Extraction and analysis of colourful eggshell pigments using HPLC and HPLC/electrospray ionization tandem mass spectrometry

Gorchein, A.; Lim, Chang Kee; Cassey, Phillip

doi:10.1002/bmc.1158

Cited by 97 publications

(84 citation statements)

References 12 publications

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“…(2015). Briefly, we took 0.200 g of pulverized eggshell and used the ethylenediaminetetraacetic acid (EDTA) pigment extraction protocol (Gorchein, Lim, & Cassey, 2009; Verdes et al., 2015), ultimately resulting in 1 ml of dissolved sample in acetonitrile–acetic acid (4:1 v/v). Within 24 hr of sample preparation, the supernatants were measured for their UV absorbance in a Cary 300 UV‐Vis spectrophotometer.…”

Section: Methodsmentioning

confidence: 99%

Eggshells as hosts of bacterial communities: An experimental test of the antimicrobial egg coloration hypothesis

Dearborn

Page

Dainson

et al. 2017

Ecology and Evolution

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Oviparous animals have evolved multiple defenses to prevent microbes from penetrating their eggs and causing embryo mortality. In birds, egg constituents such as lysozyme and antibodies defend against microbial infestation, but eggshell pigments might also impact survival of bacteria. If so, microbes could exert an important selective pressure on the evolution of eggshell coloration. In a previous lab experiment, eggshell protoporphyrin caused drastic mortality in cultures of Gram positive, but not Gram negative, bacteria when exposed to light. Here, we test this “photodynamic antimicrobial hypothesis” in a field experiment. In a paired experimental design, we placed sanitized brown, protoporphyrin‐rich chicken eggs alongside white eggs that lack protoporphyrin. We deployed eggs for 48 hr without incubation, as can occur between laying and incubation, when microbial infection risk is highest. Eggs were placed on the open ground exposed to sunlight and in dark underground storm‐petrel burrows. We predicted that the proportion of Gram‐positive bacteria on brown eggs should be lower when exposed to sunlight than when kept in the dark, but we expected no such difference for white eggs. Although our data revealed variation in bacterial community composition, the proportion of Gram‐positive bacteria on eggshells did not vary by egg color, and there was no interaction between egg color and location. Instead, Gram‐positive bacteria were proportionally more common on eggs on the ground than eggs in burrows. Overall, our experiment did not support the photodynamic antimicrobial hypothesis. The diverse range of avian egg colors is generated by just two pigments, but over 10 hypotheses have been proposed for the evolution of eggshell color. If our results are generalizable, eggshell protoporphyrin might not play a substantial role in defending eggs against microbes, which narrows the field of candidate hypotheses for the evolution of avian eggshell coloration.

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Section: Methodsmentioning

confidence: 99%

Eggshells as hosts of bacterial communities: An experimental test of the antimicrobial egg coloration hypothesis

Dearborn

Page

Dainson

et al. 2017

Ecology and Evolution

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“…Two relevant candidate genes occur within the oocyan haplotype: SLCO1C1 (solute carrier organic anion transporter family, member 1C1) found within the 42.4 Kb region and located 1.5 Kb downstream of GgaluGA022891 (Z¼6.09 traditional breeds only and Z¼5.84 with village chicken) and SLCO1B3 (solute carrier organic anion transporter family, member 1B3; LOC418187) located 5.5 Kb from GgaluGA022896. Analysis of eggshell pigments by mass spectrometry in several wild avian and Dongxiang chicken eggshells confirm the principal eggshell pigments to be protoporphyrin and the bile pigment biliverdin, with the accumulation of the latter being responsible for the oocyan phenotypes (Gorchein et al, 2009;Wang et al, 2009). Organic anion transporters have been shown to be involved in bile acid transport (for review see Klaassen and Aleksunes, 2010), adding credibility to these genes as possible candidates for the trait.…”

Section: Mendelian Traits In Traditional and Village Chickens D Wraggmentioning

confidence: 97%

Analysis of genome-wide structure, diversity and fine mapping of Mendelian traits in traditional and village chickens

et al. 2012

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Extensive phenotypic variation is a common feature among village chickens found throughout much of the developing world, and in traditional chicken breeds that have been artificially selected for traits such as plumage variety. We present here an assessment of traditional and village chicken populations, for fine mapping of Mendelian traits using genome-wide singlenucleotide polymorphism (SNP) genotyping while providing information on their genetic structure and diversity. Bayesian clustering analysis reveals two main genetic backgrounds in traditional breeds, Kenyan, Ethiopian and Chilean village chickens. Analysis of linkage disequilibrium (LD) reveals useful LD (r 2 X0.3) in both traditional and village chickens at pairwise marker distances of B10 Kb; while haplotype block analysis indicates a median block size of 11-12 Kb. Association mapping yielded refined mapping intervals for duplex comb (Gga 2:38.55-38.89 Mb) and rose comb (Gga 7:18.41-22.09 Mb) phenotypes in traditional breeds. Combined mapping information from traditional breeds and Chilean village chicken allows the oocyan phenotype to be fine mapped to two small regions (Gga 1:67.25-67.28 Mb, Gga 1:67.28-67.32 Mb) totalling B75 Kb. Mapping the unmapped earlobe pigmentation phenotype supports previous findings that the trait is sex-linked and polygenic. A critical assessment of the number of SNPs required to map simple traits indicate that between 90 and 110K SNPs are required for full genome-wide analysis of haplotype block structure/ancestry, and for association mapping in both traditional and village chickens. Our results demonstrate the importance and uniqueness of phenotypic diversity and genetic structure of traditional chicken breeds for fine-scale mapping of Mendelian traits in the species, with village chicken populations providing further opportunities to enhance mapping resolutions.

