Monitoring the variation in the gut microbiota of captive woolly monkeys related to changes in diet during a reintroduction process.

Microbiome is known to play an important role in the health of organisms and different factors such as diet have been associated with modifications in microbial communities. Differences in the microbiota composition of wild and captive animals has been evaluated; however, variation during a reintroduction process in primates has never been reported. Our aim was to identify changes in the bacterial composition of three individuals of reintroduced woolly monkeys (Lagothrix lagothricha) and the variables associated with such changes. Fecal samples were collected and the V4 region of the 16S rRNA gene was sequenced to determine gut microbial composition and functionality. Individual samples from released individuals showed a higher microbial diversity after being released compared to before liberation, associated with changes in their diet. Beta diversity and functionality analysis showed separation of samples from released and captive conditions and the major factor of variation was the moment of liberation. This study shows that intestinal microbiota varies depending on site conditions and is mainly associated with diet diversity. The intake of food from wild origin by released primates may promote a positive effect on gut microbiota, improving health, and potentially increasing success in reintroduction processes.

Effects of field conditions on fecal microbiota.

Gut microbiota can provide great insight into host health, and studies of the gut microbiota in wildlife are becoming more common. However, the effects of field conditions on gut microbial samples are unknown. This study addresses the following questions: 1) How do environmental factors such as sunlight and insect infestations affect fecal microbial DNA? 2) How does fecal microbial DNA change over time after defecation? 3) How does storage method affect microbial DNA? Fresh fecal samples were collected, pooled, and homogenized from a family group of 6 spider monkeys, Ateles geoffroyi. Samples were then aliquoted and subjected to varying light conditions (shade, sun), insect infestations (limited or not limited by netting over the sample), and sample preservation methods (FTA - Fast Technology for Analysis of nucleic acid - cards, or freezing in liquid nitrogen then storing at -20°C). Changes in the microbial communities under these conditions were assessed over 24h. Time and preservation method both effected fecal microbial community diversity and composition. The effect size of these variables was then assessed in relation to fecal microbial samples from 2 other primate species (Rhinopithecus bieti and R. brelichi) housed at different captive institutions. While the microbial community of each primate species was significantly different, the effects of time and preservation method still remained significant indicating that these effects are important considerations for fieldwork.

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

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 8weeks) prior to microbial extraction and analysis.