# Earthworm impacts on soil carbon storage: the importance of quantifying all drilosphere compartments

Hoang Xuan Dang, Quang Van Pham, Tien Minh Tran, Cornelia Rumpel, Nicolas Bottinelli

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Hoang Xuan Dang, Quang Van Pham, Tien Minh Tran, Cornelia Rumpel, Nicolas Bottinelli. Earthworm impacts on soil carbon storage: the importance of quantifying all drilosphere compartments. Applied Soil Ecology, 2025, 215, pp.106448. 10.1016/j.apsoil.2025.106448. hal-05482461

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# Earthworm impacts on soil carbon storage: the importance of quantifying all drilosphere compartments

Hoang Xuan Dang<sup>a,b</sup>, Quang Van Pham<sup>c</sup>, Tien Minh Tran<sup>b</sup>, Cornelia Rumpel<sup>a</sup>, Nicolas Bottinelli<sup>a,b,*</sup>

## ARTICLE INFO

### Keywords:

Bioturbation

Earthworms

Organic matter storage

## ABSTRACT

Earthworms play a key role in soil organic carbon (SOC) dynamics. Yet few studies have quantified SOC accumulation in specific drilosphere compartments as affected by different earthworm species and their interaction. The aim of this study was to determine the effects of the tropical species *Amynthas zenkevichi* (burrower) and *Pontoscolex corethrurus* (shallow bioturbator) on SOC accrual in the drilosphere, comprising surface casts, belowground casts, and burrow walls, over a three-month period under controlled laboratory conditions. Buffalo dung was provided as a surface food source. X-ray computed tomography combined with bulk density measurements were used to estimate the mass of burrow walls and belowground casts. Drilosphere compartments and surrounding soil were collected manually at the end of the experiment. Their SOC stocks and the incorporation of dung-derived carbon were quantified. Total SOC stocks in earthworm mesocosms were compared to those of control mesocosms without earthworms.

Although the drilosphere mass was similar between species, *A. zenkevichi* produced a higher mass of surface casts, whereas in *P. corethrurus* treatments more mass was allocated to burrow walls and belowground casts. Dung-derived carbon was more incorporated into casts than into burrow walls, with both species showing similar incorporation rates. For both species, SOC stocks in the soil mesocosms increased by 7 % relative to the control, with the majority of SOC stored in surface casts and burrow walls. In mixed species treatments, synergistic effects were observed with SOC stocks exceeding additive expectations derived from single species treatments by 3 %. Our findings highlight that two earthworm species belonging to contrasting functional groups have similar positive effects on SOC accrual in the drilosphere compartments and that their interspecific interactions may further enhance SOC stocks.

## 1. Introduction

Earthworms are key organisms in terrestrial ecosystems, given their substantial contributions to soil functioning (Blouin et al., 2013). They play a crucial role in modifying soil structure, soil organic carbon (SOC) and nutrient concentrations through their burrowing and feeding activities. Earthworms contribute to SOC accrual and turnover due to their impact on the formation of aggregates (Bossuyt et al., 2004), microbial necromass (Angst et al., 2024) and organo-mineral interactions (Vidal et al., 2019; Song et al., 2025). The impact of earthworms on SOC dynamics occurs within specific soil regions known as the drilosphere, the

part of the soil influenced by earthworm secretions, burrowing and casting activity (Lavelle, 1988). Drilosphere compartments include biogenic structures such as surface casts, belowground casts, and burrow walls, which exhibit distinct chemical, physical, and microbial properties compared to the surrounding soil (Brown et al., 2000; Johnson-Maynard and Strawn, 2016; Pham et al., 2024a). During earthworm gut passage, the ingested organic materials, e.g. plant litter are fragmented, partly digested and mixed with mineral particles, microorganisms, and mucus, before being returned to the soil as surface or belowground casts. These fresh casts are rich in readily decomposable carbon and exhibit elevated microbial and enzymatic activity (Zhang et al., 2000),

<footnote><sup>a</sup> IRD, CNRS, Sorbonne Université, Univ Paris Est Creteil, INRAe, Institute of Ecology and Environmental Sciences (iEES-Paris), F-75005, Paris, France</footnote>

