Dung Beetle Assembly Affects Nitrous Oxide Emission, Ammonia Volatilizaiton and Nutrient Cycling

doi:10.21203/rs.3.rs-2100296/v1

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Dung Beetle Assembly Affects Nitrous Oxide Emission, Ammonia Volatilizaiton and Nutrient Cycling

https://doi.org/10.21203/rs.3.rs-2100296/v1

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published 02 Mar, 2023

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This study examined how dung beetle species and assemblage affect nitrous oxide (N₂O), ammonia volatilization, and pearl millet [Pennisetum glaucum (L.) R] performance. Seven treatments were applied in pot and they were two controls (only soil and soil + dung without beetles), single species of Onthophagus taurus (1), Digitonthophagus gazella (2), or Phanaeus vindex [MacLeay] (3); and their assemblages (1 + 2 and 1 + 2 + 3) respectively. After an initial trial assessing gas emissions, pearl millet was planted to assess growth, nitrogen yield (NY), and dung beetle activity after dung application. Dung beetle species increased N₂O flow on dung on the 6th day (80 g N₂O-N ha^− 1 day^− 1) compared to soil and dung (2.6 g N₂O-N ha^− 1 day^− 1). Ammonia emissions varied with the presence of dung beetles (P < 0.05), and D. gazella had lesser NH₃-N on days 1, 6, and 12 with averages of 2061, 1526, and 1048 g ha^− 1 day^− 1, respectively. The soil N content increased with dung + beetle application. Each pot with dung application affected pearl millet herbage accumulation (HA) regardless of dung beetle application, averages ranged from of 5 and 8 g DM pot^− 1. A PCA analysis was applied to analyze variation and correlation to each variable, but it indicated a low principal component explanation (less than 80%), not enough to explain the variation in findings. The presence of dung beetles prior to planting improved pearl millet production by enhancing N cycling, although assemblages with the three beetle species enhanced N losses to the environment via denitrification.

Agricultural and animal activities contribute to the intensification of the greenhouse effect. Cattle enteric fermentation¹, fresh dung², and rice production³ are major components of methane (CH₄) emissions from agricultural, as are N fertilizers⁴ and livestock excreta (urine and dung) for nitrous oxide (N₂O) emissions. According to Jensen et al.⁵, annual greenhouse gas (GHG) emissions from N fertilizer production and usage are predicted to be 300 Tg CO₂.

Dung beetles have the potential to reduce GHG emissions by aerating the soil and breaking the anaerobic zones formed under the dung crust⁶. These activities affect the interaction of deposited excreta with soil microbial populations^7,. Dung beetles are Scarabaeidae insects that play an important role in N cycling in both temperate and tropical agricultural grasslands⁸. They may help reduce GHG emissions and improve carbon sequestration by enhancing grass growth and soil fertility^{9, 10}.

Different dung beetle taxa can alter gas fluxes to varying degrees¹¹. Dung beetle taxa differ in their nesting techniques and are classified as dwellers, tunnelers, or rollers¹². These various nesting techniques may have a major impact on ecological function, such as dung removal efficiency¹³. Various beetle species excavate the soil in distinct ways, with varying diameters and sizes, resulting in different micro-environments that may alter GHG fluxes^{14, 15}. Dung beetles may also help to enhance nutrient cycling by enhancing soil carbon transfer and bacterial soil diversity^{16, 17}.

Thus, the goal of this study was to quantify N losses via N₂O emission and NH₃ volatilization from manure over time, including ammonia volatilization and N₂O, to assess forage productivity and N concentration after dung application, and the activity of different dung beetle species assemblages. We hypothesized that: 1) the presence of dung beetles would reduce N₂O emission and NH₃ volatilization; 2) dung beetle assemblages would differ on the way they affect N₂O emission and NH₃ volatilization; 3) dung beetle would have a positive effect on subsequent crop because of enhanced N cycling and reduced N losses.

