Microbiota development in Cyanistes caeruleus nest boxes throughout their breeding season. Andy Devaynes, André Antunes, Alan Bedford, Paul Ashton
[email protected]
Edge Hill University, Ormskirk, Lancashire, L39 4QP
Introduction
Nests were swabbed across four sites (Fig. 1) at three stages; nest build completion, clutch completion and at fledging.
cDNA was extracted, amplified and purified, then an endonuclease digestion was performed using the restriction enzyme Mspl.
T-RFLP analysis was performed with each peak determining a separate Operational Taxonomic Unit (OTU) (Fig. 4).
Peaks below 50 relative fluorescence units were discarded and binning determined peaks within 2 base pairs identical (Torok et al, 2008)
OTUs were converted into binary codes, Hellinger transformed and subjected to RDA analysis.
Fig. 2: Average number of OTUs present at each stage
RDA2 (1.2%)
Number of OTUs
Method
Nest boxes are a common way of studying and enjoying birds. As in any nesting site they have the potential to harbour bacteria. Despite bacteria being potential disease agents, bird-microbe interactions within the nest have seen little research. Existing studies have focussed upon ectoparasite load, with nest boxes harbouring significantly higher numbers of ectoparasites (e.g. Moreno et al. 2009, Tomas et al. 2012). Cyanistes caeruleus (Blue Tits) are widespread and construct new nests each year, they prefer nest boxes rather than natural holes and thus are a suitable model organism. Few studies have investigated the bacterial community of the nest and those that have are restricted by culturable bias or sampled only at a single point. This study addresses the culturable bias utilising T-RFLP analysis sampling at multiple points throughout the breeding attempt. Fig. 1: sample sites
ANOVA: F = 0.19, p = 0.82, d.f. = 2, 72. Error bars = standard deviation.
RDA1 (2.4%) Fig. 3: RDA plot of OTUs by stage 2
Fig. 4: Fragment analysis output, individual peaks represent OTUs
RDA, R = 6% for breeding stage with site constrained. ANOVA revealed the model not significant; F = 1.22, p = 0.21, d.f. = 1, 70.
Results
Discussion
No significant difference in the number of OTUs at each stage, however there is a change in the OTUs present (Figs. 2 & 3). OTUs present upon nest completion and clutch completion are more closely clustered than those present upon fledging (Fig. 3). Although little difference in number of OTUs at each stage, the variance increases slightly at clutch completion and doubles upon fledging. 2 Site constrained for redundancy analysis and had minimal effect , R = 2%.
The bacterial community within the nest follows a model of ecological succession as new modes of entry arise and available nutrients change. Laying of eggs introduces bacteria associated with the birds cloacal cavity. Post hatching, food introduction and chick defecation are obvious additional bacterial sources, alongside increased adult activity. Increased modes of entry give opportunity for a greater diversity of bacteria to be present across nests/sites. Increased bacterial counts upon fledging (5-8 orders of magnitude, personal data) may restrict the introduction of additional OTUs through competition.
References Moreno, J., Merino, S., Lobato, E et al. 2009. Nest-dwelling ectoparasites of two sympatric hole-nesting passerines in relation to nest composition: an experimental study. Ecoscience, 16 (3), pp. 418--427. Tomas, G., Merino, S., Martinez-De La Puente, J et al. 2012. Interacting effects of aromatic plants and female age on nest-dwelling ectoparasites and blood-sucking flies in avian nests. Behavioural processes, 90 (2), pp. 246--253. Torok , V. Ophel-Keller, K. Loo, M. Hughes, R. 2008. Application of methods for identifying broiler chicken gut bacterial species linked with increased energy metabolism. Applied and Environmental Microbiology, 74(3), pp. 783-791.
Acknowledgements I would like to thank the land owners/managers for allowing me access to each of my study sites, the RSPB and Natural England for issue of the relevant licence.
