Pedro M. Araújo

Genetic Basis for Red Coloration in Birds

The yellow and red feather pigmentation of many bird species [1] plays pivotal roles in social signaling and mate choice [2, 3]. To produce red pigments, birds ingest yellow carotenoids and endogenously convert them into red ketocarotenoids via an oxidation reaction catalyzed by a previously unknown ketolase [4-6]. We investigated the genetic basis for red coloration in birds using whole-genome sequencing of red siskins (Spinus cucullata), common canaries (Serinus canaria), and red factor canaries, which are the hybrid product of crossing red siskins with common canaries [7]. We identified two genomic regions introgressed from red siskins into red factor canaries that are required for red coloration. One of these regions contains a gene encoding a cytochrome P450 enzyme, CYP2J19. Transcriptome analysis demonstrates that CYP2J19 is significantly upregulated in the skin and liver of red factor canaries, strongly implicating CYP2J19 as the ketolase that mediates red coloration in birds. Interestingly, a second introgressed region required for red feathers resides within the epidermal differentiation complex, a cluster of genes involved in development of the integument. Lastly, we present evidence that CYP2J19 is involved in ketocarotenoid formation in the retina. The discovery of the carotenoid ketolase has important implications for understanding sensory function and signaling mediated by carotenoid pigmentation.

Health-State Variables and Enzymatic Biomarkers as Survival Predictors in Nestling Great Tits (Parus Major): Effects of Environmental Conditions

Abstract The health state of nestlings can be a useful bioindicator of the quality of the environment in which they are reared, but, to enable detection of responses to environmental change, the variation of health parameters under natural conditions should be evaluated. We describe the variation of morphological, biochemical, and hematological variables in relation to time of sampling, hatching date, brood size and type, and year in nestlings of two populations of Great Tits (Parus major) in Choupal, Portugal, and Wytham, United Kingdom. The influence of these health variables on nestlings survival to first winter and recruitment into the breeding population was assessed in Wytham. Variation in plasma protein, total plasma cholinesterase (ChE), and acetylcholinesterase activities reflected circadian rhythms. Hatching date affected total plasma ChE and butyrylcholinesterase (BuChE) activities, and levels of red-blood-cell hemoglobin (Hb) and hematocrit (HCT). In Choupal, HCT increased with brood size. Nestlings in Choupal had significantly lower protein and Hb levels, and higher glutathione peroxidase (GSH-Px) activity, during a drier year. Second-brood nestlings had significantly lower levels of Hb and HCT. Of the studied variables, only plasma BuChE and red-blood-cell GSH-Px activities were related to nestlings survival to first winter and recruitment.

High-density lipoprotein receptor SCARB1 is required for carotenoid coloration in birds

Significance The yellow, orange, and red colors of birds are produced through the deposition of carotenoid pigments into feathers and skin, and often function as signals in aggressive interactions and mate choice. These colors are hypothesized to communicate information about individual quality because their expression is linked to vital cellular processes through the mechanisms of carotenoid metabolism. To elucidate these mechanisms, we carried out genomic and biochemical analyses of the white recessive canary breed, which carries a heritable defect in carotenoid uptake. We identified a mutation in the SCARB1 gene in this breed that disrupts carotenoid transport function. Our study implicates SCARB1 as a key mediator of carotenoid-based coloration and suggests a link between carotenoid coloration and lipid metabolism. Yellow, orange, and red coloration is a fundamental aspect of avian diversity and serves as an important signal in mate choice and aggressive interactions. This coloration is often produced through the deposition of diet-derived carotenoid pigments, yet the mechanisms of carotenoid uptake and transport are not well-understood. The white recessive breed of the common canary (Serinus canaria), which carries an autosomal recessive mutation that renders its plumage pure white, provides a unique opportunity to investigate mechanisms of carotenoid coloration. We carried out detailed genomic and biochemical analyses comparing the white recessive with yellow and red breeds of canaries. Biochemical analysis revealed that carotenoids are absent or at very low concentrations in feathers and several tissues of white recessive canaries, consistent with a genetic defect in carotenoid uptake. Using a combination of genetic mapping approaches, we show that the white recessive allele is due to a splice donor site mutation in the scavenger receptor B1 (SCARB1; also known as SR-B1) gene. This mutation results in abnormal splicing, with the most abundant transcript lacking exon 4. Through functional assays, we further demonstrate that wild-type SCARB1 promotes cellular uptake of carotenoids but that this function is lost in the predominant mutant isoform in white recessive canaries. Our results indicate that SCARB1 is an essential mediator of the expression of carotenoid-based coloration in birds, and suggest a potential link between visual displays and lipid metabolism.

Patterns of tick infestation and their Borrelia burgdorferi s.l. infection in wild birds in Portugal.