<footnote><sup>b</sup> Department of Soil sciences, Soils and Fertilizers Institute (SFI), Hanoi, Viet Nam</footnote>

<footnote><sup>c</sup> Faculty of Environmental Sciences, Vietnam National University, Hanoi, Viet Nam</footnote>

<footnote>* Corresponding author at: IRD, CNRS, Sorbonne Université, Univ Paris Est Creteil, INRAe, Institute of Ecology and Environmental Sciences (iEES-Paris), F-75005, Paris, France.</footnote>

<footnote>E-mail address: nicolas.bottinelli@ird.fr (N. Bottinelli).</footnote>
accelerating the decomposition of organic matter (OM) (Abail et al., 2017). As cast aging occurs, OM associated to minerals or trapped within pore space of the casts showed increased persistence in the soil (Bottinelli et al., 2020; Puche et al., 2022). Another effect of earthworm burrowing activity is the rapid transport of fresh OM into the deeper layers of soil (Don et al., 2008). The wall of these burrows, often coated with earthworm casts or mucus, are enriched with OM (Jégou et al., 1998; Don et al., 2008; Hoang et al., 2016; Athmann et al., 2017). Despite the importance of the drilosphere for SOC dynamics, few studies have addressed all drilosphere compartments, as most works have focused on surface casts (Van Groenigen et al., 2019) often overlooking belowground compartments. With few exceptions qualitatively reporting changes in elemental composition (Jégou et al., 1998, 2001; Lipiec et al., 2015), research on earthworm effects on SOC does not yet address the whole drilosphere (Huang et al., 2020). Therefore, our understanding of how earthworms affect SOC dynamics remains incomplete, especially because drilosphere systems may be earthworm species specific.

Indeed, earthworms can be classified according to their bioturbation activity (Capowiez et al., 2024), resulting in six functional groups that partly correspond to the ecological categories proposed by Bouché (1977): intense tunnelers (anecic), burrowers (epi-anecic), deep bioturbators (hypo-endogeic), shallow bioturbators (epi-endogeic), litter dwellers (epigeic), and intermediates. Co-occurrence of earthworm species can lead to interactions and synergistic effects, as we recently demonstrated for bioturbation activity in a laboratory study involving two tropical earthworm species commonly found in Vietnam (Pham et al., 2024a). The two species included in the present study, *Amynthas zenkevichi* and *Pontoscolex corethrurus*, exhibited contrasting burrowing behaviors but similar surface feeding activity. *A. zenkevichi*, likely endemic to South-East Asia, constructed few but continuous vertical burrows that reached the soil surface, produced surface casts, and actively consumed surface litter. This behavior is consistent with the burrower group. In contrast, *P. corethrurus*, a widespread invasive species in tropical and subtropical environments, displayed a more diffuse and shallow burrowing pattern, with discontinuous burrows filled with belowground casts, while also feeding on OM provided at the soil surface. This behavior is characteristic for shallow bioturbators. The results of the former study indicated that mesocosms containing both *A. zenkevichi* and *P. corethrurus* increased surface cast production, thereby increasing soil resistance to water loss by evaporation (Pham et al., 2024a). As casting behavior may have strong impacts on soil OM dynamics (Song et al., 2025), we hypothesized in the present study that species interactions might also influence soil OM storage in drilosphere compartments. Indeed, while physiological and behavioral differences among species may lead to contrasting cast properties (Zi et al., 2025), interspecific interactions can further modify these impacts, through behavioral adjustments to minimize competition, reuse of existing burrows, and commensal relationships enhancing resource utilization (Jégou et al., 2001; Capowiez et al., 2001; Felten and Emmerling, 2009; Pham et al., 2024a). Although relatively few studies have investigated the impact of interspecific interactions on SOC cycling, their data indicate that mixed-species treatments can enhance microbial respiration and carbon mineralization (Postma-Blaauw et al., 2006; Speratti and Whalen, 2008). However, to the best of our knowledge, up to now it is unknown how the interaction of different earthworm species affects SOC accrual in the drilosphere compartments.

