The role of dung beetle species in nitrous oxide emission, ammonia volatilization, and nutrient cycling

All procedures involving animals were conducted in accordance with the guidelines and regulations from Institutional Animal Care and Use Committee (IACUC) of the University of Florida (protocol #201509019). Tis manuscript is reported in accordance with ARRIVE guidelines.

Site description

This study was carried out at the North Florida Research and Education Center, in Marianna, FL (30°46′35″N 85°14′17″W, 51 m.a.s.l). The trial was performed in two experimental years (2019 and 2020) in a greenhouse.

The soil used was collected from a pasture of rhizoma peanut (Arachis glabrata Benth.) and Argentine bahiagrass (Paspalum notatum Flügge) as the main forages. Without plant and root material, only soil was placed into buckets, as described below in the bucket assemblage section. Soil was classified as Orangeburg loamy sand (fine-loamy-kaolinitic, thermic Typic Kandiudults), with a pH_water of 6.7, Mehlich-1-extratable P, K, Mg and Ca concentrations of 41, 59, 63, 368 mg kg⁻¹, respectively. Average of minimum and maximum daily temperature and relative humidity in the greenhouse for September and November (September for beetle trial due seasonal appearance of beetles, and October and November to the Pear Millet trial) in 2019 and 2020 were 11 and 33 °C, 81%; 10 and 35 °C, 77%, respectively.

Biological material determination

To select the species of beetles, a previous dung beetle sampling was performed in the grazing experiment in the same area (grass and legume forage mixture) to determine the number of dung beetle species according to the functional groups as described by Conover et al.^44. Beetles were pre-sampled from March 2017 to June 2018, where Tunnelers group were dominant and represented by Onthophagus taurus (Schreber), Digitonthophagus gazella (Fabricius), Phanaeus vindex (MacLeay), Onthophagus oklahomensis (Brown), and Euniticellus intermedius (Reiche). Other species were present but not abundant, including Aphodius psudolividus (Linnaeus), Aphodius carolinus (Linnaeus), and Canthon pilularius (Linnaeus) identified as Dweller and Roller groups, respectively. The pre-sampling indicated three species from the Tunneler group were more abundant, and thereby, were chosen to compose the experimental treatments (Fig. 4).

Beetles collection and experimental treatments

Three species of common communal dung beetles were used: O. taurus (1), D. gazella (2), and P. vindex (3). Treatments included two treatments containing only soil and soil + dung without beetles were considered as Control 1 (T1) and Control 2 (T2), respectively. Isolated species T3 = 1, T4 = 2, T5 = 3 and their combinations T6 = 1 + 2 and T7 = 1 + 2 + 3. Dung beetles were trapped in the pasture with grazing animals using the standard cattle-dung-baited pitfall traps, as described by Bertone et al.⁴¹. To avoid losing samples due to cattle trampling, 18 traps were randomized in nine paddocks (two traps per paddock) and installed protected by metal cages, and after a 24-h period, beetles were collected, and the traps removed. Table 1 shows the number of dung beetles, their total mass (used to standardize treatments) per treatment, and the average mass per species. To keep uniformity across treatments we kept beetle biomass constant across species at roughly 1.7 to 1.8 g per assemblage (Table 1). Twenty-four hours after retrieving the beetles from the field traps, they were separated using an insect rearing cage, classified, and thereafter stored in small glass bottles provided with a stopper and linked to a mesh to keep the ventilation and maintaining the beetles alive.

Buckets assemblage

The soil used in the buckets was collected from the grazing trial in two experimental years (August 2019 and August 2020) across nine paddocks (0.9 ha each). The 21 plastic buckets had a 23-cm diameter and 30-cm (0.034 m²) and each received 10 kg of soil (Fig. 5). At the bottom of the recipient, seven holes were made for water drainage using a metallic mesh with 1-mm diameter above the surface of the holes to prevent dung beetles from escaping. Water was added every four days to maintain the natural soil conditions at 60% of the soil (i.e., bucket) field capacity (measured with the soil weight and water holding capacity of the soil). Because soil from the three paddocks had a slightly different texture (sandy clay and sandy clay loam), we used them as the blocking factor.

The fresh dung amount used in the trial was determined based on the average area covered by dung and dung weight (0.05 to 0.09 m² and 1.5 to 2.7 kg) from cattle in grazing systems, as suggested by Carpinelli et al.⁴⁵. Fresh dung was collected from Angus steers grazing warm-season grass (bahiagrass) pastures and stored in fridge for 24 h, prior to start the experiment. A total of 16.2 kg of fresh dung was collected, in which 0.9 kg were used in each bucket. After the dung application, dung beetles were added to the bucket. To prevent dung beetles from escaping, a mobile plastic mesh with 0.5 mm diameter was placed covering the buckets before and after each evaluation. The experiment lasted for 24 days in each experimental year (2019 and 2020), with average temperature 28 °C and relative humidity of 79%, acquired information from the Florida Automated Weather Network (FAWN).

Chamber measurements

The gas fluxes from treatments were evaluated using the static chamber technique⁴⁶. The chambers were circular, with a radius of 10.5 cm (0.034 m²). Chamber bases and lids were made of polyvinyl chloride (PVC), and the lid were lined with an acrylic sheet to avoid any reactions of gases of interest with chamber material (Fig. 6). The chamber lids were covered with reflective tape to provide insulation, and equipped with a rubber septum for sampling⁴⁷. The lid was fitted with a 6-mm diameter, 10-cm length copper venting tube to ensure adequate air pressure inside the chamber during measurements, considering an average wind speed of 1.7 m s^−148,49. During measurements, chamber lids and bases were kept sealed by fitting bicycle tire inner tubes tightly over the area separating the lid and the base. Bases of chambers were installed on top of the buckets to an 8-cm depth, with 5 cm extending above ground level. Bases were removed in the last evaluation day (24th) of each experimental year.

