Fig. 2

Description of the eggshell-associated microbial communities using culture-based and sequencing-based techniques. a Boxplot tracking pre-hatch changes in the abundance (CFU/eggshell) of total recoverable anaerobically growing bacteria (red) and recoverable suspected sporulating anaerobic bacteria (blue) throughout incubation. Whiskers represent the maximum and minimum values. The interquartile range is indicated by the upper and lower boundaries of the boxes. Significance between the total recoverable anaerobic bacteria counts and the suspected sporulating bacteria counts and between weeks was determined by pairwise T-test. An asterisk (*) indicates a significant difference in bacterial counts between both the total viable anaerobe and recoverable sporulating populations within a timepoint and/or between that time point and other time points (P < 0.05). b Boxplot comparing abundance (CFU/eggshell) of total recoverable anaerobically growing bacteria (red) and recoverable suspected sporulating anaerobic bacteria (blue) at day 21 both pre-hatch and post-hatch. Significance between the total recoverable anaerobic bacteria counts and the suspected sporulating bacteria counts and between pre- and post-hatch was determined by pairwise T-test. An asterisk (*) indicates a significant difference in bacterial counts between both the total viable anaerobe and recoverable sporulating populations within a time point and between that time point and other time points for that bacterial population (P < 0.05). c Changes in eggshell microbiota alpha-diversity (Shannon index) throughout the incubation period for both trials. Week 3 post-hatch community analysis was not conducted for T1 eggs. Comparisons between Shannon indices between weeks and trials were made using an analysis of variance (ANOVA) followed by subsequent pairwise comparisons with the Tukey’s “Honest Significant Difference” method. An asterisk (*) indicates a significant difference in Shannon index between that time point and other time points within the same trial (P < 0.05). d Stacked barcharts comparing the weekly relative abundance of bacterial families representing > 0.01 (1%) of the eggshell bacterial community at any time point within either T1 or T2