How should we store avian faecal samples for microbiota analyses? Comparing efficacy and cost-effectiveness

Paula Vargas-Pellicer,Cecylia Watrobska,Sarah Knowles,Julia Schroeder,Cristina Banks-Leite

doi:10.1016/j.mimet.2019.105689

Abstract

Analyses of bacterial DNA in faecal samples are becoming ever more common, yet we still do not know much about bird microbiomes. These challenges partly lie in the unique chemical nature of their faeces, and in the choice of sample storage method, which affects DNA preservation and the resulting microbiome composition. However, there is little information available on how best to preserve avian faeces for microbial analyses. This study evaluates five widely used methods for preserving nucleic acids and inferring microbiota profiles, for their relative efficacy, cost, and practicality. We tested the five methods (in-situ bead-beating with a TerraLyzer instrument, silica-bead desiccation, ethanol, refrigeration and RNAlater buffer) on 50 fresh faecal samples collected from captive House sparrows (Passer domesticus). In line with other studies, we find that different storage methods lead to distinct bacterial profiles. Storage method had a large effect on community composition and the relative abundance of dominant phyla such as Firmicutes and Proteobacteria, with the most significant changes observed for refrigerated samples. Furthermore, differences in the abundance of aerobic or facultatively aerobic taxa, particularly in refrigerated samples and those stored in ethanol, puts limits on comparisons of bacterial communities across different storage methods. Finally, the methods that did not include in-situ bead-beating did not recover comparable levels of microbiota to the samples that were immediately processed and preserved using a TerraLyzer device. However, this method is also less practical and more expensive under field work circumstances. Our study is the most comprehensive analysis to date on how storage conditions affect subsequent molecular assays applied to avian faeces and provides guidance on cost and practicality of methods under field conditions.

Highlights

Microbial composition is determined by storage method; relative abundances of certain phyla change across treatments, especially on refrigerated and ethanol samples; this could be driven by the differentiated proportion of aerobes and anaerobes, indicating selective detection rates
The most abundant phyla across all samples were Proteobacteria and Firmicutes, which is consistent with what was previously reported for House sparrows (Kohl et al, 2019; Mirón et al, 2014); we found higher relative abundance of Proteobacteria in samples stored dried
This result suggests that consideration should be given to differences in abundance at certain taxonomic levels that have undergone this type of storage, those involving Proteobacteria and Actinobacteria which are able to grow at a range of temperatures (Weese and Jalali, 2014)

Summary

Introduction

The gut microbiome is important for host health through its impacts on the immune system (Brisbin et al, 2008; Ruiz-Rodríguez et al, 2009b; Yang et al, 2012), digestion (Dewar et al, 2013; Godoy-vitorino et al, 2010; Ruiz-Rodríguez et al, 2009a), development (Barbosa et al, 2016; Teyssier et al, 2018; Torok et al, 2011; Videvall et al, 2019) and behaviour (Cryan and Dinan, 2012). Since the intimate interaction between hosts and their microbiota is thought to have wide-ranging effects on all aspects of host biology, there is tremendous potential for knowledge about the avian microbiome to contribute to research in avian ecology, evolution, and conservation (Hird, 2017; Trevelline et al, 2019). A growing number of avian studies are capitalizing on this development and investigating interactions between host life-history traits, ecology, and the gut microbiota (Grond et al, 2018; Kohl, 2012; Teyssier et al, 2018; Trevelline et al, 2019; van Dongen et al, 2013; Videvall et al, 2019). A key question for many ecological studies, is how to best store and preserve avian faecal samples for downstream molecular work as it affects sampling strategy, experimental design and study costs

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