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. 2020 Jun 11;11:60. doi: 10.1186/s40104-020-00459-w

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© The Author(s) 2020

Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

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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