For the present study, we sampled three distinct drilosphere compartments (surface casts, belowground casts and burrow walls), as well as the surrounding soil, and investigated their contribution to SOC storage as influenced by the two earthworm species and their interaction. Additionally, stable carbon isotope analysis was performed to determine whether the SOC associated with these compartments originated from newly added or native OM, in order to investigate the effect of single species and their combination on aboveground and belowground SOC accrual. We hypothesized, firstly, that the drilosphere

compartments would exhibit higher SOC enrichment than the surrounding bulk soil, with distinct differences between surface casts, belowground casts, and burrow walls reflecting varying mechanisms of OM incorporation. Moreover, given that both species ingested similar amounts of OM provided at the soil surface (Pham et al., 2024a), we hypothesized that their contrasting burrowing behaviors may lead to differences in the spatial distribution of SOC within the drilosphere, rather than changes in its total amount or biochemical quality. Additionally, we expected interactions between *A. zenkevichi* and *P. corethrurus* to substantially modify SOC stocks through alterations in their burrowing patterns and casting activities.

## 2. Materials and methods

### 2.1. Soil, earthworms and experimental design

Soil was collected from large outdoor mesocosms (clay = 22 %, silt = 35 %, sand = 43 %, SOC = 12.0 mg g⁻¹, nitrogen = 1.3 mg N g⁻¹ and pH<sub>water</sub> = 5.3) of our study site in Hanoi, northern Vietnam (21°07′ N, 105°75′ E), which were previously used for growing maize (Doan et al., 2015). The soil was air dried and sieved at 2 mm to remove any remaining earthworms and cocoons. Mature specimens of *Pontoscolex corethrurus* (Rhinodrilidae) and *Amynthas zenkevichi* (Megascolecidae) were collected in a cropland, which was located <1 km away from the site, where the soil used for incubation originated (20°57′ N 105°29′ E). While both species are often found on the same site in Vietnam in the present study they were collected from separate fields. After collection, earthworm species were kept in a dark room in the same soil used in the experiment. Three days prior to start of the incubation, earthworms were transferred to moistened tissues to empty their guts. Buffalo dung with a 8¹³C signature of -27.2 ‰ was collected in a farm close to where the soil (8¹³C signature of -21.9 ‰) and earthworms were collected. We investigated the influence of earthworms on SOC storage using the experimental setup described in detail by Pham et al. (2024b). Briefly the experimental setup consisted of mesocosms made of polyvinyl chloride cores (height: 36 cm, diameter: 20 cm) filled with sieved soil and packed with a hydraulic press to reach a dry bulk density of 1 g cm⁻³. The soil was remoistened at 40 % gravimetric water content. Individuals of single or mixed species were introduced at the surface of the soil mesocosms with a total fresh earthworm biomass of approximately 3 g per mesocosm. Consequently, we added to the single-species mesocosms two *A. zenkevichi* (mean individual weight of 1.8 g) or six *P. corethrurus* (mean individual weight of 0.5 g). To the mixed-species mesocosms, we added one *A. zenkevichi* and three *P. corethrurus*. Food in the form of 2 g air-dried and then remoistened buffalo dung was added to the top of the mesocosms and replenished whenever food at the surface ran out. Each time another 2 g were added. Buffalo dung was used because it closely mimics the natural diet available in the field, where the earthworms were collected, and also presented a 13C label allowing for stable isotope tracing of added food to study recent carbon accrual in drilosphere compartments. Three soil mesocosms without earthworm additions were kept as a control. In total, we prepared 17 soil mesocosms including: single-species mesocosms for *A. zenkevichi* (n = 4) and *P. corethrurus* (n = 4), mixed species assemblages (n = 6), and control replicates (n = 3). The incubation lasted 92 days, and mesocosms were kept in the dark at 25 °C.

### 2.2. Collection and mass determination of the drilosphere and surrounding soil

We defined three drilosphere compartments within each column: (i) the burrow wall up to 2 mm from the lumen of the burrow (Bouché, 1977), (ii) the surface casts and (iii) the belowground casts. In addition, we considered the surrounding soil, situated at least at 20 mm from the burrow wall. Surface casts were collected at the end of the experiment, air-dried and weighed. To sample the belowground drilosphere
compartments (casts, burrow walls) and surrounding soil, the PVC columns were cut in half vertically, and the two halves were gently separated to expose the soil profile. This method preserved the structure of the soil and minimized mixing between compartments. Casts and burrow walls were then carefully identified and collected along the entire depth of the column using sterile spatulas, while surrounding soil was collected at least 20 mm from the burrows to prevent mixing. The samples were airdried and stored before analyses of their properties.

