Exposure to crop production alters cecal prokaryotic microbiota, inflates virulome and resistome in wild prairie grouse☆
Graphical abstract
Introduction
Agriculture could be the single most important threat to biodiversity globally (Dudley and Alexander, 2017). Its current detrimental impacts on wildlife may be more profound than those of global climate change (Dudley and Alexander, 2017; Maxwell et al., 2016). Although habitat loss due to agricultural conversion is a pressing issue in developing countries, agrochemical pollution associated with intensification of crop production is causing the greatest concern in developed nations (Green et al., 2005; Stanton et al., 2018). Recent studies have demonstrated that populations of 74% of farmland birds declined in the USA and Canada since the 1960s despite slight decreases in farm area (Stanton et al., 2018). Furthermore, grasslands suffered the greatest decline of avian abundance since 1970 among North American biomes (Rosenberg et al., 2019).
Agrochemicals may affect non-target wildlife directly by exposure to lethal doses, but, more frequently, the effects are indirect when the exposure is sub-lethal and animals suffer chronic fitness consequences leading to population declines over time (Gibbons et al., 2015; Stanton et al., 2018; van der Sluijs et al., 2015). These indirect effects include depletion of wildlife food resources by indiscriminate kill of terrestrial and aquatic invertebrates, plants, or fungi, (van der Sluijs et al., 2015) and adverse effects on animal growth, development, health, survival, and reproduction (Gibbons et al., 2015; Pandey and Mohanty, 2015). Agrochemicals also suppress wildlife immune systems, facilitating the emergence and spread of infectious diseases with unusually high mortality rates - the proposed leading cause of continuing global wildlife declines (Mason et al., 2013).
Wildlife microbiota - the community of microbes that primarily inhabit the animal digestive tract (gut) is crucial for its host response to environmental stressors. Gut microbiota participates in food digestion and detoxification, nitrogen recycling, protects the host from pathogens and environmental toxins, stimulates host's immune system, produces vitamins and hormones, and regulates brain function, behavior, and reproduction (Clarke et al., 2014; Heintz-Buschart and Wilmes, 2018; Hird, 2017; Jiménez and Sommer, 2017; Liang et al., 2018; Mohajeri et al., 2018; Rosenberg and Zilber-Rosenberg, 2018). Microbiota richness, composition, and hence functionality are sensitive to dietary changes and environmental pollution (Fackelmann and Sommer, 2019; Jiménez and Sommer, 2017; Rosenfeld, 2017). Surprisingly, the effect of agrochemicals on gut microbiota has not been studied in free-ranging wildlife despite recent studies demonstrating significant negative impacts of common agrochemicals on microbiota of humans and laboratory rodents (Defois et al., 2018; Liang et al., 2019; Lukowicz et al., 2018; Yuan et al., 2019).
To evaluate the effects of wildlife exposure to modern crop production, we compared cecal microbiota of populations of the sharp-tailed grouse (Tympanuchus phasianellus) and greater prairie chicken (T. cupido) residing in areas with and without crop production in Nebraska. North-central Nebraska contains the largest intact, uncultivated grassland in North America, the Sandhills (>50,000 km2), while other parts of the state are extensively used for crop production (Fig. 1). Although individual prairie grouse (Tympanuchus spp.) can occasionally move tens of kilometers, the vast majority have small home ranges. Annual home ranges of the greater prairie chicken vary from 0.36 to 104.33 km2 with a median of 5.44 km2 (Patten et al., 2011). Home ranges of the sharp-tailed grouse vary from 0.13 to 2.00 km2 during the breeding period and from 0.22 to 7.52 km2 during winter, with an average distance of 2.2 km (3.2 km maximum) between the two ranges, whereas daily movements do not exceed 1.2 km (Connelly et al., 2020). Diet of both species, sexes and age groups (<1 year and >1 year) is similar and is comprised of seeds, including spilled and wasted grain where available, insects, fruit, and foliage including alfalfa (Connelly et al., 2020; Johnson et al., 2020). The sedentary habits and diet of prairie grouse are well suited for a comparative study of possible effects of exposure to crop production on their microbiota.
