Data from: Eggshells as hosts of bacterial communities: an experimental test of the antimicrobial egg coloration hypothesis

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 hrs 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 colour, and there was no interaction between egg colour 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 colours is generated by just two pigments, but over 10 hypotheses have been proposed for the evolution of eggshell colour. 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.卵生动物（Oviparous animals）演化出了多重防御机制，以阻止微生物侵入卵内并造成胚胎死亡。在鸟类中，溶菌酶（lysozyme）与抗体等卵内成分可抵御微生物侵染，但卵壳色素或许也会影响细菌的存活情况。若果真如此，微生物便可能对卵壳色彩的演化施加重要的选择压力。 此前一项实验室研究发现，卵壳原卟啉（protoporphyrin）在光照条件下，可使革兰氏阳性（Gram positive）细菌的培养物出现大幅死亡，但对革兰氏阴性（Gram negative）细菌并无此效果。本文中，我们通过野外实验检验这一“光动力抗菌假说（photodynamic antimicrobial hypothesis）”。我们采用配对实验设计，将经过消毒的富含原卟啉的棕色家鸡卵，与缺乏原卟啉的白色家鸡卵一同放置。我们将卵放置48小时且不进行孵化，以模拟产卵至孵化前这一微生物感染风险最高的时段。实验卵被放置于暴露在阳光下的开阔地面，以及黑暗的地下风暴海燕（storm-petrel）巢穴中。 我们预测，在光照条件下，棕色卵表面的革兰氏阳性细菌占比应低于黑暗环境中的样本，但白色卵不会出现此类差异。 尽管我们的数据显示细菌群落组成存在差异，但卵壳表面的革兰氏阳性细菌占比并未随卵色变化，且卵色与放置位置之间也不存在交互效应。相反，地面放置的卵表面的革兰氏阳性细菌占比，高于地下巢穴中的卵。总体而言，本实验并未支持光动力抗菌假说。 鸟类多样的卵色仅由两种色素生成，但学界已提出超10种关于卵壳色彩演化的假说。若我们的研究结果具备普适性，那么卵壳原卟啉或许并未在抵御微生物的卵防御中发挥实质性作用，这一结论将缩小鸟类卵壳色彩演化候选假说的研究范围。