As it was not feasible to determine the mass of belowground casts and burrow walls through manual collection, we assessed their volume using X-ray computed tomography (Pham et al., 2024b) and estimated their mass using their bulk density. The volume of burrow walls was calculated as follows: first, we assessed the length of the burrow from the volume measured using X-ray computed tomography, considering that burrows are tube-like; second, we calculated the new volume of burrows assuming the radius extends by 2 mm. Finally, the difference between this extended volume and the original burrow volume represented the volume of burrow walls. The bulk density of casts (both surface and belowground) and burrow walls was measured using the kerosene saturation method (McIntyre and Stirk, 1954) whereas the bulk density of the surrounding soil outside the drilosphere and in control soil without earthworm was determined by extracting and weighing a cylinder (diameter × height = 3.8 × 4 cm) after oven drying at 105 °C. To avoid including burrows in the bulk density measurements of the surrounding soil, we visually inspected the exposed soil profile after cutting the columns in half and selected areas that showed no visible signs of burrow structures. The cylinders were then inserted only into portions of the soil that were clearly outside the drilosphere, ensuring accurate bulk density measurements of undisturbed surrounding soil. Because *A. zenkevichi* produced only very small amounts of belowground casts, the collected material was insufficient to do any analyses and thus we considered the contribution of belowground casts from *A. zenkevichi* negligible.

## 2.3. Elemental analyses and stable carbon and nitrogen isotope ratios

The total carbon and nitrogen concentrations of air-dried samples (< 200 µm) of drilosphere compartments, and (surrounding) soil were analyzed using an elemental analyzer (Leco CN-TruSpec®, St. Joseph, MI, USA). Total carbon concentrations are representing soil organic carbon (SOC), as no carbonates were present in this tropical soil. SOC stocks were calculated by multiplying the mass of each drilosphere compartment by their respective SOC concentrations. Stable isotopes of SOC were analyzed using a continuous-flow Elementar® VarioPyro cube analyzer coupled with a Micromass® Isoprime Isotope Ratio Mass Spectrometer (EA-IRMS). The results were expressed as δ¹³C relative to tyrosine standards. We then calculated the rate of buffalo dung (F value) incorporated into the drilosphere components by using the isotopic mass balance (Eq. (1)):

$$F = \frac{(\delta^{13}C_{\text{t}} - \delta^{13}C_{\text{control soil}})}{(\delta^{13}C_{\text{buffalo dung}} - \delta^{13}C_{\text{control soil}})} \quad (1)$$

These data were used to calculate the SOC stocks derived from buffalo dung in the drilosphere compartments.

## 2.4. Statistical analysis

Statistical analyses were performed with R version 4.2.0 (R Core Team, 2024), and the figures were produced with the package "ggplot2" (Wickham et al., 2016). Due to the unbalanced nature of the dataset, as belowground casts from *A. zenkevichi* could not be obtained, differences in soil properties (bulk density, carbon concentration, C/N ratio, δ¹³C and C derived from buffalo dung) in earthworm treatments were tested using two-way ANOVA without interaction. Soil compartments (surface casts, belowground casts, burrow walls, and surrounding soil) and earthworm treatments (*A. zenkevichi*, *P. corethrurus*, and mixed species)

were the main factors. Following ANOVA, post-hoc analyses were conducted using Tukey's HSD test, implemented via the "multcomp" package (Hothorn et al., 2016). For mass and SOC stock, Kruskal-Wallis tests were applied to assess differences among soil compartments and earthworm treatments due to violations of normality and homogeneity assumptions, followed by Dunn's post-hoc test. Additionally, one-way ANOVA was used to compare the total drilosphere mass and SOC stock between different earthworm treatments and the control mesocosms. To assess species interactions, we compared observed values in the mixed-species mesocosms to expected values under the assumption of no interaction. Since the earthworm mass was standardized at 3 g in all mesocosms, the expected values in mixed-species mesocosms without interaction corresponded to the average values from single-species mesocosms for each parameter. Earthworm interaction was defined as significant deviation in experimental results from the expected additive values obtained by summing the results from single-species treatments, and this was tested using a one-sample t-test. Before conducting t-tests and ANOVA, parameters were tested for normality and homogeneity of variances using the Shapiro-Wilk test and Levene's test. Log 10-transformations were applied when data failed to meet the assumptions of normality or homogeneity of variances. Log 10-transformed data were used solely for statistical analysis to ensure valid application of t-tests and ANOVA. Differences among treatments were considered significant at p < 0.05.