Wild birds may act as reservoirs for zoonotic pathogens and may be mechanical carriers of pathogen infected vector ticks through long distances during migration. The aim of this study was to assess tick infestation patterns in birds in Portugal and the prevalence of tick infection by Borrelia burgdorferi s.l. using PCR techniques. Seven tick species were collected from birds including Haemaphysalis punctata, Hyalomma spp., Ixodes acuminatus, Ixodes arboricola, Ixodes frontalis, Ixodes ricinus and Ixodes ventalloi. We found that I. frontalis and Hyalomma spp. were the most common ticks infesting birds of several species and that they were widespread in Portugal. Turdus merula was the bird species that presented the highest diversity of infesting ticks and had one of the highest infestation intensities. B. burgdorferi s.l. was detected in 7.3% (37/505) of Ixodidae ticks derived from birds. The most common genospecies was Borrelia turdi (6.9%), detected in ticks collected from Parus major, T. merula and Turdus philomelos, but Borrelia valaisiana (0.2%) and one Borrelia sp. (0.2%) similar to Borrelia bissettii (96% of similarity of the flaB gene in Blastn) were also detected. This study contributed to a better knowledge of the Ixodidae tick fauna parasitizing birds in Western Europe and to the assessment of the prevalence of B. burgdorferi s.l. associated with birds and their ticks.

Characterization Through Multilocus Sequence Analysis of Borrelia turdi Isolates from Portugal

Borrelia turdi is a spirochete from the Borrelia burgdorferi complex, first reported in Japan, that has been increasingly detected in Europe. This genospecies is mostly associated with avian hosts and their ornithophilic ticks such as Ixodes frontalis. In this study, we isolated B. turdi from five I. frontalis feeding on Turdus merula, Turdus philomelos, Parus major and Troglodytes troglodytes, and one Ixodes ricinus feeding on a T. merula in Portugal. These isolates were genetically characterised according to their 5S-23S rRNA intergenic spacer, 16S rRNA and through typing of seven housekeeping genes (multilocus sequence typing). Multilocus sequence analyses revealed that the strains isolated in our study, although belonging to B. turdi genospecies, are not identical to the B. turdi reference strain Ya501. Instead, our strains are separated into a clear defined group, suggesting that the European samples diverged genetically from the strain originally detected in Japan. Population analysis of 5S-23S rRNA sequences can further resolve subpopulations within B. turdi, but more samples from a large geographical scale and host range would be needed to assess potential phylogeographical patterns within this genospecies.

Reed warblers migrating through Portugal: climatic influence on stopover ecology over the last decade.

The arrival and breeding dates of small migrant birds have advanced throughout Europe. This study evaluates the hypothesis of a faster migration along the migratory route, which should lead to a decrease in stopover duration in staging areas over the last decades. Several climatic predictors were analysed as proxies to understand the stopover ecology of reed warbler Acrocephalus scirpaceus migrating through Central Portugal. The minimum stopover duration of migratory reed warblers decreased significantly over the last decade during both the spring and autumn migrations. Warmer conditions en route should increase food availability, increasing the body condition of departing birds and the quality of departing sites en route to Portugal, such that migrants will reduce the stopover duration at Portuguese reedbeds.

Experimental infection by microparasites affects the oxidative balance in their avian reservoir host the blackbird Turdus merula

By draining resources, microparasites can negatively affect the host fitness, which in turn can result in reduced transmission when virulence leads to reductions in host population size. Therefore, for a microparasite to persist in nature, the level of harm it can do to its host is expected to be limited. We tested this hypothesis for tick-borne Borrelia burgdorferi sensu lato (s.l.) infections in the blackbird Turdus merula, one of the most important avian reservoir hosts in Europe. Experimental and observational data were combined to examine the physiological effects caused by B. burgdorferi s.l. infection in blackbirds. Pathogen-free blackbirds were exposed to B. burgdorferi s.l.-infected Ixodes ricinus and I. frontalis nymphs, and compared with a control group (exposed to naïve laboratory-derived I. ricinus nymphs). Their physiological status was evaluated before and after infection with B. burgdorferi s.l., through a set of immunological (erythrocyte sedimentation rate, haptoglobin, white blood cell count and heterophil/lymphocyte ratio), oxidative stress (glutathione peroxidase activity, protein carbonyls and nitric oxide) and general body condition variables (body condition, glucose and haematocrit). Infected males showed higher levels of oxidative damage to proteins (increased levels of protein carbonyls), decreased glutathione peroxidase activity and increased body mass. Infected females had higher levels of glutathione peroxidase activity after infection by B. burgdorferi s.l. than the control group. No significant effects of B. burgdorferi s.l. infection were detected on erythrocyte sedimentation rate, haptoglobin, heterophil/lymphocyte ratio, nitric oxide, glucose and haematocrit. The first experimental study on the effects of B. burgdorferi s.l. on its avian reservoir hosts shows that these bacteria may inflict non-negligible physiological costs. We speculate that during energetically demanding periods, these physiological costs may reduce host fitness and affect pathogen transmission.

