DOI: 10.20350/digitalcsic/17766
title: Data from: Short-term microbial dynamics and changes in greenhouse gas emissions in cattle dung treated with ivermectin

description: [Description of methods used for collection/generation of data] The experiments were conducted at two sites within Doñana National Park (Andalusia, southern Spain) located 10 km apart: the Doñana Biological Reserve, which is traditionally grazed by organic livestock that does not receive veterinary medical products, and Los Sotos, characterised by the long-term presence of livestock treated with ivermectin (“site” factor). At both sites, short-term temporal variation in CO2 and CH4 emissions from dung pats was measured under two conditions: allowing or preventing dung beetle colonisation (“beetle” factor), and using dung pats either with or without ivermectin (“dung treatment” factor). Eight artificial dung pats were placed on the ground to assess CO2 and CH4 emissions at four different time points (“time” factor): immediately after placement (T0), 6 hours (T1), 24 hours (T2), and 48 hours after placement (T3). In total, 256 artificial dung pats were placed (8 dung pats x 4 time levels x 2 beetle levels x 2 dung treatment level x 2 site levels. After estimating CO2 and CH4 emissions using a portable gas analyser, all dung beetles from pats where colonisation was allowed were manually collected and transferred to pots for taxonomic identification, allowing the calculation of species richness, abundance, and biomass (fresh weight). CO2 concentrations were corrected to avoid overestimation by subtracting the contributions of soil respiration (microbiota, edaphic mesofauna, etc.), vegetation respiration, and dung beetle respiration. To estimate microbiota, ten artificial dung pats were arranged in two rows spaced 5 meters apart. One row contained dung pats with ivermectin, and the other without (“dung treatment” factor). Dung samples of 2 cm3 were extracted from non-contiguous portions of the centre of the pats at the same four time points (“time” factor). Of the potential 80 samples (10 dung pats x 2 dung treatment levels x 4 time levels), 14 were damaged. Molecular and bioinformatic analyses were performed to characterize archaea and bacteria present in the dung samples. DNA was extracted and purified, followed by multiplex PCR amplification and Illumina MiSeq sequencing. The V4 variable region of the 16S rRNA gene was amplified, and raw sequences were processed using the UPARSE pipeline. After merging paired reads, approximately 70.5% of the original reads were retained (2,477,067 reads out of 3,512,012). These retained reads were dereplicated (collapsing sequences at 100% identity) and clustered into zero-radius operational taxonomic units (zOTUs), excluding singletons. Over 85.05% of the merged sequence pool (2,912,356 reads) was mapped back to the zOTUs, resulting in 3,427 prokaryotic zOTUs. Taxonomic assignments were carried out using the SILVA_138 database via the SINA reference-based multiple sequence alignment tool. The final curated dataset comprised 3,403 prokaryotic zOTUs. The complete gene sequence dataset has been deposited in the NCBI Sequence Read Archive under BioProject ID PRJNA515520 and is available upon request. Environmental ontology descriptive terms (EnvO terms) were extracted from the closest sequences matches (99% identity) in the GenBank global databases using the SEQenv pipeline.
description: This dataset contains the quantified measurements of greenhouse gas emissions (CO2 and CH4) from dung pats collected to evaluate the effects of ivermectin on dung beetles and prokaryotic assemblages in Mediterranean pastures, based on field experiments conducted under controlled conditions. The treatments either allowed or excluded dung beetle colonisation of dung pats treated with ivermectin or left untreated, while also examining temporal changes in the richness and taxonomic composition of dung-associated prokaryotic assemblages during the first 48 hours after dung deposition. The data show that greenhouse gas emissions at least double in the absence of dung beetles, reinforcing the causal link between ivermectin use and increased CO2 and CH4 emissions. The dataset also shows that the bacterial phyla Firmicutes, Bacteroidota, and Proteobacteria dominated the dung microbiota, with ivermectin potentially favouring certain anaerobic and facultative taxa.
description: File List: This dataset consists of three tables and two figures. Table S1, comprising 512 rows and 11 columns, includes the number of species (Species Richness), number of individuals (Abundance), and Biomass (measured in grams of fresh weight) of dung beetles collected in the artificial dung pats, according to the four experimental factors considered (Site, Time, Beetle and Dung treatment). When dung beetle colonization was prevented, richness, abundance, and biomass values are zero. The “Emission” column reports methane (CH4) or carbon dioxide (CO2) emitted by dung pats (in g m-2 day-1). “Corrected Emissions-Beetles” values exclude emissions attributable to dung beetles, whereas “Corrected Emissions-(Beetles+Soil+Vegetation)” values exclude emissions from dung beetles, soil and vegetation, thereby isolating those directly attributable to the dung pats. Table S2, composed of 3,403 rows and 73 columns, shows the zOTUs' taxonomy and their sequence read counts for each sample. Samples are designated with three letters indicating the absence (ECO) or presence (IVM) of ivermectin in the dung pats, the time point of collection (T0, T1, T2, and T3), and an identification number. Table S3 contains 33 rows and 9 columns and presents the results of a two-way factorial ANOVA evaluating the effects of dung treatment (DT; dung pats with and without ivermectin) and time (T; from T0 to T3), as well as of the DT × T interaction, on the relative abundance of prokaryotic families representing more than 0.5% of the total sequences. Only relationships with probability values lower than the threshold set by a strict Bonferroni correction (0.05/33 = 0.002) are shown. The first number is the F3,58 value while the second number is the associated p-value. The table also reports the relative abundance (RA) of each family. An arrow in the last column indicates whether the relative abundance of zOTUs increases or decreases at the specified time point(s). In cases of statistically significant interactions, arrows refer to changes observed in dung pats containing ivermectin (IVM). Figure S1 presents box plots comparing the relative abundance (%) of prokaryotic families representing more than 0.5% of the total abundance (n = 33) across the four time levels: T0 (at dung pat deposition), T1 (6 hours after deposition), T2 (24 hours after deposition), and T3 (48 hours after deposition). Families are classified according to their main metabolic characteristics (i.e., anaerobic, aerobic/facultatively anaerobic, and aerobic) and are compared independently. Results of pairwise hypothesis contrast tests are presented in the figure. *P &lt; 0.05, **P &lt; 0.01, ***P &lt; 0.001, ****P &lt; 0.0001. Bacteroi: Bacteroidota; Bdellovi.: Bdellovibrionota; Cyano: Cyanobacteria; Fi.;B: Firmicutes;Bacilli; Fi.;C: Firmicutes;Clostridia; Fi.: Firmicutes; Alpha-P: Alphaproteobacteria; Gamma-P: Gammaproteobacteria; Verru.: Verrucomicrobiota. Figure S2 shows box plots comparing the relative abundance (%) of families corresponding to methanogenic Archaea in dung pats with and without ivermectin across the four time points.