Cloacal microbiota of barn swallows from Northern Italy

<p>Bird gut microbiota shows large variation among geographical populations of the same species – probably because, differently from mammals, gut microbiota of birds is largely affected by extrinsic factors such as diet and environmental conditions. We analysed the cloacal microbiota (CM) of 12 barn swallows (<i>Hirundo rustica</i>) from a colony in Northern Italy by high-throughput DNA sequencing of the 16S rRNA gene. The CMs, dominated by bacteria of the phyla Proteobacteria, Actinobacteria, Firmicutes and Bacteroidetes, did not significantly differ between adult females, adult males and fledglings. This first description of barn swallow CM in Italy, together with two previous studies from Europe, suggests that CMs of geographically different barn swallow populations are dominated by bacteria belonging to the same phyla but different genera. The intra-colony similarity of the CMs may be due to the exposure of individuals to the same local environmental conditions while on their breeding grounds.</p

Sex allocation and maternal survival

Sex allocation and maternal surviva

TSR of yearlings in the population in any (<i>t</i>+1)-th study year in relation to adult survival between year <i>t</i> and year <i>t</i>+1.

<p>The analysis was restricted to the years when more than 180 yearlings were sampled (see <i>Statistical analyses</i>).</p

Confocal images of Neuro2A cells transfected with the wild-type or the mutant construct.

<p>To investigate intracellular distribution of CRH, cell were fixed in PFA and probed with mouse polyclonal anti-GM130 (red) for Golgi visualization and rabbit polyclonal anti-CRH (green) antibodies.</p

Ability to express CRH in Neuro2A cells transiently transfected with wild-type or mutant preproCRH construct.

<p>A) CRH levels of expression detected by realtime quantitative PCR in not transfected (NT) or transfected cells (wt or p.Pro30Arg) at three different times: 24 h, 48 h and 72 h. Each bar represents the mean ± S.E.M. (<i>n</i> = 3) of mRNA levels normalized to the basal CRH expression in Neuro2A cells (NT values) and to a housekeeping control gene (b-Actin). * t = −3.676 and p = 0.020 compared with wt at 24 h; ** t = 5.274 and p = 0.002 compared with wt at 24 h. B) Densitometric analysis of CRH immunoreactive proteins in subcellular fractions of the Neuro2A cells. Each bar represents the mean ± S.E.M. (<i>n</i> = 3) and protein content is expressed in arbitrary units. C) Levels of secreted CRH protein measured by ELISA. The ability of cells to secrete the CRH hormone was evaluated by measuring the protein level in cultured media of cells transfected either with the wild-type or the mutant construct at 24 h or 48 h after the transfection. Each bar represents the mean ± S.E.M. (<i>n</i> = 2) and protein content is expressed as % in respect to the mean value of wt 24 h (assumed equal to 100%).* t = −7.403 and p = 0.005 compared with wt at 24 h; ** t = 7.796 and p = 0.004 compared with wt at 24 h.</p

Length of the growing R₄ in relation to time since feather removal.

<p>Length of the growing R₄ relative to the length of the original R₄ in relation to time since removal of the original R₄. The continuous lines represent the Gompertz functions fitted to the data.</p

A Trade-Off between Reproduction and Feather Growth in the Barn Swallow (<i>Hirundo rustica</i>)

<div><p>Physiological trade-offs mediated by limiting energy, resources or time constrain the simultaneous expression of major functions and can lead to the evolution of temporal separation between demanding activities. In birds, plumage renewal is a demanding activity, which accomplishes fundamental functions, such as allowing thermal insulation, aerodynamics and socio-sexual signaling. Feather renewal is a very expensive and disabling process, and molt is often partitioned from breeding and migration. However, trade-offs between feather renewal and breeding have been only sparsely studied. In barn swallows (<i>Hirundo rustica</i>) breeding in Italy and undergoing molt during wintering in sub-Saharan Africa, we studied this trade-off by removing a tail feather from a large sample of individuals and analyzing growth bar width, reflecting feather growth rate, and length of the growing replacement feather in relation to the stage in the breeding cycle at removal and clutch size. Growth bar width of females and length of the growing replacement feather of both sexes were smaller when the original feather had been removed after clutch initiation. Importantly, in females both growth bar width and replacement feather length were negatively predicted by clutch size, and more strongly so for large clutches and when feather removal occurred immediately after clutch completion. Hence, we found strong, coherent evidence for a trade-off between reproduction, and laying effort in particular, and the ability to generate new feathers. These results support the hypothesis that the derived condition of molting during wintering in long-distance migrants is maintained by the costs of overlapping breeding and molt.</p></div

Ecological features of feather microbiota in breeding common swifts

<p>We provide the first-ever investigation of feather microbiota by high throughput DNA sequencing for any bird species by describing bacteria found on the innermost tertial feather of 22 adult common swifts (<i>Apus apus</i>). We found feather microbiomes with large abundance of Bacillales, Actinomycetales, Burkholderiales, Sphingobacteriales, Sphingomonadales, Rhizobiales, Pseudomonadales, Clostridiales, Rubrobacterales and Lactobacillales. Bacterial communities did not change with any feature of individual swifts. Network and cluster analysis of feather microbiomes disclosed three clusters, characterized by bacteria typical of seawater, plants and soil and unrelated to conditions at the breeding grounds. We hypothesize that feather microbiomes reflect, at least partly, airborne bacterial communities of the environments where individuals spent non-breeding periods, or of those that they crossed during migration, rather than breeding environment. If confirmed, this evidence may disclose the possibility to use feather bacteria as proxies for tracing non-breeding origin and routes of migratory birds.</p

Strength of the association between growing R₄ length and clutch size during the breeding cycle.

<p>Contouring of <i>t</i>-values associated to the effect of clutch size on LengthRe (see Methods) of females obtained in multiple regression models also including breeding stage and date at original feather removal (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096428#pone-0096428-t005" target="_blank">Table 5</a>). <i>t</i>-values were obtained from models including ranges of breeding stages centered at different median values and differing in width (see SI.5). Thus, for example, the yellow dot indicates a model where breeding stages spanning from breeding stage 10 and 21, and centered on breeding stage 15.5 were considered. Red isopletes indicate breeding stage ranges where unsigned <i>t</i>-values were significant. The arrow indicates the range where the unsigned <i>t</i>-value was largest. The apex of the triangle denotes the t-value of the models including the entire range of breeding stages (i.e. all data points) (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096428#pone-0096428-t005" target="_blank">Table 5</a>).</p