# 3. Results

## 3.1. Bulk density and mass of drilosphere compartments

Bulk density of surrounding soil and drilosphere components varied between 1.1 and 1.7 g cm⁻³ in mesocosms containing earthworms (Table 1). On average, these values were higher than those in control mesocosms without earthworms (Table 1). Bulk density was lowest in surrounding soil and significantly increased in burrow walls (by a factor of 1.3), belowground casts (by a factor of 1.4) and surface casts (by a factor of 1.6) (p < 0.001). The bulk density of drilosphere compartments was significantly affected by earthworm treatment (p < 0.05), with *A. zenkevichi* producing compartments with lower bulk density compared to those formed by *P. corethrurus*, while the mixed species treatment showed intermediate values. The total drilosphere mass (Fig. 1) varied between 356 and 516 g and was not influenced by earthworm treatment (p > 0.05). The total mass of the drilosphere, including all three compartments, in mixed species mesocosms was similar to the expected additive value. The mass of burrow walls was higher than those of belowground casts and surface cast (p < 0.001), without significant differences among earthworm treatments. However, we found that the masses of surface casts in the mixed species treatment were greater than the expected additive values (p < 0.001).

## 3.2. Soil organic carbon (SOC) concentrations, C/N ratios and SOC stocks

The SOC concentrations ranged between 12.4 and 50.3 mg g⁻¹, and the ratio between carbon and nitrogen concentrations (C/N ratio) varied between 10.5 and 13.5 in mesocosms with earthworms (Table 1). In casts and burrow walls, these values were significantly higher than those of control mesocosms without earthworms (12.0 ± 0.2 mg SOC g⁻¹ and 9.7 ± 0.2 for C/N). The SOC concentrations increased in the order: control mesocosms = surrounding soil < burrow walls (greater by a factor of 1.4) < belowground casts (greater by a factor of 2.1) < surface casts (greater by a factor of 4.1) (p < 0.001), regardless of the earthworm treatment (p > 0.05). The C/N ratios were higher in surface casts (by a factor of 1.3) and belowground casts (by a factor of 1.1), than in burrow walls and surrounding soil. C/N ratios were not influenced by earthworm treatment (p > 0.05). The SOC stocks (Fig. 2) ranged from 119.8 to 125.0 g in mesocosms with earthworms and were higher (p <
<caption>**Table 1**</caption>

Means and SD of soil bulk density, SOC concentration, C/N ratio, δ¹³C and SOC derived from buffalo dung (δ¹³C = -27.2 ‰) for control soil and drilosphere compartments separated from mesocosms with *A. zenkevichi*, *P. corethrurus*, and mixed treatments. Difference between soil compartments and earthworm treatments were tested by additive two-way ANOVA. Values bearing different letters are statistically significantly different within each property. ns = not significant. F indicates the variance ratio.

