Avian incubation inhibits growth and diversification of bacterial assemblages on eggs

Abstract

Microbial infection is a critical source of mortality for early life stages of oviparous vertebrates, but parental defenses against infection are less well known. Avian incubation has been hypothesized to reduce the risk of trans-shell infection by limiting microbial growth of pathogenic bacteria on eggshells, while enhancing growth of commensal or beneficial bacteria that inhibit or competitively exclude pathogens. We tested this hypothesis by comparing bacterial assemblages on naturally incubated and experimentally unincubated eggs at laying and late incubation using a universal 16S rRNA microarray containing probes for over 8000 bacterial taxa. Before treatment, bacterial assemblages on individual eggs from both treatment groups were dissimilar to one another, as measured by clustering in non-metric dimensional scaling (NMDS) ordination space. After treatment, assemblages of unincubated eggs were similar to one another, but those of incubated eggs were not. Furthermore, assemblages of unincubated eggs were characterized by high abundance of six indicator species while incubated eggs had no indicator species. Bacterial taxon richness remained static on incubated eggs, but increased significantly on unincubated eggs, especially in several families of Gram-negative bacteria. The relative abundance of individual bacterial taxa did not change on incubated eggs, but that of 82 bacterial taxa, including some known to infect the interior of eggs, increased on unincubated eggs. Thus, incubation inhibits all of the relatively few bacteria that grow on eggshells, and does not appear to promote growth of any bacteria.

Figure 1. Scatterplots showing placement within non-dimensional metric scaling ordination space of bacterial assemblages on shells of unincubated and incubated eggs at laying and after 12 days.

NMDS is a nonparametric ordination technique that maps ranked data non-linearly onto ordination space using both taxa composition and abundance . Here, the assemblage data (composition and relative abundance of taxa) were used to assign a position in ordination space to each sample. Samples with similar assemblages were positioned close to one another in ordination space, while samples with dissimilar assemblages were positioned further apart. To test whether assemblage composition changed over time on incubated or unincubated eggs, we compared positions in ordination space of samples taken before and after treatment in each experimental group. We tested for significant dissimilarity of these positions using a multi-response permutation procedure (MRPP), a nonparametric method for testing group differences that is not constrained by distributional assumptions . The MRPP provides a measure of effect size (A) from 0–1 for within-group homogeneity. Significance of A is tested using a randomization test. A and p values from multi-response permutation procedures are presented at the top of each panel.

Figure 2. Boxplots of taxa richness of Kingdom Bacteria and individual Families within Bacteria on incubated or unincubated eggs at laying and after 12 days.

Significant (p<0.05) differences are indicated with an asterisk. The line within each box represents the median richness, the lower and upper borders of each box are the 25th and 75th percentiles, and the lower and upper bars are the 10th and 90th percentiles. N = 6 in each group.

Figure 3. Boxplots of abundance of Kingdom Bacteria (measured as DNA concentration) or individual taxa within Bacteria (measured as hybridization fluorescence intensity) on incubated or unincubated eggs at laying and after 12 days.

Significant (p<0.05) differences are indicated with asterix. The line within each box represents the median richness, and the lower and upper borders of each box are the 25th and 75th percentiles are the lower and upper bars are the 10th and 90th percentiles. N = 6 in each case.