Gas fluxes measurements

The gas fluxes were measured at 1000 h following sampling recommendations by Parkin & Venterea⁵⁰, on seven occasions from August 28th to September 22nd in both years (2019 and 2020), being days 0, 1, 2, 3, 6, 12, and 24 after dung application. For each chamber, gas samples were taken using a 60-mL syringe at 15-min intervals (t0, t15, and t30). The gas was immediately flushed into pre-evacuated 30-mL glass vials equipped with a butyl rubber stopper sealed with an aluminium septum (this procedure was made twice per vial and per collection time). Time zero (t0) represented the gas collected out of the buckets (before closing the chamber). Immediately thereafter, the bucket lid was tightly closed by fitting the lid to the base with the bicycle inner tube, followed by the next sample deployment times.

Gas sample analyses were conducted using a gas chromatograph (Trace 1310 Gas Chromatograph, Thermo Scientific, Waltham, MA). For N₂O, an electron capture detector (350 °C) and a capillary column (J&W GC packed column in stainless steel tubing, length 6.56 ft (2 M), 1/8 in. OD, 2 mm ID, Hayesep D packing, mesh size 80/100, pre-conditioned, Agilent Technologies) were used. Temperature of the injector and columns were 80 and 200 °C, respectively. Daily flux of N₂O-N (g ha⁻¹ day⁻¹) was calculated as described in Eq. (1):

where F is flux of N₂O (g ha⁻¹ day⁻¹), A is the area of the chamber, and dC/dt is the change of concentration in time calculated using a linear method of integration by Venterea et al.⁴⁹.

Ammonia volatilization measurement

Ammonia volatilization was measured using the open chamber technique, as described by Araújo et al.⁵¹. The ammonia chamber was made of a 2-L volume polyethylene terephthalate (PET) bottle. The bottom of the bottle was removed and used as a cap above the top opening to keep the environment controlled, free of insects and other sources of contamination. An iron wire was used to support the plastic jar. A strip of polyfoam (250 mm in length, 25 mm wide, and 3 mm thick) was soaked in 20 ml of acid solution (H₂SO₄ 1 mol dm⁻³ + glycerine 2% v/v) and fastened to the top, with the bottom end of the foam remaining inside the plastic jar. Inside each chamber there was a 250-mm long wire designed with a hook to support it from the top of the bottle, and wire basket at the bottom end to support a plastic jar (25 mL) that contained the acid solution to keep the foam strip moist during sampling periods (Fig. 7). The ammonia chambers were placed installed in the bucket located in the middle of each experimental block after the last gas sampling of the day and removed before the start of the next gas sampling.

Nutrient cycling

Photographs of the soil and dung portion of each bucket were taken twenty-four hours after the last day of gas flux measurement sampling to determine the dung removal from single beetle species and their combination. In the section on statistical analysis, the programming and statistical procedures are described. After this procedure, seeds of pearl millet were planted in each bucket. After 5 days of seed germination plants were thinned, maintaining four plants per bucket. Additionally, plants were clipped twice in a five-week interval, with the first cut occurring on October 23rd and the second cut occurring on November 24th, in both experimental years. Before each harvest, plant height was measured twice in the last week. In the harvest day all plants were clipped 10 cm above the ground level. Samples were dried at 55 °C in a forced-air oven until constant weight and ball-milled using a Mixer Mill MM 400 (Retsch, Newton, PA, USA) for 9 min at 25 Hz, and analyzed for total N concentration using a C, H, N, and S analyzer by the Dumas dry combustion method (Vario Micro Cube; Elementar, Hanau, Germany).

Statistical analysis

Treatments were distributed in a randomized complete block design (RCBD), with three replications. Data were analyzed using the Mixed Procedure from SAS (ver. 9.4., SAS Inst., Cary, NC) and LSMEANS compared using PDIFF adjusted by the t-test (P < 0.05). Gas sampling day and harvest were used as repeated measures. Treatments were considered the fixed effect, while block and experimental year were considered random effects. Polynomial contrasts were used to test the effect of dung absence (Soil vs. Soil + Dung), the effect of dung beetle (Soil + Dung vs. Soil + Dung + Dung beetle species) and the effect of each species and their combinations (Species 1 vs. 2, 3, 1 + 2, 1 + 2 + 3) on N₂O emission and nutrient cycling. A principal component analysis (PCA) was used to o get a better understanding about the effect of dung beetle treatments, using a biplot graph with the PRCOMP functions from the stats package (R Core Team)⁵² and a ggbiplot from the ggbiplot package⁵³, with the assistance of the vegan, tidyverse and devtools packages⁵⁴.

The following steps were taken to determine the proportions of interest, “soil” and “dung,” after segmenting the original images for the exclusive analysis of the portion corresponding to the contents of the buckets: conversion of colored images to grayscale images using the RGB model⁵⁵; smoothing boundary transitions using defocusing and the Multidimensional Gaussian Filter technique⁵⁶; Manual thresholding involves analyzing the histogram of grayscale images to generate binary images and determining the proportion of “soil” and “dung” in the binary images by counting the pixels of interest. The Python programming language⁵⁷, as well as the imageio⁵⁸ numpy⁵⁹ matplotlib⁶⁰ , and scikit-image libraries⁶¹, were used to analyze the images and create the figures that resulted from the process.

Source: Ecology - nature.com