<table><thead><tr><th></th><th>Treatment</th><th>Bulk density (g cm<sup>-3</sup>)</th><th>SOC (mg g<sup>-1</sup>)</th><th>C/N</th><th>δ¹³C (‰)</th><th>SOC derived from buffalo dung (%)</th></tr></thead><tbody><tr><td rowspan="5">Soil compartments</td><td>Control soil</td><td>1.00 (0.01)<sup>a</sup></td><td>12.03 (0.19)<sup>d</sup></td><td>9.72 (0.22)<sup>d</sup></td><td>-21.95 (0.06)<sup>a</sup></td><td>0.94 (0.98)<sup>c</sup></td></tr><tr><td>Surface cast</td><td>1.72 (0.05)<sup>a</sup></td><td>50.31 (17.8)<sup>a</sup></td><td>13.47 (1.09)<sup>a</sup></td><td>-24.16 (0.47)<sup>c</sup></td><td>41.83 (8.67)<sup>a</sup></td></tr><tr><td>Belowground cast<sup>(1)</sup></td><td>1.57 (0.06)<sup>b</sup></td><td>26.54 (6.53)<sup>b</sup></td><td>12.19 (0.84)<sup>b</sup></td><td>-23.14 (0.47)<sup>b</sup></td><td>23.13 (8.54)<sup>a</sup></td></tr><tr><td>Burrow wall</td><td>1.39 (0.04)<sup>c</sup></td><td>17.19 (4.16)<sup>c</sup></td><td>11.05 (0.63)<sup>c</sup></td><td>-23.07 (0.42)<sup>b</sup></td><td>21.58 (7.77)<sup>b</sup></td></tr><tr><td>Surrounding soil</td><td>1.09 (0.01)<sup>d</sup></td><td>12.35 (0.51)<sup>d</sup></td><td>10.52 (0.92)<sup>cd</sup></td><td>-22.15 (0.12)<sup>a</sup></td><td>4.65 (2.27)<sup>c</sup></td></tr><tr><td rowspan="3">Earthworm treatments</td><td><i>A. zenkevichi</i></td><td>1.40 (0.26)<sup>b</sup></td><td>30.78 (26.11)</td><td>12 (1.74)</td><td>-23.35 (0.89)</td><td>26.87 (16.53)</td></tr><tr><td><i>P. corethrurus</i></td><td>1.45 (0.25)<sup>a</sup></td><td>23.53 (10.86)</td><td>11.58 (1.14)</td><td>-23.13 (0.86)</td><td>22.77 (16.01)</td></tr><tr><td>Mixed</td><td>1.44 (0.25)<sup>ab</sup></td><td>26.57 (17.52)</td><td>11.8 (1.55)</td><td>-23.01 (0.8)</td><td>20.72 (14.79)</td></tr><tr><td rowspan="2">ANOVA</td><td>Soil compartments</td><td>(F = 939.9)<sup>***</sup></td><td>(F = 87.5)<sup>***</sup></td><td>(F = 43.9)<sup>***</sup></td><td>(F = 92.4)<sup>***</sup></td><td>(F = 92.8)<sup>***</sup></td></tr><tr><td>Earthworm treatments</td><td>(F = 22.6)<sup>***</sup></td><td>(F = 0.50) ns</td><td>(F = 0.80) ns</td><td>(F = 0.87) ns</td><td>(F = 0.84) ns</td></tr></tbody></table>

<figure><img src="image_2.png" /><figcaption>**Fig. 1.** Means and SD of the mass of the drilosphere for *A. zenkevichi*, *P. corethrurus*, mixed-species treatments and expected values. Differences between drilosphere compartments and earthworm treatments were tested using a Kruskal-Wallis test (lowercase letters), whereas differences in the total mass of the drilosphere among earthworm treatments were tested using one-way ANOVA (uppercase letters). Lower/uppercase letters indicate significant pairwise differences. Differences between observed and expected mass of drilosphere compartments of mixed species treatments were tested using t-tests. The star indicates a significant difference between observed and expected additive values.</figcaption></figure>

0.05) than those in control mesocosms without earthworms (112.8 g). *A. zenkevichi* was not sampled for belowground casts, so no SOC stock was recorded for this compartment. While similar SOC stocks were recorded among earthworm treatments, their distribution in soil compartments varied. The SOC stocks were higher in surface casts (by a factor of 6.7) and burrow walls (by a factor of 6.1), than in belowground casts in *P. corethrurus* and mixed species treatments. The total SOC stocks in drilosphere compartments of mesocosms with mixed-species were 3 % higher than expected values based on single-species treatments (p < 0.05). This increase was attributed to the additional amount of SOC stored in surface casts (p < 0.05).

