Effect of preservation method on spider monkey (Ateles geoffroyi) fecal microbiota over 8 weeks

Highlights

• We examine differences in microbial communities due to fecal preservation method.

• Fresh, frozen, and ethanol-preserved samples have most similar microbial communities.

• 8 weeks of preservation had little effect on microbial composition and diversity.

• DNA amount/purity did not correlate to microbe amplification, diversity, and composition.

• Preservation methods exhibit bias toward/against some bacterial phyla.

Abstract

Studies of the gut microbiome have become increasingly common with recent technological advances. Gut microbes play an important role in human and animal health, and gut microbiome analysis holds great potential for evaluating health in wildlife, as microbiota can be assessed from non-invasively collected fecal samples. However, many common fecal preservation protocols (e.g. freezing at − 80 °C) are not suitable for field conditions, or have not been tested for long-term (greater than 2 weeks) storage. In this study, we collected fresh fecal samples from captive spider monkeys (Ateles geoffroyi) at the Columbian Park Zoo (Lafayette, IN, USA). The samples were pooled, homogenized, and preserved for up to 8 weeks prior to DNA extraction and sequencing. Preservation methods included: freezing at − 20 °C, freezing at − 80 °C, immersion in 100% ethanol, application to FTA cards, and immersion in RNAlater. At 0 (fresh), 1, 2, 4, and 8 weeks from fecal collection, DNA was extracted and microbial DNA was amplified and sequenced. DNA concentration, purity, microbial diversity, and microbial composition were compared across all methods and time points. DNA concentration and purity did not correlate with microbial diversity or composition. Microbial composition of frozen and ethanol samples were most similar to fresh samples. FTA card and RNAlater-preserved samples had the least similar microbial composition and abundance compared to fresh samples. Microbial composition and diversity were relatively stable over time within each preservation method. Based on these results, if freezers are not available, we recommend preserving fecal samples in ethanol (for up to 8 weeks) prior to microbial extraction and analysis.

Introduction

The gastrointestinal (GI) tract is home to trillions of microbes that play an important role in shaping diet and digestion (Backhed et al., 2004, Ley et al., 2008, Martin et al., 2007, Turnbaugh et al., 2006), host immunity (Cho and Blaser, 2012, Chung et al., 2012, Hooper et al., 2012, Littman and Pamer, 2011), and disease processes (Petersen and Round, 2014, Round and Mazmanian, 2009, Sekirov et al., 2010). Recent advances in next-generation sequencing and bioinformatics have allowed us to analyze and compare entire gut microbial communities efficiently and effectively. As a result, the number of gut microbial studies published over the last 15 years has grown dramatically (Sekirov et al., 2010). Studies of the gut microbiome have also begun expanding to wildlife (Amato et al., 2013, Nelson et al., 2013, Uenishi et al., 2007, Villers et al., 2008, Xenoulis et al., 2010). These studies hold great potential for evaluating health in wildlife, as microbiota can be assessed from non-invasively collected fecal samples. However, there is limited information available on long-term (greater than 2 weeks) fecal microbial preservation methods under field conditions (Frantzen et al., 1998, Vlčková et al., 2012). Preserving fecal samples via freezing is commonly considered the ‘gold-standard’ for microbial analysis (Rochelle et al., 1994, Wu et al., 2010, but see Bahl et al., 2012), and most protocols focus on short term (less than 2 weeks) human or animal fecal preservation in highly controlled conditions including laboratories or hospitals with electricity and − 20 °C or − 80 °C freezers (Carroll et al., 2012, Dominianni et al., 2014, Lauber et al., 2010, Nechvatal et al., 2008, Ott et al., 2004, Roesch et al., 2009, Wu et al., 2010). Evaluation of methods for long-term storage under field conditions (i.e. without electricity/freezers) is necessary to understand if or how fecal microbial communities are affected by preservation method and time.

Feces are already used for many different types of wildlife studies including monitoring reproductive status (Dehnhard et al., 2008, Stoops et al., 1999), physiological stress (Cavigelli, 1999, Shutt et al., 2012), parasite load (Muller-Graf et al., 1999, Rietmann and Walzer, 2014), and genetic relatedness of populations (Adams et al., 2003, Mowry et al., 2011). Each of these types of studies have differing requirements in terms of fecal preservation, and much research has been devoted to optimizing fecal preservation in wildlife (for fecal steroid analysis (Khan et al., 2002, Shutt et al., 2012); for microsatellite amplification (Bubb et al., 2011, Murphy et al., 2002, Vallet et al., 2008); for parasite detection (Nielsen et al., 2010, Rietmann and Walzer, 2014)). Gut microbial studies have only recently begun in wildlife, and field-friendly microbial preservation methods still need to be validated, particularly with host species and dietary ecology in mind. For example, feces from herbivorous or folivorous host species (e.g. Barbary macaques, lowland gorillas) may contain high concentrations of secondary compounds that inhibit DNA extraction or PCR success (Vallet et al., 2008). Feces from animals that practice geophagy (consumption of soil directly or incidentally as a part of their diet) may contain large quantities of soil microbes (Delsuc et al., 2013). Preservation and analysis of such samples require thought regarding the transience or biological relevance of soil microbes within the gut.

To guide future fecal collection and preservation protocols for gut microbial studies in wildlife, particularly in herbivorous primates we assessed the effect of different preservation methods on the fecal microbiome of Ateles geoffroyi, the spider monkey, at the Columbian Park Zoo (Lafayette, IN, USA). Our study compared 5 methods of fecal preservation: freezing at − 20 °C, freezing at − 80 °C, immersion in 100% ethanol, application to FTA cards, and immersion in RNAlater.

These methods were selected because they are relatively common fecal preservation techniques with varying advantages and disadvantages. Freezing, one of the most common preservation methods, inhibits microbial growth, limits opportunities for contamination, and effectively preserves DNA over time (Rochelle et al., 1994, Wu et al., 2010). However, few studies have examined differences between freezing at − 20 °C and − 80 °C. Additionally, freezing is often not a viable method for field studies. Chemical means of fecal preservation such as ethanol, RNAlater or FTA cards are more “field friendly” methods of preservation. Like freezing, ethanol is recognized as another common and effective fecal DNA preservation method (Murphy et al., 2002), but restrictions apply to ethanol transport due to its status as a ‘hazardous chemical.’ RNAlater is a nonhazardous liquid that preserves both RNA and DNA (Nechvatal et al., 2008). While RNAlater faces fewer transport restrictions than ethanol, both RNAlater and ethanol pose another challenge; carrying large quantities of liquid into remote locations can be logistically difficult. FTA cards are the easiest to transport and most convenient to use in the field, but convenience comes at a price: FTA cards and RNAlater are the most expensive preservation methods.

We hypothesized that freezing would be the most effective method for preserving microbial DNA. Specifically, we predicted that frozen (at − 20 °C and − 80 °C) samples would be the most stable in terms of DNA concentration, purity, and microbial composition over 8 weeks. Previous studies have found that freezing results in greater DNA concentrations with higher purity values compared to RNAlater, ethanol, and FTA card preservation (Nechvatal et al., 2008, Vlčková et al., 2012). We also predicted that the microbial communities of frozen samples would most closely resemble the microbial communities of fresh, never-preserved, immediately-extracted fecal samples.