A non-coding region near Follistatin controls head colour polymorphism in the Gouldian finch

Discrete colour morphs coexisting within a single population are common in nature. In a broad range of organisms, sympatric colour morphs often display major differences in other traits, including morphology, physiology or behaviour. Despite the repeated occurrence of this phenomenon, our understanding of the genetics that underlie multi-trait differences and the factors that promote the long-term maintenance of phenotypic variability within a freely interbreeding population are incomplete. Here, we investigated the genetic basis of red and black head colour in the Gouldian finch (Erythrura gouldiae), a classic polymorphic system in which naturally occurring colour morphs also display differences in aggressivity and reproductive success. We show that the candidate locus is a small (approx. 70 kb) non-coding region mapping to the Z chromosome near the Follistatin (FST) gene. Unlike recent findings in other systems where phenotypic morphs are explained by large inversions containing hundreds of genes (so-called supergenes), we did not identify any structural rearrangements between the two haplotypes using linked-read sequencing technology. Nucleotide divergence between the red and black alleles was high when compared to the remainder of the Z chromosome, consistent with their maintenance as balanced polymorphisms over several million years. Our results illustrate how pleiotropic phenotypes can arise from simple genetic variation, probably regulatory in nature.

Metabolic plasticity for subcutaneous fat accumulation in a long distance migratory bird traced by 2H2O

ABSTRACT The migrant black-tailed godwit (Limosa limosa) traditionally used natural wetlands in the Iberian Peninsula to prepare for migratory flights by feeding mainly in estuaries. In recent decades, this species has become increasingly dependent on rice fields, thereby relying on a plant-based diet for fuelling. Dietary fatty acids (FA) seem to be determinant to the composition of accumulated subcutaneous fat in migratory birds. It is still unclear whether metabolic plasticity allows for modification and/or synthesis of FA, contributing to a lipid profile that enables a successful migratory performance. Deuterated water was administered to captive black-tailed godwits submitted to two diets (fly larvae versus rice) and the incorporation of deuterium (2H) into subcutaneous triglycerides was analyzed by NMR. A recently developed localized biopsy method for sampling subcutaneous fat was employed with later successful release of all birds into the wild. The average chemical structure reflected mostly a mixture of saturated and monounsaturated 16- and 18-carbon FA, a profile frequently found in migrant birds. Significantly higher levels of polyunsaturated FA, as well as detectable levels of n-3 FA, were observed in fly-larvae-fed birds. Excess 2H-enrichments in FA revealed significantly higher rates of fractional de novo lipogenesis and FA desaturation capacity in rice-fed birds. This novel and non-lethal tracer method revealed the capacity of this species to alter its lipid metabolism to compensate for a poorer dietary lipid contribution. Because of its versatility, adapting this method to other scenarios and/or other migratory species is considered feasible and cost-effective. Summary: A novel and non-lethal tracer method using deuterated water revealed alteration in lipid metabolism of migrant black-tailed godwits subjected to different diets.

The Importance of Reedbeds and Riparian Areas for Cetti’s Warbler Cettia cetti throughout its Annual Cycle

This study describes the importance of reedbed and riparian habitats for the annual cycle of Cetti’s warbler Cettia cetti. We examined the seasonal variation in population structure and body condition using a long-term data set (11xa0years) from bird ringing. Because seasonal variations in the differential use of each wetland type should be influenced by food resources and shelter conditions for roosting, a short term study (1xa0year) was performed to assess the diet, trophic niche and health condition of Cetti’s warbler, and to examine differences in the abundance of food resources and temperature between the two habitats. Results revealed that reedbeds are very important for Cetti’s warbler, supporting large numbers of (mainly) juvenile females during the late summer and autumn periods. Fecal analysis showed that during late summer predator insects (mostly Coleoptera) were more important for Cetti’s warblers on the reedbed, while Phytophagous insects (Hemiptera) were more important for Cetti’s warblers on the riparian habitat. Stable isotope mixing models from blood and feathers samples corroborated these results and revealed the opportunistic foraging behaviour of Cetti’s warbler. The variation on the δ13C values of juvenile secondary feathers was higher for the reedbed, indicating a wider provenance for birds that move into the reedbed in late summer/early autumn. The δ13C values for the riparian area had a smaller variation suggesting that these birds were less dispersive. Overall, the higher abundance of higher trophic level food resources, and higher minimum temperatures for roosting on the reedbed, makes this habitat particularly attractive for Cetti’s warbler after the breeding season.