### 3.3. C transfer from the food source at the soil surface into the drilosphere

δ¹³C values in the drilosphere compartments and surrounding soil ranged from -22.1 to -24.2 ‰ in mesocosms with earthworms (Table 1). These values were lower than those of control mesocosms without earthworms (-21.9 ‰). δ¹³C values decreased in the following order: surrounding soil < belowground casts = burrow walls < surface casts (p < 0.001). No differences in δ¹³C were observed among earthworm treatments (p > 0.05). Depleted ¹³C values in the drilosphere

<figure><img src="image_3.png" /><figcaption>**Fig. 2.** Means and SD of SOC stock for control soil, *A. zenkevichi*, *P. corethrurus*, mixed-species treatments, and expected additive values. Differences between soil compartments and earthworm treatments were tested using a Kruskal-Wallis test (lower case letters), whereas differences between the total SOC stock of earthworm treatments and control mesocosms were tested by one-way ANOVA (upper case letters). Lower/uppercase letters indicate significant pairwise differences. Differences between observed and expected SOC stock of soil compartments of mixed species treatments were tested using t-tests. The star indicates a significant difference between observed and expected additive values.</figcaption></figure>

compartments are related to contribution of SOC derived from buffalo dung provided as a food source at the soil surface. Isotopic mass balance calculations indicated that the proportion of SOC derived from buffalo dung ranged from 4.6 to 41.8 % and increased in the following order: surrounding soil < burrow walls < belowground casts < surface casts (p < 0.05).

<footnote>*** p < 0.001.</footnote>

<footnote>(1) Data for *A. zenkevichi* are not included as no belowground casts could be collected.</footnote>
< 0.001), regardless of earthworm treatments (p > 0.05).

# 4. Discussion

## 4.1. Effects of *A. zenkevichi* and *P. corethrurus* on drilosphere formation

Our experiment showed that *A. zenkevichi* constructs few vertical burrows and deposits casts at the soil surface. Belowground casts were not collected for *A. zenkevichi* due to insufficient material, likely resulting from their compaction into burrow walls during repeated passage (Jégou et al., 2001). In contrast, *P. corethrurus* forms burrows close to the surface (Pham et al., 2024b), most of which are backfilled with casts. For both species, burrow walls represented the largest component of the drilosphere, accounting for more than 60 % of the mass. In mixed species treatments, the mass of surface casts was 53 % higher than in the single species treatment, which might be attributed to the greater activity observed for *P. corethrurus* (Pham et al., 2024b).

The drilosphere had a higher bulk density than the surrounding soil, confirming local compaction resulting from earthworm activity during the formation of casts and burrows (Pham et al., 2024a). Surface casts had greater bulk density than those formed belowground, likely because the soil near the surface experiences more frequent drying and rewetting, unlike the more stable moisture conditions deeper in the mesocosm. Contrary to our expectations, the bulk density of burrow walls did not differ significantly between species, despite their distinct burrowing strategies. This similarity may be explained by the initial bulk density of the soil mesocosms, which was too high to detect any species-specific differences, or by the similar body diameter of both species.

## 4.2. SOC accrual in the drilosphere compartments

Compared to the surrounding soil, SOC concentrations increased by a factor of 4.1 in surface casts, 2.1 in belowground casts, and 1.4 in burrow walls. These values are consistent with findings from numerous other studies, highlighting carbon enrichment in drilosphere compartments formed by temperate and tropical earthworm species (e.g. Jégou et al., 1998; Lipiec et al., 2015; Van Groenigen et al., 2019).

Stable carbon isotope analyses revealed greater incorporation of carbon derived from buffalo dung into surface and belowground casts than into burrow walls. This distribution is consistent with other studies on epi-anecic, anecic and endogeic species, which have reported a preferential incorporation of carbon derived from surface litter into casts rather than burrows (Jégou et al., 1998, 2000). This may be related to the contrasting incorporation pathways of litter/dung derived carbon in the two drilosphere compartments. Casts are formed after gut passage and may contain undigested litter/dung compounds, while the origin of carbon in burrow walls may be external mucus being composed of digestion products deposited by the earthworms. The higher C/N ratios of casts support this interpretation, indicating that both *A. zenkevichi* and *P. corethrurus* incorporated relatively fresh, undigested OM during casting activity and strongly decomposed materials during burrow wall formation. These findings showed that both species actively fed on soil surface in accordance with their behavior in the natural environment. However, burrow walls exhibited an intermediate level of incorporation of carbon derived from buffalo dung, positioned between those of the casts and the surrounding soil. This could indicate that burrow walls also contain belowground cast material as shown previously by observation of soil thin sections (Jégou et al., 2000). However, the significantly lower C/N ratio of burrow walls compared to belowground casts suggests that the deposition of mucus, a nutrient rich lubricant that facilitates earthworm movement through soil and adheres to mineral surfaces (Guhra et al., 2020) may have contributed to buffalo-derived SOC allocation to the burrow walls. In summary, our results showed a contrasting SOC content within the drilosphere compartments, which was highly influenced by incorporation of new carbon derived from buffalo dung, supporting the important role of earthworms for SOC

sequestration (Angst et al., 2019; Lavelle, 2022).