There was an evaluation × treatment interaction on fluxes of N₂O (P < 0.05), with average emissions ranging from 2 g N₂O-N ha^-1day^-1 to all treatments in day 0 (after feces and dung beetle application) and significantly greater peak with 46 and 80 g N₂O-N ha^-1day^-1 on day 6 to pot with just dung and pot with dung + dung beetle species, respectively (Fig. 1). The fluxes of N₂O from pot with dung beetle species were the greatest and differed significantly to pot with dung and pot with just soil over time (P < 0.001), except in day 2, when N₂O flux was greater to just dung than dung + beetles.

Fluxes of N₂O were relatively high over time for pot with dung + beetles and were greater than the ones observed for soil and soil + dung (P < 0.001). However, O. taurus and D. gazella had the least N₂O flux compared with other beetle treatments over time (Fig. 2). The N₂O flux from the pot with dung (treatment 2) increased over time, however, since day 6 the fluxes decreased considerably until day 24, and the values ranged from 45 to 2.9 g N₂O-N ha^-1day^-1, respectively. The treatment 3 (O. taurus) and treatment 4 (D. gazella) showed the most depleted N₂O-N among the dung beetle treatments in days 0, 1, and 12, with averages of -3, 12.3, 25.8 g N₂O-N ha^-1day^-1 and − 1, 17.5, 23.5 g N₂O-N ha^-1day^-1, respectively. Treatment 7 (O. taurus + D. gazella + P. vindex) presented the greatest pick of N₂O-N in day 6 with average of 145.7 g N₂O-N ha^-1day^-1; however, in day 12 the N₂O flux decreased considerable, not differing (P > 0.05) from T3, and T4 Fig. 2). Treatment 1 (control pot with just soil) had the least N₂O flux and did not show significant variation over time. Treatment 5 showed a progressive increase over time, with the greatest pick of N₂O-N in day 12 and day 24.

There was an evaluation × treatment interaction (P < 0.05) on ammonia volatilization, which varied from a maximum of 6431 g NH₃-N ha^− 1 for T6 in day 2, decreasing over time, showing the least value in day 24, with average of 241 g NH₃-N ha^− 1. The treatment T3 and T4 presented the least values with 1536 and 1575 g of NH₃-N ha^− 1, respectively, when compared to other beetle treatments and T2 (Fig. 4). The T4 presented the most depleted NH₃-N emission from day 6, 12, and 24 with average of 1526, 1048, and 245 g of NH₃-N ha^− 1 when compared to T2 and to other beetle treatments. The T5 showed a peak on day 6, which was greater (P < 0.05) than T1, T2, T3, and T4. The T1 presented the least NH₃-N emission, and it did not significantly vary over time (Fig. 3).

T1: just soil, T2: soil + dung, T3: soil + dung + O. taurus (OT), T4: soil + dung + D. gazella (DG), T5: soil + dung + P. vindex (PV), T6: soil + dung + OT + DG, T7: soil + dung + OT + DG + PV. (*): Indicates significant difference at the 0.05 probability level among treatments in the same month, according to orthogonal contrast test.

At the end of the experiment, the dung removal efficiency was not statistically analyzed. However, according to a visual observation, the pot with just dung did not show any changes over time, losing humidity and getting a superficial crust (Fig. 4). On the other hand, the dung beetle species on the pot surface buried almost all the dung into the soil as much as the single and assemblage species.

T2: soil + dung, T3: soil + dung + O. taurus (OT), T4: soil + dung + D. gazella (DG), T5: soil + dung + P. vindex (PV), T6: soil + dung + OT + DG, T7: soil + dung + OT + DG + PV.

There was a treatment effect (P < 0.05) on soil nitrogen content. The two control treatments (soil and soil + dung) did not differ (P > 0.05) among them. The soil from pot with dung + beetle application (treatment 3, 4, 5, 6 and 7) presented greater nitrogen concentration compared with treatment 1 and 2 (Fig. 5).

T1: just soil, T2: soil + dung, T3: soil + dung + O. taurus (OT), T4: soil + dung + D. gazella (DG), T5: soil + dung + P. vindex (PV), T6: soil + dung + OT + DG, T7: soil + dung + OT + DG + PV. Different letters in lowercase indicate statistically significant differences among treatments, according to student test.

