Shifts in Bacterial Communities of Eggshells and Antimicrobial Activities in Eggs during Incubation in a Ground-Nesting Passerine

Abstract

Microbial invasion of egg contents is a cause of embryonic death. To counter infection risks, the embryo is protected physically by the eggshell and chemically by antimicrobial proteins. If microbial pressure drives embryo mortality, then females may have evolved, through natural selection, to adapt their immune investment into eggs. Although frequently hypothesized, this match between immune allocation and microorganisms has not been explored yet. To examine if correlations between microbes on eggs and immunity in eggs exist, we collected eggs from red-capped larks (Calandrella cinerea) and simultaneously examined their bacterial communities and antimicrobial components—pH, lysozyme and ovotransferrin—during natural incubation. Using molecular techniques, we find that bacterial communities are highly dynamic: bacterial abundance increases from the onset to late incubation, Shannon’s α-diversity index increases during early incubation stages, and β-diversity analysis shows that communities from 1 day-old clutches are phylogenetically more similar to each other than the older ones. Regarding the antimicrobials, we notice a decrease of pH and lysozyme concentration, while ovotransferrin concentration increases during incubation. Interestingly, we show that two eggs of the same clutch share equivalent immune protection, independent of clutch age. Lastly, our results provide limited evidence of significant correlation between antimicrobial compounds and bacterial communities. Our study examined simultaneously, for the first time in a wild bird, the dynamics of bacterial communities present on eggshells and of albumen-associated antimicrobial components during incubation and investigated their relationship. However, the link between microorganisms and immunity of eggs remains to be elucidated further. Identifying invading microbes and their roles in embryo mortality, as well as understanding the role of the eggshell microbiome, might be key to better understand avian strategies of immune maternal investment.

Microbiome assembly of avian eggshells and their potential as transgenerational carriers of maternal microbiota

“…Changes in bacterial abundances between second laid eggs (<12 h post laying) and first laid eggs (24-36 h post laying), as a means of taxon-specific growth or thriving survivors, revealed that proliferating taxa on eggshells were not indicative for maternal cloacal microbiomes but for soil communities, and occasionally for nest material, feather and skin communities. The minimal success of gut symbionts on eggshells adds to the current understanding of egg microbiome dynamics: incubation induces an increase of bacterial abundance but a decrease of diversity of eggshell communities [29,30,35,64] enhancing egg viability through reduction of the probability of trans-shell infection [25,28]. Shawkey et al [65] showed that mainly potential pathogenic bacteria were inhibited by incubation.…”

“…Without handling eggs, we marked each first laid egg with a small dot using a water-resistant marker at the blunt end to distinguish it from second eggs. We collected both eggs when the second egg had been laid (first eggs within 36 h and second eggs within 12 h post laying), minimising egg age and incubation effects [35]. We aseptically stored eggs individually in sterile plastic bags (Whirl-Pak ® , Nasco, Fort Atkinson, WI, USA), and replaced them by ethanolsterilised homemade self-hardening clay eggs to encourage clutch completion.…”

Different incubation patterns affect selective antimicrobial properties of the egg interior: experimental evidence from eggs of precocial and altricial birds

“…Among them, variation in the eggshell microstructure (D'Alba et al, 2014;Grellet-Tinner et al, 2017), eggshell pigmentation (Ishikawa et al, 2010), deposition of egg white antimicrobial proteins (AMPs) into the eggshell membranes and cuticle (Bain et al, 2013;Gautron et al, 2011;Wellman-Labadie et al, 2008a), and cuticle nanostructuring (D'Alba et al, 2016(D'Alba et al, , 2017 have been documented to significantly reduce the eggshell microbial load. Among behavioural mechanisms, partial and full incubation of a clutch have been shown to significantly reduce the abundance (Bollinger et al, 2018;Cook et al, 2005a;D'Alba et al, 2010a;Ruiz-De-Castaneda et al, 2011 but also the diversity and community structure of the eggshell microbiota (Grizard et al, 2014(Grizard et al, , 2015Lee et al, 2014). In contrast, defence mechanisms in species of the family Megapodiidae, which do not incubate their clutches, lie in the covering of the eggshells with an inorganic layer of nanometer-scale spheres of hydroxyapatite, making the eggshells super-hydrophobic and preventing attachment by bacteria (D'Alba et al, 2014).…”

“…While lysozyme has significant bactericidal activity against Gram-positive as well as Gram-negative bacteria (Ibrahim et al, 2001;Wellman-Labadie et al, 2008a), ovotransferrin is primarily bacteriostatic via its iron-binding potential, forming an iron-deficient environment, inhibiting the growth of many microorganisms (Wu and Acero-Lopez, 2012). However, the concentration and antimicrobial activity of egg white lysozyme and ovotransferrin have been documented to vary during the incubation cycle (Fang et al, 2012a, b;Qiu et al, 2012;Grizard et al, 2015;Liu et al, 2015). These changes in AMP activity are considered to be linked with alterations in egg white pH (Smolelis and Hartsell, 1952;Ibrahim et al, 1994;Halbrooks et al, 2005).…”

Experimental old nest material predicts hoopoe Upupa epops eggshell and uropygial gland microbiota

“…Parental and breeding activity, as well as nest material and ectoparasites, determine bacterial environment of nesting cavities and of eggshells (González-Braojos et al 2012, Peralta-Sánchez et al 2012, Grizard et al 2015, Tomás et al 2018. These bacteria can have positive, negative or no effects on the offspring (Singleton and Harper 1996, Moreno et al 2003, González-Braojos et al 2012, Soler et al 2017, Devaynes et al 2018.…”