Candidate genes for carotenoid coloration in vertebrates and their expression profiles in the carotenoid-containing plumage and bill of a wild bird.

Carotenoid-based coloration has attracted much attention in evolutionary biology owing to its role in honest, condition-dependent signalling. Knowledge of the genetic pathways that regulate carotenoid coloration is crucial for an understanding of any trade-offs involved. We identified genes with potential roles in carotenoid coloration in vertebrates via (i) carotenoid uptake (SR-BI, CD36), (ii) binding and deposition (StAR1, MLN64, StAR4, StAR5, APOD, PLIN, GSTA2), and (iii) breakdown (BCO2, BCMO1). We examined the expression of these candidate loci in carotenoid-coloured tissues and several control tissues of the red-billed quelea (Quelea quelea), a species that exhibits a male breeding plumage colour polymorphism and sexually dimorphic variation in bill colour. All of the candidate genes except StAR1 were expressed in both the plumage and bill of queleas, indicating a potential role in carotenoid coloration in the quelea. However, no differences in the relative expression of any of the genes were found among the quelea carotenoid phenotypes, suggesting that other genes control the polymorphic and sexually dimorphic variation in carotenoid coloration observed in this species. Our identification of a number of potential carotenoid genes in different functional categories provides a critical starting point for future work on carotenoid colour regulation in vertebrate taxa.

A novel method for screening a vertebrate transcriptome for genes involved in carotenoid binding and metabolism.

Carotenoid-based colour signals are widespread in the animal kingdom and common textbook examples of sexually selected traits. Carotenoid pigments must be obtained through the diet as all animals lack the enzymatic machinery necessary to synthesize them from scratch. Once ingested, carotenoids are metabolized, stored, transported and deposited, and some or all of these processes may be limiting for signal production and thus subjected to social or sexual selection on phenotypic coloration. Very little is known about which genes and physiological pathways are involved in carotenoid pigmentation which is unfortunate, as genetic information would allow us to investigate the biochemical consequences of sexual selection. In this study, we present a transcriptome-screening technique and apply it to a carotenoid-signalling bird species, the southern red bishop Euplectes orix, to uncover the gene(s) responsible for the conversion of dietary ß-carotene (orange) to canthaxanthin (bright red). The transcriptome, extracted from the liver of a male entering his breeding moult, is expressed within bacterial cells genetically modified to synthesize beta-carotene. Effects of expressed E. orix proteins on the structure or amount of ß-carotene are initially detected by eye (based on colour change) and subsequently confirmed by high-performance liquid chromatography. Here, we demonstrate the validity of the technique and provide a list of candidate genes involved in the carotenoid pigmentation pathway. We believe that this method could be applied to other species and tissues and that this may help researchers uncover the genetic basis of carotenoid coloration in vertebrates.

An investigation of the levels of N-nitrosodimethylamine, apparent total N-nitroso compounds and nitrate in beer.

Over 170 retail samples of beer have been analysed for N-nitrosodimethylamine (NDMA), apparent total N-nitroso compounds (ATNC) and nitrate. Levels of NDMA ranged from below 0.1 up to 1.2 micrograms/kg with a mean of 0.2 micrograms/kg. ATNC was detected in 42% of the samples in concentrations of up to 569 micrograms (N-NO)/kg. The levels of nitrate ranged from less than 0.2 up to 143 mg/kg with a mean of 16.8 mg/kg. There was no correlation between the amounts of NDMA and ATNC found in the retail beers. Samples taken during the course of fermentation showed that NDMA was unaffected by the bacterial reduction of nitrate which causes ATNC formation. HPLC studies using a photolysis/chemiluminescence detector revealed that the ATNC in beer are highly polar species of as yet unknown identity.

The determination of nitrate and nitrite in cured meat by HPLC/UV.

A rapid strong anion exchange HPLC/UV procedure has been developed for the determination of nitrate and nitrite in a wide variety of cured meats. The accuracy of this technique has been confirmed by the good agreement achieved with the existing British Standard colorimetric method. The applicability and repeatability of the procedure has been established in a survey of over 200 samples. The agreement between duplicate determinations and their respective means averaged +/- 3.4% for nitrite and +/- 4.3% for nitrate as defined by the term [(a - b)/(a + b)] X 100% where a and b are the repeat determination values.

Investigation of ethyl carbamate levels in some fermented foods and alcoholic beverages.

An analytical procedure has been developed for the determination of trace amounts of ethyl carbamate in fermented foodstuffs and alcoholic beverages. Concentrations were generally below the 1-5 micrograms/kg detection limit in bread, cheese, yoghurt, beer, gin and vodka. Higher concentrations were found in the other alcoholic beverages examined, which included whisky, fruit brandy, liqueur, wine, sherry and port.