All treatments with dung beetle species resulted in taller pear millet than just dung on the pot surface, with both groups differing significantly (P < 0.10). Millet plants under dung beetle effect had 41.8 cm, significantly greater than millet plants with just dung and no beetle, showing 39.9 cm (Fig. 6).

*Different letters in lowercase indicate statistically significant differences at the 0.05 probability level among treatments in the same month, according to single degree of freedom polynomial contrast test.

There was a harvest × treatment interaction (P < 0.05) on HA of pear millet. Dung application had a positive effect on the HA. The greatest HA was observed in all treatments with dung application in the first harvest with average of 8 g of DM pot^− 1 (pot area of 0.32 m²), greater (P < 0.05) than T1 (control with just soil), which averaged 5 g of DM pot^− 1. In the second harvest, treatments did not differ among them, presenting the least DM values (Fig. 7

There was a treatment effect (P < 0.05) on N yield (Fig. 8). Treatments T3, T6, and T7 resulted in greater N yield for the pearl millet than T1, T2, T4, and T5. The T3, T6, and T7 also had more soil N available, with average of 0.34, 0.30, and 0.31 g N pot^− 1, respectively.

T1: just soil, T2: soil + dung, T3: soil + dung + O. taurus (OT), T4: soil + dung + D. gazella (DG), T5: soil + dung + P. vindex (PV), T6: soil + dung + OT + DG, T7: soil + dung + OT + DG + PV. Different letters in lowercase indicate statistically significant differences among treatments, according to orthogonal contrast test.

In this experiment, we hypothesized that dung beetle increased the N₂O-N and NH₃-N volatilization because of faster dung N mineralization in the soil as a result of dung incorporation and breakdown of dung pat crust, enhancing availability of oxygen for nitrification to occur (height and HA; Figs. 7 and 8). On the other hand, to simplify the response of the treatments of this experiment on gas emissions and nutrient cycling variables, a principal component analysis was used (Fig. 9).

Our results indicate that dung beetle presence on dung from livestock increased the N₂O-N fluxes over time. The beetle species O. taurus and D. gazella showed less N fluxes in day 0, 1, and 12 (Fig. 2) compared with P. vindex and other beetle assemblages. Previous studies have indicated an increase on fluxes of N₂O from cow dung resulted from dung beetle activity. Penttila et al.¹⁷ observed episodic high flux on days 15, 20, and 30, with 17, 110, and 40 g N₂O-N ha^− 1day^− 1 in dung pats with dung beetles, respectively. These results differ from ours in the days of the emission events, with lower values than the ones found in this study (Fig. 2). This might have occurred because of the N₂O dynamics during denitrification is particularly related to soil depth, labile organic carbon, and microbial biomass C¹⁸. Dung beetles have been suggested to increase NO₃⁻ levels by aerating the substrate¹⁹. However, our trial did not measure NO₃⁻ soil levels to attest this fact. May the reason why N₂O flux increase with dung beetle addition was due dung balls and its anaerobic inside, with great N concentration and available carbon. These are perfect conditions for N₂O emission to occur, as well by providing optimal conditions for denitrifying bacteria to function in the beetle gut as the earthworms²⁰. In our study, dung beetle activity increased N₂O-N flux by 71% and 79% when compared to pot with dung and pot with just soil in day 2, respectively. The increase occurred mainly on days 1, 2, and 6 (Figs. 1 and 2).

The NH₃-N emission is dependent on soil pH, moisture, texture, CEC, and soil temperature, as well as on the wind speed and air temperature²¹. In this experiment, N₂O-N and NH₃-N showed the highest peak with the presence of dung beetles. Soil temperature and humidity averaged 27ºC and 37% in pot without dung nor beetles, and 26ºC and 78% in pot with dung + dung plus beetles, respectively. An explanation for the lesser NH₃ emission in pot with just dungs as compared to pot with dung + beetle is the formation of a superficial crust on the dung. This crust can act as a physical barrier to the wind, preventing NH₃ volatilization. Furthermore, NH₃ tends to diffuse between fecal matter making soil surface contact, in which it will be converted into NH₄+, making NH₃ emission even more difficult²².