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“…The red, brown, and black eggshell colors are caused by protoporphyrin pigment (Kennedy and Vevers 1976, Mikšík et al 1996, Gorchein et al 2009), which is a metabolite intermediate in the biosynthesis of heme (Baird et al 1975), or may derive from the transformation of heme (Wang et al 2009). Several hypotheses have been proposed to explain the pigmentation of bird eggshells (Kilner 2006, Reynolds et al 2009, Maurer et al 2011a); these hypotheses may not be mutually exclusive and could share similar predictions.…”

Section: Introductionmentioning

confidence: 99%

Darker eggshell spotting indicates lower yolk antioxidant level and poorer female quality in the Eurasian Great Tit (Parus major)

Hargitai

Herényi

Nyíri

et al. 2016

The Auk

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Protoporphyrin pigment causes the red-brown eggshell colors; however, for many species, the function of this pigment is unknown. It has been proposed that eggshell pigmentation may strengthen the shell or that it may be a sexually selected signal, which advertises the quality of the female and that of her offspring to the male parent. In this study, we aimed to discover whether protoporphyrin-based eggshell pigmentation patterns of Eurasian Great Tits (Parus major) were related to female or egg quality. Additionally, we tested whether different methods of eggshell pigmentation estimation could be reliable predictors of eggshell protoporphyrin levels. We found that spot intensity, spot size, spotting coverage, and brown spot chroma indicated the protoporphyrin pigment concentration of the eggshell. Our results revealed that Eurasian Great Tit females that laid eggs with darker pigmentation had more lymphocytes in their circulation and had paler yellow breast and lower UV plumage reflectance, possibly indicating poorer health and individual quality. However, we did not find evidence that eggshell pigmentation patterns indicated the body condition, body size, or plasma oxidative status of females. Furthermore, we found that eggs with darker spots contained lower concentrations of antioxidants in the yolk. High protoporphyrin levels may be detrimental to females as they may cause oxidative damage, and this may be why birds that laid eggs with darker spots deposited lower amounts of antioxidants into the egg yolk. Shell spot darkness may also indicate territory quality, as females that laid smaller clutches also laid eggs with higher eggshell pigmentation levels. Thus, our results suggest that shell spot darkness may indicate the state of health of the female, egg yolk antioxidant level, and possibly also the quality of the territory. Keywords: eggshell color, female condition, lymphocyte count, plumage coloration, protoporphyrin pigmentation, oxidative status, sexual signalling hypothesis Manchas má s oscuras de la cá scara de huevo indican un nivel má s bajo de antioxidantes y una menor calidad de la hembra en Parus major RESUMEN El pigmento protoporfirina causa el color marrón rojizo de la cáscara de huevo; sin embargo, para muchas especies, la función de este pigmento es desconocida. Se ha propuesto que la pigmentación de la cáscara de huevo fortalece la cáscara o que puede ser una señal seleccionada sexualmente, la cual publicita la calidad de la hembra y la de sus polluelos al macho paterno. En este estudio, nuestro objetivo fue determinar si el patrón de pigmentación de la cáscara de huevo basado en protoporfirina de Parus major estuvo relacionado a la calidad de la hembra o del huevo. Además, evaluamos si diferentes métodos de estimación de la pigmentación de la cáscara de huevo podrían ser predictores confiables del nivel de protoporfirina en la cáscara de huevo. Encontramos que la intensidad de las manchas, el tamaño de las manchas, la cobertura de las manchas y el color marrón de las manchas indica...

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Extraction and analysis of colourful eggshell pigments using HPLC and HPLC/electrospray ionization tandem mass spectrometry

Cited by 97 publications

References 12 publications

Eggshells as hosts of bacterial communities: An experimental test of the antimicrobial egg coloration hypothesis

Eggshells as hosts of bacterial communities: An experimental test of the antimicrobial egg coloration hypothesis

Analysis of genome-wide structure, diversity and fine mapping of Mendelian traits in traditional and village chickens

Darker eggshell spotting indicates lower yolk antioxidant level and poorer female quality in the Eurasian Great Tit (Parus major)

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