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Shifts in bacterial communities of eggshells and antimicrobial activities in eggs during incubation in a ground-nesting passerine.

Grizard S, Versteegh MA, Ndithia HK, Salles JF, Tieleman BI - PLoS ONE (2015)

Bottom Line: Regarding the antimicrobials, we notice a decrease of pH and lysozyme concentration, while ovotransferrin concentration increases during incubation.Lastly, our results provide limited evidence of significant correlation between antimicrobial compounds and bacterial communities.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.

View Article: PubMed Central - PubMed

Affiliation: Animal Ecology Group, Centre for Ecological and Evolutionary Studies, University of Groningen, Groningen, The Netherlands; Department of Microbial Ecology, Centre for Ecological and Evolutionary Studies, University of Groningen, Groningen, The Netherlands.

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.

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Egg-related bacteria and antimicrobials associated with clutch age.The clutch age is the number of days that the completed clutch spent in the nest; day 1 is the day of clutch completion. Incubation starts between day 1 and day 2 (arrow). ‘r2’ corresponds to the coefficient of determination between clutch age and bacterial/antimicrobial data. (A) Bacterial abundance is examined from day 1 to day 11 (full line) and from day 2 to day 11 (dashed line). (B) Shannon‘s diversity index. (C) Relative abundance of Operational Taxonomic Units (OTUs) of three main classes of Proteobacteria: Alphaproteobacteria (Alpha-) (dotted line), Betaproteobacteria (Beta-) (dashed line) and Gammaproteobacteria (Gamma-) (full line). (D) Albumen pH. (E) Lysozyme (dashed line) and ovotransferrin (full line) concentrations.
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pone.0121716.g001: Egg-related bacteria and antimicrobials associated with clutch age.The clutch age is the number of days that the completed clutch spent in the nest; day 1 is the day of clutch completion. Incubation starts between day 1 and day 2 (arrow). ‘r2’ corresponds to the coefficient of determination between clutch age and bacterial/antimicrobial data. (A) Bacterial abundance is examined from day 1 to day 11 (full line) and from day 2 to day 11 (dashed line). (B) Shannon‘s diversity index. (C) Relative abundance of Operational Taxonomic Units (OTUs) of three main classes of Proteobacteria: Alphaproteobacteria (Alpha-) (dotted line), Betaproteobacteria (Beta-) (dashed line) and Gammaproteobacteria (Gamma-) (full line). (D) Albumen pH. (E) Lysozyme (dashed line) and ovotransferrin (full line) concentrations.

Mentions: The log copy number of the 16S rRNA gene on eggshells increased from day 1, i.e. day of clutch completion, to day 11 but this increase was not significant (r2 = 0.15, F1,16 = 2.83, P = 0.11) (Fig 1A, Table 1). In fact, eggs collected on the day of clutch completion have not been incubated yet; only eggs sampled from day 2 and onwards have been incubated. Therefore, while investigating changes in abundance from day 2 to day 11, we found that the increase in log copy number was steeper and significant (r2 = 0.37, F1,8 = 6.94, P = 0.03) (Fig 1A, Table 1). The difference between the two models was explained by a drop in abundance from day 1 to days 2–3 (t = 2.19, df = 11.3, P = 0.050).


Shifts in bacterial communities of eggshells and antimicrobial activities in eggs during incubation in a ground-nesting passerine.

Grizard S, Versteegh MA, Ndithia HK, Salles JF, Tieleman BI - PLoS ONE (2015)

Egg-related bacteria and antimicrobials associated with clutch age.The clutch age is the number of days that the completed clutch spent in the nest; day 1 is the day of clutch completion. Incubation starts between day 1 and day 2 (arrow). ‘r2’ corresponds to the coefficient of determination between clutch age and bacterial/antimicrobial data. (A) Bacterial abundance is examined from day 1 to day 11 (full line) and from day 2 to day 11 (dashed line). (B) Shannon‘s diversity index. (C) Relative abundance of Operational Taxonomic Units (OTUs) of three main classes of Proteobacteria: Alphaproteobacteria (Alpha-) (dotted line), Betaproteobacteria (Beta-) (dashed line) and Gammaproteobacteria (Gamma-) (full line). (D) Albumen pH. (E) Lysozyme (dashed line) and ovotransferrin (full line) concentrations.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4400097&req=5

pone.0121716.g001: Egg-related bacteria and antimicrobials associated with clutch age.The clutch age is the number of days that the completed clutch spent in the nest; day 1 is the day of clutch completion. Incubation starts between day 1 and day 2 (arrow). ‘r2’ corresponds to the coefficient of determination between clutch age and bacterial/antimicrobial data. (A) Bacterial abundance is examined from day 1 to day 11 (full line) and from day 2 to day 11 (dashed line). (B) Shannon‘s diversity index. (C) Relative abundance of Operational Taxonomic Units (OTUs) of three main classes of Proteobacteria: Alphaproteobacteria (Alpha-) (dotted line), Betaproteobacteria (Beta-) (dashed line) and Gammaproteobacteria (Gamma-) (full line). (D) Albumen pH. (E) Lysozyme (dashed line) and ovotransferrin (full line) concentrations.
Mentions: The log copy number of the 16S rRNA gene on eggshells increased from day 1, i.e. day of clutch completion, to day 11 but this increase was not significant (r2 = 0.15, F1,16 = 2.83, P = 0.11) (Fig 1A, Table 1). In fact, eggs collected on the day of clutch completion have not been incubated yet; only eggs sampled from day 2 and onwards have been incubated. Therefore, while investigating changes in abundance from day 2 to day 11, we found that the increase in log copy number was steeper and significant (r2 = 0.37, F1,8 = 6.94, P = 0.03) (Fig 1A, Table 1). The difference between the two models was explained by a drop in abundance from day 1 to days 2–3 (t = 2.19, df = 11.3, P = 0.050).

Bottom Line: Regarding the antimicrobials, we notice a decrease of pH and lysozyme concentration, while ovotransferrin concentration increases during incubation.Lastly, our results provide limited evidence of significant correlation between antimicrobial compounds and bacterial communities.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.

View Article: PubMed Central - PubMed

Affiliation: Animal Ecology Group, Centre for Ecological and Evolutionary Studies, University of Groningen, Groningen, The Netherlands; Department of Microbial Ecology, Centre for Ecological and Evolutionary Studies, University of Groningen, Groningen, The Netherlands.

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.

Show MeSH
Related in: MedlinePlus