Even though our experimental design was based on the same total beetle biomass in each experimental unit, the single species P. vindex had a tendency (P < 0.05) to release more N₂O-N and NH₃ volatilization than the other single species (Fig. 2, 3), even when this species was included in other assemblages as in treatment 7. A possible explanation for this response is the fact that the species P. vindex has longer lifetime (can live over a year²³) than the other species. Furthermore, P. vindex presents in its gut 24% of bacteria that belong to Enterecoccace amilyliy²⁴. Some bacteria belonging to this family could contribute to denitrifying process as Enterococcus casseliflavus²⁵. These are the likely reasons why the N₂O fluxes were greater in pot with P. vindex. Evans et al.²⁶ indicated that dung beetle affects N₂O flux during the late summer season by modifying the moisture-dependent gas transport processes. The least value of N₂O from all treatments with dung in the first day of evaluation (Fig. 2) might be related to the fact that organic N needs to go through several processes before producing N₂O²⁷. Furthermore, according to de Castro et al.²⁸, smaller dung beetles remove more dung than bigger beetles, due to shorter legs and head that helps to bury and make holes. This explanation makes sense with our study due to the dung removal observed in pot with D. gazella (DG), when this species was able to remove and bury all dung on the soil surface (Fig. 4).

O. taurus, despite being the smallest beetle in the trial, did not remove dung as D. gazella. Furthermore, when it grouped with D. gazella, the dung removal apparently was reduced considerably (Fig. 4). Because D. gazella is a beetle species with strong invasion capacity, it might have impacted negatively the abundance and nesting behavior of other beetle species. As a result, there was inefficient dung removal in this group for intense intraspecific competition for dung²⁹.

This result can be explained to the fact that 85 to 95% of N consumed by cattle returns to the soil via excreta³⁰. This fecal N could be buried and mineralized in short period by dung beetle activity and break N and P available to the plant³¹. May it be the reason why the soil N concentration was greater with the dung beetle activity (Fig. 5) and the key to increase the N₂O emission, due more N available as substrate to denitrifying bacteria. Maldonado et al.¹⁹ performed an experiment in a grazed pasture in Mendoza Province, Argentine, to evaluate four dung beetle species (Sulcophanaeus imperator, Eucranium arachnoides, D. gazella and Malagoniella puncticollis) and their effect on nutrient cycling. All dung beetle species increased soil fertility, but D. gazella was more efficient, increasing soil organic matter by 159 g in 600 m² (equivalent to 2647 kg ha^− 1). Perhaps a similar effect occurred in our experiment in the pot with beetles. Although we did not measure organic matter content, dung beetles removed and buried the dung (Fig. 4) from the soil surface. This could promote the action of soil microbial respiration and affect the decomposition rate of soil organic matter³². Greater soil N concentration (Fig. 5) in the presence of dung beetles affect nitrogen mineralization, producing nitrate from the dung applied, and making it available for plant uptake and growth.

Dung has N and other nutrients required for plant development, increasing tillering and forage mass^{33, 34}. Badenhorst et al.³⁵ performed a study with mixed pastures (Eragrotis tef Trotter, Chloris gayana Kunth, and Digitaria eriantha Steud), cattle dung, and dung beetles. The authors observed that plant average biomass yield was 120 g DM m^− 2 and 200 g DM m^− 2 over 1 and six months of evaluation to treatments with dung beetles [(Euoniticellus intermedius (Reiche, 1849), D. gazella (Fabricius, 1787), and Onitis alexis (Klug, 1835)] respectively, greater (P < 0.05) than the treatment without beetles with 50 g DM m^− 2 and 110 g DM m^− 2 in the first and sixth month of evaluation, respectively. These results differ from the findings in this current experiment when treatment with just dung and dung + beetles did not differ from each other. The low HA in the second harvest for all treatments might have happened because of the low regrowth capacity of pear millet due to the frequent harvest (each 30 days), more frequent than the recommend 45 or 90 days³⁶.

