Earthworms do not increase greenhouse gas emissions (CO2 and N2O) in an ecotron experiment simulating a realistic three-crop rotation system

Earthworms are known to stimulate soil greenhouse gas (GHG) emissions, but the majority of previous studies have used simplified model systems or lacked continuous high-frequency measurements. To address this, we conducted a two-year study using large lysimeters (5 m2 area and 1.5 m soil depth) in an ecotron facility, continuously measuring ecosystem-level CO2, N2O, and H2O fluxes. We investigated the impact of endogeic and anecic earthworms on GHG emissions and ecosystem water use efficiency (WUE) in a simulated agricultural setting. Although we observed transient stimulations of carbon fluxes in the presence of earthworms, cumulative fluxes over the study indicated no significant increase in CO2 emissions. Endogeic earthworms reduced N2O emissions during the wheat culture (-44.6%), but this effect was not sustained throughout the experiment. No consistent effects on ecosystem evapotranspiration or WUE were found. Our study suggests that earthworms do not significantly contribute to GHG emissions over a two-year period in experimental conditions that mimic an agricultural setting. These findings highlight the need for realistic experiments and continuous GHG measurements.

Methods
The CO2 concentration was measured at the inlet and outlet of each dome (every 12 min) using a multiplexer system coupled with two LI-7000 CO2/H2O analysers (LI-COR Biosciences, Lincoln, NE, USA). The Reichstein et al. (2005) C flux partitioning algorithm was used to estimate the daytime ecosystem respiration based on an exponential regression model (Lloyd et al., 1994). This allowed for the estimation of ecosystem respiration over 24 h (Reco = Reco_night + Reco_day) and gross primary production (GPP = NEE_day – Reco_day).

Ecosystem-level N2O fluxes were measured continuously as an open system using a TILDAS Compact Single analyser (N2O Aerodyne Research, Inc., USA). The analyzer was coupled to a multiplexer system allowing N2O fluxes measurement every 72 min for each Macrocosm. Evapotranspiration (ET) was computed as the lysimeter weight difference between two consecutive days. Four shear beam load cells per lysimeter (CMI-C3, Precia-Molen, Privas CEDEx France), with an accuracy of ±200 g, were used to measure the changes in mass. Ecosystem WUE was estimated as the ratio of GPP to ET derived from measurements by lysimeter weight changes over 24h.

Earthworms were sampled by utilizing the non-invasive octet electric method (Schmidt et al., 2001), which enabled sampling of a 1 m² surface area for a duration of 50 minutes using a customized version of the octet device manufactured by Electrotechnik Schuller (Darmstadt, Germany). To achieve coverage of the entire 5 m² surface area, five devices were simultaneously deployed.

References:

Reichstein, M. et al. On the separation of net ecosystem exchange into assimilation and ecosystem respiration: review and improved algorithm. Glob. Change Biol. 11, 1424–1439 (2005).

Lloyd, J. & Taylor, J. A. On the Temperature Dependence of Soil Respiration. Funct. Ecol. 8, 315–323 (1994).

Schmidt, O. Appraisal of the electrical octet method for estimating earthworm populations in arable land. Ann. Appl. Biol. 138, 231–241 (2001).

已知蚯蚓可促进土壤温室气体（GHG）排放，但既往多数研究均采用简化模型系统，或未开展连续高频监测。为此，我们在生态控制舱（ecotron）设施中开展了为期两年的研究，采用大型渗漏计（lysimeter，面积5 m²、土层深度1.5 m），持续监测生态系统尺度的CO₂、N₂O及H₂O通量。我们在模拟农业生境中，探究了内栖蚯蚓（endogeic）与深栖蚯蚓（anecic）对温室气体排放及生态系统水分利用效率（WUE）的影响。尽管我们观测到蚯蚓存在时碳通量出现短暂提升，但研究周期内的累积通量数据显示，CO₂排放并无显著增加。内栖蚯蚓在小麦种植期内降低了N₂O排放（降幅达44.6%），但该效应并未在整个实验周期中持续。本研究未发现其对生态系统蒸散发（ET）或水分利用效率存在一致影响。本研究表明，在模拟农业生境的实验条件下，两年周期内蚯蚓对温室气体排放并无显著贡献。上述研究结果凸显了开展真实情境实验与连续温室气体监测的必要性。

研究方法
CO₂浓度监测：采用多通道采样系统搭配两台LI-7000型CO₂/H₂O分析仪（LI-COR Biosciences，美国内布拉斯加州林肯市），于每个气室的进气口与出气口处监测CO₂浓度，监测频率为每12分钟一次。基于指数回归模型（Lloyd等，1994），采用Reichstein等（2005）提出的碳通量分配算法，估算日间生态系统呼吸速率。据此可计算24小时生态系统总呼吸量（Reco = 夜间呼吸量Reco_night + 日间呼吸量Reco_day）与总初级生产力（GPP = 日间净生态系统交换量NEE_day – 日间呼吸量Reco_day）。

N₂O通量监测：采用开放式系统搭配TILDAS Compact Single型分析仪（美国Aerodyne Research公司N₂O分析仪），持续监测生态系统尺度的N₂O通量。该分析仪连接至多通道采样系统，可实现每个实验微宇宙（Macrocosm）的N₂O通量每72分钟监测一次。

蒸散发与水分利用效率计算：蒸散发（ET）通过相邻两日渗漏计的重量差计算得到。每个渗漏计配备4个剪切梁式称重传感器（CMI-C3型，Precia-Molen公司，法国普里瓦CEDEx市），测量精度为±200 g，用于监测重量变化。生态系统水分利用效率通过24小时内基于渗漏计重量变化测得的总初级生产力与蒸散发的比值估算得到。

蚯蚓采样方法：采用无创八电极电学法（octet electric method，Schmidt等，2001）开展蚯蚓采样：使用德国达姆施塔特Electrotechnik Schuller公司定制的八电极装置，可在50分钟内完成1 m²地表面积的采样。为覆盖全部5 m²的地表面积，需同时部署5台该装置。

参考文献
1. Reichstein, M. 等. 净生态系统交换量分解为同化作用与生态系统呼吸量的方法：综述与改进算法. 全球变化生物学（Glob. Change Biol.）, 11, 1424–1439 (2005).
2. Lloyd, J. & Taylor, J. A. 土壤呼吸的温度依赖性. 功能生态学（Funct. Ecol.）, 8, 315–323 (1994).
3. Schmidt, O. 评估八电极电学法估算耕地蚯蚓种群数量的适用性. 应用生物学年报（Ann. Appl. Biol.）, 138, 231–241 (2001).