Performing Department
(N/A)
Non Technical Summary
Antibiotic compounds are frequently used to promote the health and growth of livestock. As with many medicines, the un-metabolized, active compounds are eliminated through animal waste products and introduced to the surrounding environment. How these compounds affect soil food webs is poorly resolved. This proposed project aims to understand how a commonly administered livestock antibiotic interacts with manure to shape soil communities, ecosystem function and antibiotic resistance. Using antibiotic-laden manure from dairy cattle, I will conduct a field experiment to assess how antibiotic compounds affect soil ecosystems and their function. I hypothesize that antibiotics change the microbial community in manure and agricultural soils, slowing decomposition rates. Furthermore, I hypothesize that the addition of antibiotics creates a metabolically stressful environment for microbiota, increasing their production of greenhouse gases and the prevalence of antibiotic resistant genes. Finally, I hypothesize that antibiotic-laden manure decreases invertebrate diversity and abundance by lowering microbial biomass and via deleterious effects on invertebrate microbiome. Through field experiments, invertebrate collections, and microbial analysis, I will assess how the introduction of antibiotic-laden manure disrupts the soil food web community and the ecosystem services it provides. The results of this study will help shape future agricultural practices, while providing insight into what governs the health of economically important agricultural pasturelands. The proposed project addresses the AFRI priority areas of Bioenergy, Natural Resources and Environment; Animal health and production and animal products; and Agriculture Systems and Technology. This project will help develop the research, education, and mentorship credentials of the Project Director.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
0%
Goals / Objectives
This project explores how antibiotics present in livestock waste influence microbial and invertebrate communities. We predict that the introduction of these biologically active products can disrupt the natural function and health of agricultural ecosystems. Through the disruption of natural microbial communities, the ability for soils to retain and recycling importantnutrients may be diminished. We also may find that carbon storage is reduced due to the increased metabolic stress of microbial communities under antibiotic attack. Similarly, if antibiotics harm the native invertebrate community, we may find that soils have reduced rates of decomposition and lower moisture retention. The soil texture may also be disrupted through the removal of key ecosystem engineers like Annelida. This project will fill a critical gap in our knowledge of how both antibiotic compounds and manure inputs shape soil function, while also linking soil health to human health.Primary Objectives(1) Determine how antibiotic additions affect ecosystem function in agricultural soils(2) Examine how microbial and invertebrate communities respond to antibiotics(3) Assess how antibiotic additions influence antibiotic resistant gene abundance anddiversity
Project Methods
Work already accomplished:The field portion of the proposed project will be conducted at two paired locations (1) the remnant Palouse prairie research reserve at Paradise Ridge, ID at the University of Idaho's Moscow campus and (2) the University of Idaho's experimental Kambitsch Farm. These paired sites will allow me to examine whether historically managed fields respond differently from uncultivated grasslands.Manure will be collected and homogenized from 10 dairy cattle at the University of Idaho's Dairy Center. None of the cattle inour study will have received a previous course of antibiotics. I will then add our antibiotic to the manure, at a rate equal to the average excretion rate. I will use Monensin, a broad-spectrum antibiotic that blocks bacterial cell protein transport, as it is one of the most frequently used antibiotics in livestock management and has a high active-compound excretion rate. A subset of the initial manure collections, and the antibiotic-manure mixtures will be collected and kept at -80C to preserve DNA. Their microbial communities will be assessed alongside samples.I will create plots by placing PVC collars (20 cm in diameter, inserted 5 cm into the soil), with open tops to allow for aboveground invertebrates to colonize. I will sample soil from each collar to establish a "starting" microbial community. Due to the destructive nature of invertebrate sampling, we will create 4 additional starting plots and sample them to establish the local invertebrate community. Antibiotics and/or manure will be added to collars in a 2 x 2 factorial design with six replicate collars per treatment per location. Treatments will be supplied monthly for a period of 4 months. Throughout the 4 months, grass-house gas emissions, decomposition rates and soil microbial and invertebrate communities will be sampled.To examine the effects of antibiotics and manure on green-house gas emissions and decomposition, each PVC collar will contain two decomposition bags filled with pre-weighed local plant litter. Bags will be collected after 60 and 120 days, and decomposition rates will be determined by measuring the amount of mass loss compared to initial weights. Gas fluxes of CO2 will be measured in the field using a portable Licor LI-8100A CO2 Gas Analyzer with a 20-cm closed, dynamic survey chamber (Licor, Nebraska, USA) attached to the PVC collar, with previously developed protocols.Gas emissions will be taken at day 1, 2, 4, 8, 16, 32, 64, 94, 120. At day 60 and 20, we will take a subsample of soil (~10 g) to perform C-mineralization (i.e., bioavailable C) assays and microbial biomass assessments. Combined, these measurements will allow us to quantify rates of microbial efficiency, an essential metric for understanding soil health.Soil from each PVC collar plot will be sampled at 0, 1, 4, 8, and 16 days after monthly treatment to examine microbial communities. At the time of sampling, we will measure temperature, moisture level, and pH in each plot. We will sample microbes using Zymo Xpeditiontm Soil/Fecal DNA Miniprep kits and protocol. We will analyze microbial communities by Illumina© sequencing libraries generated by PCR amplification of 16S bacterial and ITS fungal sequences. All microbial sequences will be processed, cleaned and taxonomically assigned using the pipeline DADA2. Subsequent analysis will be done in R v. 3.5.2.Invertebrate communities in half of the PVC will be sampled at day 60 and the second half at day 120 via the Berlese method. Once the invertebrate communities are sorted, we will determine the dominant taxa present across treatments. We will examine changes in the microbiome of these dominant invertebrates by sampling them as described above after surface sterilizing the invertebrates with a bleach solution. All further microbial analysis will be the same as described above.To be finished:I will use an emerging sequence-capture technique to examine the influence of antibiotics and manure on levels of ARGs. Broadly, this technique, name ResCap, allows researchers to amplify specific genes of interest across samples. Unlike traditional methods, sequence-capture allows for all known ARGs (~70,000) to be examined in one sequencing event. Sequence-capture is preferable to whole metagenome sequencing (WMS) which is often costly and inefficient due to the large portions of genetic material that are not of interest (i.e., DNA synthesis genes, structural coding genes). This technique also provides real-time measurements of gene abundance, allowing us to assess genetic diversity and abundance levels in unison. While commonly used in human medicine, ResCap has yet to be applied to soil ecosystems. Our novel approach of this technique could shape how we assess genetic and functional diversity in our soilsystems.Using the DNA extracted from soil microbial and invertebrate communities as describe above, I will use NimbleGen's SeqCap EZ (Roche Nimblegen, Inc) kits, probes and protocol to conduct analysis of ARGs. The genes for this study willbe compiled using a combination of ARG databases. After cleaning, "captured" DNA will then proceed through an additional brief amplification (ligation mediated PCR) prior to being purified and quantified. The DNA libraries will be sequenced on an Illmina© HiSeq at Cornell University's Institute of Biotechnology. Sequence processing will be conducted, and we will use the ResCap database to predict ARGs. Subsequent statistical analysis will be done in Rand will include linear models to compare levels of gene abundance across treatments. We will also examine important correlations between environmental variables, microbial and invertebrate community identity and ARGs using network analyses.