These results indicate that dung beetle activity as O. taurus and their mixture with other beetles might accelerate the nitrogen mineralization coming from the applied dung. According to Badenhorst et al.³⁵, nutrient concentration in the vegetation increases significantly when dung beetles were active on a site. In this experiment, the activity of dung beetles did not have a significant effect (P > 0.05) on pearl millet N concentration for any of the treatments, with average of 1.65%. However, the N yield (NY) was strongly related with herbage accumulation (HA), which means that NY in this experiment corresponded to timing when the harvest occurred. An increasing trend (but not statistically significant; P = 0.27) was observed for O. taurus, O. taurus + D. gazella, and O. taurus + D. gazella + P. vindex in the first harvest on the HA of Pearl millet (Fig. 7). This could be explained by the relationship between N uptake and soil pH, whereby N needs to be mineralized to inorganic N for plant uptake and this process will be reduced if the pH of the soil is low (< 5.5³⁷).

The first and second principal component (PC1, PC2) explained 29.5% and 21.4% of the variability in the data set, respectively. The PC1 provided the highest variation, and HA had a positive correlation with plant height (PH). Treatment 3 (O. taurus) showed more relationship with soil nitrogen (SN) and NY. Treatment 5 (P. vindex) had a positive association with nitrous oxide emission (N₂O), as well the Treatment 2 and other beetle treatments, but a wide variation, and that is confirmed in the greater fluxes of N₂O when P. vindex was present (Fig. 2). Treatment 1 did not have dung nor beetles and that might explain its negative correlation with all the variables. From all beetle treatments, Treatment 4 was least associated with N₂O. Both PC explained 50.9% of the variability in the data set; this percentage is not enough to provide a better description. According to Cruz et al.³⁸, the accumulated variability for both PC must be greater than 80% to account for the variability manifested among the characteristics and have a better interpretation of the response of each variable.

Presence of dung beetles in cattle dung provided important ecosystem service by improving nutrient cycling and increasing retention of soil nitrogen. Greater soil N resulted in greater plant biomass and N concentration. Dung beetles, however, provided some disservices as they increased nitrous oxide emission and ammonia volatilization from cattle dung. Among single and dung beetle species combination, D. gazella tended to reduce the total N losses as N₂O and NH₃ from cattle dung and it was more efficient in removal of dung from the pot surface. This species has potential to enhance nutrient cycling in grassland ecosystems. On the other hand, the largest species, P. vindex and the combined species with this one, need to be more studied to get a better understanding about their contribution on greenhouse gases.

Site description

This study occurred at the North Florida Research and Education Center, in Marianna, FL (30°46′35″N 85°14′17″W, 51 m above sea level). The trial was performed in 2019 and repeated in 2020.

The experiment was a greenhouse study, and the soil used was collected from a pasture containing rhizoma peanut (Arachis glabrata Benth.) and Argentine bahiagrass (Paspalum notatum Flügge) as the main forages, and placed into buckets (see in pot building section). Soils at the experimental site were 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) of 2019 and 2020 were 11 and 33ºC, 81%; 10 and 35ºC, 77%, respectively.

Biological material

Three of the most abundant species (Fig. 10) caught in the experimental site where the soil was sampled were used in this trial. According to previous research, the species classified as nesting tunnellers were used: O. taurus Schreber, D. gazella Fabricius and P. vindex Macleay³⁹. Dung beetles were collected on August 28th 2019 and August 24th 2020, using the standard cattle-dung-baited pitfall traps described by⁴⁰, located in a pasture with grazing animals. A total of 18 traps were randomized in nine paddocks (two traps per paddock) and installed inside metal cages to avoid losing samples by cattle trampling. The beetles were collected after a 24-h period. The number of dung beetles, their total mass per treatment, and average mass per species are described in Table 1.

Table 1

Total number of dung beetles per treatment.
Treatment	Ot	Dg	Pv	Total mass (g)
1	-	-	-	-
2	-	-	-	-
3	43	-	-	1.72
4	-	30	-	1.75
5	-	-	7	1.82
6	25	13	-	1.76
7	9	6	4	1.75
Dung beetle species		Average mass (mg)/beetle
Phaneus vindex (Pv)		261.15
Digitonthophagus gazella (Dg)		58.57
Onthophagus taurus (Ot)		40.12

The beetle collection happened 24 hours before starting the experiment in the greenhouse. After retrieving the beetles from the field traps, they were separated using an insect rearing cage, classified, and thereafter stored in a small glass bottle provided with a stopper linked to a mesh to keep the ventilation and maintaining the beetles alive.