We chose to focus on the cecal microbiota because of their crucial role in grouse adaptation to processing large quantities of poor-quality foods (Remington, 1989). During feeding bouts, grouse store large quantities of food in their crops. A collective function of most gut regions posterior to the crop is to separate large indigestible fragments of ingested food from finely ground, soluble, and readily digestible forage employing physical and chemical means. The former are rapidly excreted as dry feces but the latter are shunted to the ceca, which serve as microbial fermentation chambers, where non-starch polysaccharides and uric acid are broken into volatile fatty acids and ammonia, and salts and water are reabsorbed (Clench and Mathias, 1995; Svihus, 2014; Svihus et al., 2013). Grouse ceca are also responsible for detoxification of secondary phytocompounds (Kohl et al., 2016). Also, the variation in richness and composition of microbiota in the ceca is significantly lower than in other gut regions (Drovetski et al., 2019, 2018) allowing for efficient detection of environmentally induced changes in microbiota (Drovetski et al., 2019). The goals of this study were to determine (1) whether exposure to crop production affects cecal microbiota richness and composition in prairie grouse, (2) whether the differences between exposed and non-exposed birds are consistent with predictions for dysbiosis, i.e., whether changes in richness and composition of microbiota are associated with decline of beneficial host microbes and their replacement by pathogens, and (3) whether the exposure effects are consistent between host species.
Section snippets
Sampling
This project was approved by the Institutional Animal Care and Use Committees of the National Museum of Natural History (permit 2019-02), USGS Eastern Ecological Science Center (permit 2019-04), and conducted in accordance with the approved protocol, guidelines, and Nebraska hunting regulations. Voucher specimens were deposited at the National Museum of Natural History, Smithsonian Institution (USNM662731 - USNM662741, USNM663461 - USNM663499).
Birds were collected using a 12-gauge shotgun in
Prokaryotic microbiota
Our dataset included 398,634,662 sequences obtained from 49 samples (Suppl. Table A.1; Drovetski et al., 2022). The number of sequences obtained from individual samples ranged from 3,978,854 to 10,674,390 with a median of 8,326,092. There were 453 prokaryotic strains in our complete dataset (Table A.2), and the number of strains in a sample varied from 73 to 172 with a median of 109. The richness and abundance of DNA viruses and eukaryotic microbes were low and they were omitted from this
Discussion
Understanding the breadth and mechanism of effects of crop production on non-target wildlife is key for developing sustainable agricultural practices. We report results of the first study evaluating effects of exposure to crop production on prokaryotic microbiota, virulome, and resistome in wild birds. Our data demonstrate that exposure to crop production profoundly affected composition of cecal microbiota in two prairie grouse species (Fig. 2). The effect of the exposure on cecal microbiota
Conclusions
Our results suggest that exposure to modern crop production may be associated with dysbiosis of cecal microbiota in both prairie grouse species. Despite the considerable host-specific differences in microbiota composition, virulome, and resistome of the unexposed birds, they all converged in birds exposed to crop production. This convergence points to the similarity of the exposure effects in both host species. Future studies could address potential mechanisms that may be responsible for these
Data statement
The raw sequences archive has been deposited to GenBank: BioProject ID PRJNA788491 (https://www.ncbi.nlm.nih.gov/sra/PRJNA788491). GenBank biosample accession numbers for individual samples are listed in Table A.1. All other data generated or analyzed during this study are included in this published article and its Supplementary Tables A.1 - A.8 and are also available as a U.S. Geological Survey data release (Drovetski et al., 2022).
Credit author statement
Serguei V. Drovetski: conceptualization, methodology, formal analysis, investigation, resources, data curation, writing - original draft, visualization, supervision, project administration, funding acquisition; Brian K. Schmidt: methodology, investigation, resources, data curation, writing - review and editing; Jonas E. Lai: investigation, data curation, writing - review and editing; Michael S. Gross: methodology, formal analysis, investigation, resources, data curation, writing - review and
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
This study was funded by the U.S. Geological Survey, Environmental Health Program and by the Shealy family through the Basis Foundation (https://thebasisfoundation.weebly.com) Seed Grant to Gary Graves, Department of vertebrate Zoology, National Museum of National History. The funding sources had no involvement with any aspects of this study or manuscript preparation. Gary Graves encouraged us to pursue this project and provided support with logistics, supplies, and funding for fieldwork. We
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This paper has been recommended for acceptance by Professor Christian Sonne.