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.
Highlights
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
non-metric dimensional scaling (NMDS) and multi-response permutation procedure (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)
Summary
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. 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]. Antibiotic-producing gram-positive Enterococcus spp. occur in the preen gland of hoopoes Upupa epops [7] and red-billed woodhoopoes Phoeniculus purpureus [8], and it has been suggested that application of oil containing these bacteria to eggs may help defend them against pathogens [7]
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