Treatments and Experimental design

Three species of common communal dung beetles were used: O. taurus [Shreber] (1), D. gazella [Fabricius] (2), and P. vindex [Macleay] (3). Treatments included isolated species and assemblages combining species 1 + 2 and 1 + 2 + 3. In addition, there were two controls: soil only and soil + dung without beetles. Therefore, a total of seven treatments were allocated in a randomized complete block design (RCBD), with three replications. To keep uniformity across treatments we kept beetle biomass constant across species at roughly 1.7 to 1.8 g per assemblage (Table 1). Treatment identification for the single species 1, 2, and 3 were named as treatment 3, 4, and 5. The combination of species 1 + 2 was named treatment 6, and the combination of species 1 + 2 + 3 was named as T7; soil only and soil + dung are referred as treatment 1 and 2, respectively.

Pot building

The soil used to build the pot was collected from a grazing trial August of 2019 and 2020 across nine paddocks (0.9 ha each) with same management history. The pot chamber area used in this experiment was 0.034 m². To build the pot, 21 plastic buckets were made of PVC. The buckets had a 23-cm diameter and 30-cm height. Each recipient received 10 kg of soil. At the bottom of the recipient, three 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 (Fig. 11). Water was added every four days to maintaining 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 described in the section above.

The fresh dung amount used in the trial was determined using the average area covered by dung and the average dung weight (0.05 to 0.09 m² and 1.5 to 2.7 kg) from cattle in grazing systems, according to the method suggested by Carpinelli et al.⁴¹. Angus steers grazing warm-season grass (bahiagrass) pastures were brought to chute to fresh dung collection. Fresh dung was collected and stored in fridge 24 h prior to start the experiment, always in in accordance with the guidelines and regulations from Institutional Animal Care and Use Committee (IACUC) of the University of Florida, protocoled #201509019. A total of 16.2 kg of fresh dung was collected, with 0.9 kg placed on each bucket. After the dung application dung beetles were added into the bucket. To prevent dung beetles from escaping by flying, a mobile plastic mesh with 0.5 mm diameter was used, removed, and placed before and after each evaluation. The experiment lasted for 24 days, with average temperature 28ºC and relative humidity of 79%.

Chamber measurements

The gas fluxes from pot were evaluated using the closed static chamber technique. The chambers were circular, with a radius of 10.5 cm (0.034 m²). Chamber bases and tops were made with polyvinyl chloride (PVC), and the tops were lined with an acrylic sheet to avoid any reactions of gases of interest with chamber material⁴² (Fig. 12). The chamber tops were covered with reflective tape to provide insulation, and they were equipped with a rubber septum for sampling⁴³. The top 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^{− 1 44, 45}. During measurements, chamber tops and bases were kept sealed by fitting bicycle tire inner tubes tightly over the area separating top and base. Bases of chambers were installed on top of the pot to an 8-cm depth, with 5 cm extending above ground level. Bases were removed in the last evaluation day.

Gas flux measurement

The gas fluxes were measured at 10:00 am following sampling recommendations by Parkin & Venterea⁴², on seven occasions from August 28th to September 22nd 2019 and 2020 (days 0, 1, 2, 3, 6, 12, and 24). On each specific day, gas fluxes were measured in three simultaneously blocks, with each block encompassing one replicate of each treatment (T1, T2, T3, T4, T5, T6, and T7). The three blocks were initiated simultaneously. For each chamber, samples were taken with a syringe of 60-mL capacity at 15-min intervals (t0, t15, and t30). The syringe content was immediately flushed into 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 pot (before closing the pot with the chamber). After t0 collection, the top of the chamber was placed on the pot with two more gas samples collected every 15 min (t15 and t30). The contents of N₂O were analysed by the Dumas dry combustion method (Vario Micro Cube; Elementar, Hanau, Germany) coupled to an isotope ratio mass spectrometer (IsoPrime 100, Manchester, UK). Flux of N₂O-N (g ha^− 1 day^− 1) was calculated as described in Eq. 1:

F = A*dC/dt

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.⁴⁵.

