt der rot gefärbten Haut hinter dem Auge. Daraufhin ermittelten wir den individuellen Gesundheitszustand aller Männchen indem wir differenzielles Blutbild, den Anteil unreifer Erythrozyten, Malaria-Prävalenz und die entzündliche Immunantwort auf Phytohämagglutinin (PHA) untersuchten. Sowohl die melaninbasierte Ornamentik, als auch die Rotfärbung der auf Karotin basierten Merkmale waren mit dem Verhältnis Heterophile zu Lymphozyten (H/L) korreliert. Damit können beim Rebhuhn beide Schmuckkomponenten, die Rotintensität der Haut, und Gefiederpartien als ehrliche Indikatoren der individuellen Gesundheit und von Langzeitstress dienen. Allerdings ließ sich kein Korrelation zwischen den beiden Komponenten feststellen, und die Zusammenhänge beider mit dem H/L Verhältnis waren gegenteilig. Wir vermuten daher, dass beim Rebhuhn größere melaninbasierte Gefiederornamente mit höheren Stresswerten in Verbindung stehen (möglicherweise verursacht durch intensivere soziale Interaktionen mit anderen Männchen), wohingegen die Stärke der karotinbasierten Hautornamentik zuverlässiger den individuellen Gesundheitsstatus signalisieren. Unsere Ergebnisse sind potentiell aus der Sicht von Rebhuhnschutzmaßnahmen von Nutzen, da sie eine einfache Methode zur Bestimmung individueller Qualität bei dieser Art aufzeigen.

The relationship between health and cell-mediated immunity measured in ecology: Phytohaemagglutinin skin-swelling test mirrors blood cellular composition

The phytohaemagglutinin (PHA) skin-swelling test, a common ecological method for measuring immune responsiveness, uses a subcutaneous injection of PHA to induce an inflammatory response with metrically measurable swelling. Both the immunological basis of this response and the role of pre-exposure physiology remain poorly understood, however, preventing predictions of costs and benefits of immunity investments revealed by the test and their relationship to life-history traits. In this study, we measure PHA-triggered tissue swelling, cellular infiltration into inflamed tissue, and haematological changes in zebra finches Taeniopygia guttata, with the aim of identifying those cell types involved in activation of PHA-induced skin swelling. We show that the inflammation underlying the swelling response is dependent on initial haematological state and alters the composition of circulating leukocytes. Injection of PHA decreased heterophil and monocyte frequency and increased basophil frequency, suggesting changes in leukocyte trafficking and haematopoiesis. Higher initial basophil and lymphocyte frequencies in peripheral blood were associated with increased swelling. High pre-treatment blood lymphocyte levels led to intense lymphocyte migration into inflamed tissue, while high initial basophil levels resulted in lower cellular infiltration. Our results show two possible directions in the association between pre-exposure health and swelling response: (i) a lymphocyte-based pathway that leads to higher responsiveness in high-quality healthy individuals capable of higher energy and resource investment, or (ii) higher responsiveness in diseased individuals with initially elevated basophil levels and pre-activated immunity. Haematological investigation, therefore, facilitates interpretation of PHA skin-swelling test results in ecological immunology, explaining ca. 20 % of variation in skin-swelling data. J. Exp. Zool. 323A: 767-777, 2015.

Contrasting patterns of polymorphism and selection in bacterial-sensing toll-like receptor 4 in two house mouse subspecies

Detailed investigation of variation in genes involved in pathogen recognition is crucial for understanding co-evolutionary processes between parasites and their hosts. Triggering immediate innate response to invading microbes, Toll-like receptors (TLRs) belong presently among the best-studied receptors of vertebrate immunity. TLRs exhibit remarkable interspecific variation and also intraspecific polymorphism is well documented. In humans and laboratory mice, several studies have recently shown that single amino acid substitution may significantly alter receptor function. Unfortunately, data concerning polymorphism in free-living species are still surprisingly scarce. In this study, we analyzed the polymorphism of Toll-like receptor 4 (Tlr4) over the Palearctic range of house mouse (Mus musculus). Our results reveal contrasting evolutionary patterns between the two recently (0.5 million years ago) diverged house mouse subspecies: M. m. domesticus (Mmd) and M. m. musculus (Mmm). Comparison with cytochrome b indicates strong directional selection in Mmd Tlr4. Throughout the whole Mmd western Palaearctic region, a single variant of the ligand-binding region is spread, encoded mainly by one dominant haplotype (71% of Mmd). In contrast, Tlr4 in Mmm is much more polymorphic with several haplotypes at intermediate frequencies. Moreover, we also found clear signals of recombination between two principal haplogroups in Mmm, and we identified eight sites under positive selection in our dataset. Our results suggest that observed differences in Tlr4 diversity may be attributed to contrasting parasite-mediated selection acting in the two subspecies.