Avian Incubation Inhibits Growth and Diversification of Bacterial Assemblages on Eggs Matthew D. Shawkey1��, Mary K. Firestone1,2, Eoin L. Brodie2, Steven R. Beissinger1* 1 Department of Environmental Science, Policy and Management, Ecosystem Science Division, University of California, Berkeley, California, United States of America, 2 Ecology Department, Earth Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, United States of America 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. Citation: Shawkey MD, Firestone MK, Brodie EL, Beissinger SR (2009) Avian Incubation Inhibits Growth and Diversification of Bacterial Assemblages on Eggs. PLoS ONE 4(2): e4522. doi:10.1371/journal.pone.0004522 Editor: Ryan L. Earley, University of Alabama, United States of America Received October 8, 2008 Accepted January 2, 2009 Published February 19, 2009 Copyright: �� 2009 Shawkey et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by NSF grant IOB-0517549 to S. R. Beissinger and M. K. Firestone. Additional work was performed under the auspices of the U.S. Department of Energy by the University of California, Lawrence Berkeley National Laboratory, under Contract DE-AC02- 05CH11231. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: beis@nature.berkeley.edu �� Current address: Department of Biology and Integrated Bioscience Program, University of Akron, Akron, Ohio, United States of America Introduction Microbial infection is a primary source of mortality for early life stages of oviparous vertebrates [1], and this selection pressure has driven the evolution of a suite of morphological and behavioral defenses in parents. Eggs themselves can be viewed as matrices of defense against microbial infection. The tough outer layer provides physical defense and the inner contents, such as albumen in birds [2,3] and egg jelly in frogs [4], provide chemical defense via antimicrobial proteins. Antimicrobial peptides have been identi- fied in eggs of a wide variety of animal taxa [2,4,5]. A complementary parental strategy to reduce the risk of infection is to inhibit growth of pathogenic bacteria on the outer surface of the egg, while enhancing growth of commensal or beneficial bacteria that inhibit or competitively exclude pathogens. Some crustaceans chemically enhance the growth of bacteria that inhibit fungal infection of their eggs [6]. Evidence for comparable manipulations of bacteria has been hypothesized for birds. Antibiotic-producing gram-positive Enterococcus spp. occur in the preen gland of hoopoes Upupa epops [7] and red-billed woodhoo- poes Phoeniculus purpureus [8], and it has been suggested that application of oil containing these bacteria to eggs may help defend them against pathogens [7]. Avian incubation can dramatically inhibit total culturable microbial growth on eggshells [9]. These authors found that incubation primarily inhibited gram-negative enterics that can penetrate the shell and infect egg contents, but either promotes or does not inhibit the growth of gram-positive rods that infect eggs less frequently [10,11,12,13,14,15]. However, Cook et al. [9] did not address the effects of incubation on complete microbial assemblages because they used standard culture-based microbio- logical methods, which identify less than 1% of environmental microbes [16]. Here we use culture-independent methods to test whether birds selectively inhibit and promote bacterial growth during incuba- tion. We use PhyloChips, high-density oligonucleotide microarrays containing multiple DNA probes for over 8,000 bacterial taxa [17,18], to compare change over time in the composition and relative abundance of bacteria on naturally incubated and experimentally unincubated eggs. Based on previous work [9], we predicted that relative abundance and diversity of bacteria known to infect eggs such as enterics (family Enterobacteriaceae [10,11,12,13,14,15]) would increase on unincubated but not incubated eggs, and predicted the opposite pattern for apparently harmless bacteria like Gram-positive rods and cocci [10,11,12,13,14,15]. PLoS ONE | www.plosone.org 1 February 2009 | Volume 4 | Issue 2 | e4522

Results Moisture was more common on unincubated than incubated eggshells. All eggs in both groups were dry at laying. At late incubation, however, all unincubated eggs were wet and all incubated eggs were dry (Fisher���s exact test, n = 12, p,0.01). We detected 1492 unique taxa in 315 subfamilies, 256 families, 138 orders, 72 classes, and 38 phyla in at least one of the 24 total samples. A complete listing of these taxa is presented in the Supplementary Material (Results S1). NMDS and MRPP analyses revealed that bacterial assemblages remained constant over time on incubated eggs (Figure 1 A = 20.03, p = 0.75), but changed significantly on unincubated eggs (Figure 1 A = 0.07, p = 0.04). Assemblages on incubated eggs did not change their random arrangement in NMDS ordination space over time, while unincubated eggs were randomly arranged before treatment and became less variable after treatment (Figure 1). This shift towards a uniform bacterial assemblage on unincubated eggs was reflected by the presence of 6 significant Dufrene-Legendre indicator taxa on unincubated eggs after treatment, and none on incubated eggs (Table 1). Thus, unincubated eggs have some characteristic taxon abundances that were not exhibited in incubated eggs. All of these indicator species were significantly more abundant on unincubated than on incubated eggs (Table 1). Temporal changes in taxon richness also differed between incubated and unincubated eggs. Taxon richness of incubated eggs did not significantly differ between early and late incubation at the Kingdom or Family levels (paired t-test: all p.0.10 table 2 figure 2a���d). However, taxon richness of unincubated eggs was significantly higher at late incubation than at laying for Kingdom Bacteria (paired t-test: t = 22.76, p = 0.042 table 2 figure 2a) and for Families Enterobacteriaceae (t = 24.17, p = 0.009 table 2 figure 2b), Micrococcaceae (t = 23.08, p = 0.031, table 2 figure 2c), Frankiaceae (t = 22.71, p = 0.041 table 1), Xanthobacteraceae (t = 22.71, p = 0.041 table 1), and Caulobacteraceae (t = 22.60, p = 0.047 table 2 figure 2d). Taxon richness of the remaining 251 families did not differ significantly between laying and late incubation (all p.0.11). Temporal changes in bacterial abundance did not differ between incubated and unincubated eggs at the Kingdom level, but differed at the taxon level. Total bacterial abundance, measured as DNA concentration, did not significantly differ between laying and late incubation for incubated eggs (paired t- test: t = 20.98, p = 0.37 figure 3a), or for unincubated eggs (t = 21.36, p = 0.23 figure 3a). However, relative abundance of some individual taxa increased over time on unincubated, but not incubated, eggs. We analyzed change in relative abundance (PhyloChip fluorescence intensity) of 350 individual taxa. In the incubated group, abundance did not differ significantly between laying and late incubation for any taxa (all p.0.10 see figure 3a��� d full data are presented in Results S1, S2). However, in the unincubated group, relative abundance was significantly higher at late incubation than at laying for 81 bacterial taxa (Results S1, S2, figure 3a���d) but did not differ for the remaining 269 taxa (all p.0.09). The largest proportions of significant taxa were in the Families Enterobacteriaceae (23.2%), Comamonadaceae (11.0%), Caulo- bacteraceae (8.5%) and Sphingomonadaceae (8.5%). 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 [33]. 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 [34]. 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. doi:10.1371/journal.pone.0004522.g001 Incubation Inhibits Bacteria PLoS ONE | www.plosone.org 2 February 2009 | Volume 4 | Issue 2 | e4522