Ammonia volatilization measurement

Ammonia volatilization was measured using the open chamber technique, described by Araújo et al.⁴⁷. This chamber was assembled with a 2-L volume polyethylene terephthalate (PET) bottle. The bottom of the bottle was removed and used as a cap above the top opening just to keep the environment controlled and free of insects and other sources of contamination. Was used an iron wire to support a plastic jar. Fastened to the top of the wire support was a strip of polyfoam (250 mm in length, 25 mm wide, and 3 mm thick) that had been soaked in 20 ml of acid solution (H₂SO₄ 1 mol dm^− 3 + glicerina 2% v/v), with the bottom end of the foam remaining inside the plastic jar. Plastic jars were used to carry the acid immersed foam strips from the experimental building to storage in the freezer until starting NH₃ sampling procedures. 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. 13). The chambers were installed in the surface middle bucket after the last flux gas measurement of the day and removed before to start the next evaluation day.

Nutrient cycling

One day after the last day of gas flux measurement, seeds of pearl millet forage (Pennisetum glaucum cv. ‘Tifleaf III’) were planted in each treatment and replicate. After five days of seed germination, the plants were thinned, leaving four plants per bucket. Plant height evaluations started on September 24th. Plants were clipped twice at five weeks interval, with the first cut occurring on October 23th and the second cut occurring on November 24th of 2019 and 2020. Plant height was measure two times per week, in the last week of plant height measurements (day 30), all plants were harvested 10 cm above ground level. Samples were dried at 55°C until constant weight then ball milled using a Mixer Mill MM400 (Retsch) at 25 Hz for 9 min, thereafter, to start N and ¹⁵N sampling procedures. The total N from the samples were measured by dry combustion method (Vario Micro Cube, Elementar) coupled to an isotope ratio mass spectrometer (IsoPrime 100, IsoPrime).

Statistical analysis

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). Period of evaluation and harvest as were analyzed as repeated measures. Block and year were considered random effects and treatments were considered fixed effect. Single degree of freedom polynomial contrasts were used to test the effect of dung absence (contrast Soil vs. Soil + Dung), the effect of dung beetle (contrast Soil + Dung vs. Soil + Dung + Dung beetle species) and the effect of each species and their combinations (contrast Species 1 vs. 2, 3, 1 + 2, 1 + 2 + 3) on greenhouse gases 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 Team⁴⁸) and a ggbiplot from the ggbiplot package⁴⁹, together with the assistance of the vegan, tidyverse and devtools packages⁵⁰.

AUTHOR CONTRIBUTION

C.C.V.G.: data curation, formal analysis, investigation, methodology, software, writing, review and editing. J.C.B.D.J.: conceptualization, funding acquisition, investigation, methodology, project administration, resources, supervision, review and editing. X.M.: investigation, validation, methodology. D.C.: investigation, validation, methodology. E.R.S.S.: investigation, validation, review and editing. B.G.C.H.: investigation, validation, review and editing. M.R.M.: investigation, methodology, supervision. I.A.G.S.: investigation, validation, review and editing. D.S.A.: investigation, validation, review and editing. L.M.D.Q.: investigation, validation, review and editing. F.O.S.V.C.: investigation, validation, review and editing. M.V.F.S.: investigation, validation, review and editing. G.G.M.F.: investigation, validation, review and editing.

DATA AVAILABILITY

The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.

ADDITIONAL INFORMATION

Additional information Correspondence and requests for materials should be addressed to C.C.V.G.

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No competing interests reported.

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Dung Beetle Assembly Affects Nitrous Oxide Emission, Ammonia Volatilizaiton and Nutrient Cycling

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Materials And Methods

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Biological material

Treatments and Experimental design

Pot building

Chamber measurements

Gas flux measurement

Ammonia volatilization measurement

Nutrient cycling

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DATA AVAILABILITY

ADDITIONAL INFORMATION

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