Best practice for wildlife gut microbiome research: A comprehensive review of methodology for 16S rRNA gene investigations

Abstract

Extensive research in well-studied animal models underscores the importance of commensal gastrointestinal (gut) microbes to animal physiology. Gut microbes have been shown to impact dietary digestion, mediate infection, and even modify behavior and cognition. Given the large physiological and pathophysiological contribution microbes provide their host, it is reasonable to assume that the vertebrate gut microbiome may also impact the fitness, health and ecology of wildlife. In accordance with this expectation, an increasing number of investigations have considered the role of the gut microbiome in wildlife ecology, health, and conservation. To help promote the development of this nascent field, we need to dissolve the technical barriers prohibitive to performing wildlife microbiome research. The present review discusses the 16S rRNA gene microbiome research landscape, clarifying best practices in microbiome data generation and analysis, with particular emphasis on unique situations that arise during wildlife investigations. Special consideration is given to topics relevant for microbiome wildlife research from sample collection to molecular techniques for data generation, to data analysis strategies. Our hope is that this article not only calls for greater integration of microbiome analyses into wildlife ecology and health studies but provides researchers with the technical framework needed to successfully conduct such investigations.

Genetic evidence on the causality between gut microbiota and various asthma phenotypes: a two-sample Mendelian randomization study

“…The use of large datasets increases the statistical power of the MR analysis, enhancing our ability to detect and estimate causal effects accurately. Due to the absence of standardized sampling methods and longitudinal study, microecological studies often yield contradictory results ( Knight et al., 2018 ; Combrink et al., 2023 ). Moreover, confounding factors and reverse causality can often hinder the results of observational studies.…”

“…These studies aim to compare the alterations in the microbial composition between individuals with a specific disease and healthy individuals. However, conflicting results are often observed in similar studies due to the absence of standardized sampling methods and longitudinal study designs ( Knight et al., 2018 ; Combrink et al., 2023 ). Moreover, these observational studies are often hindered by confounding factors (e.g., antibiotic or corticosteroid use) and reverse causality.…”

Assessing Methodological Variability in Gut Microbiome Studies: Lessons from Southeast Asian for Effective Conservation Strategies

“…To date, the majority of microbiome studies have primarily focused on humans and domesticated animals, and for wildlife, efforts are now becoming more widespread to address this research gap [1]. This coincides with the advancement of technologies such as high-throughput sequencing which has enabled faster and cost-effective identification of microbiomes among and within wildlife [2,3]. Generally, gut microbiome is a diverse set of microbial taxa inhabiting the gastrointestinal tract system, including their genetic material [4].…”

“…Currently, there is no standardized set of best practices guiding the gut microbiome among wildlife. This necessitates a critical analysis of methodological and technical variability [1,2]. Furthermore, current evidence suggests that the impacts of the gut microbiome on host species and environments differ across various aspects.…”

“…They achieve this through direct interactions with intestinal epithelial cells, acting as our first line of defense against pathogens and toxins. Hence, subsequent investigations in this area could contribute to a more effective conservation and management plan for the wildlife species, especially in cases where understanding can lead to the development of strategies and protocols to mitigate the risk of extinction for endangered species [2,6].…”