## 4.3. Consequences for SOC stocks

Our results showed that both earthworm species individually or in combination increased the total SOC stocks by approximately 7 % compared to the control soil without earthworms. This finding contrasts with two previous meta-analyses (Lubbers et al., 2013; Huang et al., 2020), which reported no significant effects of earthworms on SOC stocks when comparing treatments with and without earthworms under laboratory or field conditions. The reasons may be that the studies included in the meta-analyses (1) did not consider anecic species separately (Lubbers et al., 2013) and (2) focused on SOC concentrations rather than SOC stock changes (Huang et al., 2020). However, similar to our study, a positive effect of earthworms on SOC storage was observed by Qiu et al. (2025) following six months of semi-field incubation with the earthworm *Pheterima guillelmi*.

In our study, the additional SOC was mainly stored in surface casts and burrow walls, while belowground casts of *P. coruthurus* showed lowest SOC stocks or were absent in *A. zenkevichi* treatments. Surface casts stored 6.7 times more SOC and burrow walls 6.1 times more SOC than recorded in belowground casts of *P. corethrurus*. The SOC stored in surface casts may be more exposed to environmental perturbations, such as rainfall and drying, making it potentially less stable than SOC stored deeper in the soil (Song et al., 2024). In contrast, SOC incorporated into belowground cast or burrow walls, especially those extending into deeper soil layers as the ones constructed by *A. zenkevichi*, is more likely to be physically protected and may therefore be more persistent over time. Further studies are needed to elucidate the lifetime of SOC in the contrasting drilosphere compartments, especially under field conditions. These studies should select earthworm species, which produce belowground casts to account for all drilosphere compartments. As earthworms contribute to the protection of fresh OM in aggregates and the formation of mineral-associated SOC (Vidal et al., 2019; Song et al., 2025), the nature of SOC and its allocation into pools of contrasting stability should also be studied. Moreover, the effect of species interactions on SOC stability still needs to be assessed.

The mixed-species treatment exhibited synergistic effects, with SOC stocks exceeding those predicted from the additive contributions of single species treatments by approximately 3 %. This effect was explained by the greater production of surface casts. As single species showed similar production of surface casts, we attribute increased cast production in mixed treatments to interaction of both species. This finding suggests that interspecific interactions can significantly alter the spatial distribution and probably dynamics of SOC storage. In addition to the increase in SOC stocks, we previously presented improved soil resistance to water loss under mixed-species treatments in this experiment (Pham et al., 2024b). Although species behavior is most likely impacted by the laboratory conditions, our observations indicate the possible ecological benefits of species mixtures and thus support fieldwork showing the importance of earthworm diversity for soil functioning (Mulia et al., 2021).

### CRediT authorship contribution statement

**Hoang Xuan Dang:** Writing – original draft, Investigation, Formal analysis, Data curation. **Quang Van Pham:** Writing – review & editing, Investigation, Data curation, Conceptualization. **Tien Minh Tran:** Writing – review & editing, Supervision, Resources, Project administration. **Cornelia Rumpel:** Writing – review & editing, Validation, Supervision, Resources, Project administration, Funding acquisition, Conceptualization. **Nicolas Bottinelli:** Writing – review & editing, Validation, Supervision, Project administration, Methodology, Investigation, Funding acquisition, Data curation, Conceptualization.
Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

Data will be made available on request.

Acknowledgements

This project was financially supported by the ANR project U2 worm (ANR-20-CE01-0015-01) and the ANR project PRECIOUS (ANR-19- CE01-0003). HXD received funding from the IRD and the French Em- bassy in Hanoi.

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