# Let's get functional: Relationship between earthworm traits and physicochemical cast properties

Yacouba Zi, Nicolas Bottinelli, Malalatiana Razafindrakoto, Yvan Capowiez, Alessandro Florio, Chao Song, Cornelia Rumpel, Marie-France Dignac

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Yacouba Zi, Nicolas Bottinelli, Malalatiana Razafindrakoto, Yvan Capowiez, Alessandro Florio, et al. Let's get functional: Relationship between earthworm traits and physicochemical cast properties. Soil Biology and Biochemistry, 2025, 207, pp.109809. 10.1016/j.soilbio.2025.109809. hal-05052536

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# Let's get functional: Relationship between earthworm traits and physicochemical cast properties

Yacouba Zi<sup>a, b, c</sup>, Nicolas Bottinelli<sup>a, b, c</sup>, Malalatiana Razafindrakoto<sup>c</sup>, Yvan Capowiez<sup>d</sup>,
Alessandro Florio<sup>e, f</sup>, Chao Song<sup>a, f</sup>, Cornelia Rumpel<sup>a, *, e</sup>, Marie-France Dignac<sup>a</sup>

## ARTICLE INFO

### Keywords:

- Earthworms

- Functional trait

- Ecological category

- Cast properties

## ABSTRACT

Although earthworms play a crucial role in soil biogeochemical processes, the importance of their traits in shaping the physicochemical properties of their casts remains poorly understood. This study aimed (1) to evaluate the influence of earthworm species and soil types on cast properties and (2) investigate the relationship between earthworm morphological, anatomical, physiological and behavioral traits and the physicochemical properties of their casts. Nine temperate earthworm species were introduced under controlled conditions in two contrasting soil types (Luvisol and Cambisol), and 21 traits were determined for each species. Casts were analyzed for their physicochemical properties, total and available organic carbon and nitrogen, and compared to control soil incubated under similar conditions without earthworms. Results showed that earthworm activity changed soil pH depending on soil type, with pH increasing in Cambisol, and decreasing in Luvisol. Species-specific effects on cast properties revealed a physicochemical gradient: the epigeic *L. castaneus* and epi-anecic *L. terrestris* produced casts with the highest nitrate, dissolved organic carbon, total organic carbon, and moisture levels, whereas cast from endogeic species showed the lowest values. Moreover, earthworm species exerted a stronger overall influence on cast properties than soil type (59 % vs. 24 %), underscoring the dominant role of species-specific traits in shaping cast characteristics. We identified nine key traits related to the earthworms' morphology, anatomy, physiology and behavior, that influenced cast properties directly or indirectly. Direct effect traits included mouth area, gizzard size and location, typhlosole complexity, intestinal mucus and cast production. Indirect effect traits, such as pigmentation, litter ingestion and litter-to-cast ratio, reflected the ecological behavior of earthworm species. This trait-based approach provides a promising avenue for future studies on the role of earthworms in soil biogeochemical cycling and a framework for improving our understanding of their impacts on soil organic matter dynamics.

## 1. Introduction

Earthworms are among the most abundant faunal groups in terrestrial ecosystems (Edwards, 2004) and are widely recognized as ecosystem engineers (Lavelle, 1997). Through their bioturbation activity, they play a crucial role in pedogenesis and soil functioning (Blouin et al., 2013; Lavelle et al., 1998; Le Bayon et al., 2021), notably through the formation of casts with distinct chemical, physical, and biological properties (Bottinelli et al., 2015; van Groenigen et al., 2019). These

casts, enriched with carbon and nutrients in their available forms, directly enhance soil fertility (Oyedele et al., 2006; Song et al., 2024; Teng et al., 2012; van Groenigen et al., 2019; Vos et al., 2019). The physicochemical properties of casts are influenced by both the soil material ingested and earthworm species, with interaction between these factors playing a significant role (Babiy et al., 2021; Clause et al., 2014; Jana et al., 2010; Schrader and Zhang, 1997). The relative importance of both factors and/or the specific earthworm traits influencing cast properties have to the best of our knowledge not been

<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 Research Institute (SFRI), Hanoi, Viet Nam</footnote>

<footnote><sup>c</sup> Université D'Antananarivo, Laboratoire des Radio-Isotopes (LRI), Antananarivo, Madagascar</footnote>

<footnote><sup>d</sup> INRAE, UMR1114, Environment Méditerranéen et Modélisation des Agro-Hydrosystèmes, Site Agroparc, Avignon, 84914, cedex 09, Paris, France</footnote>

<footnote><sup>e</sup> INRAE, Université Claude Bernard Lyon 1, CNRS, VetAgro Sup, UMR Ecologie Microbienne, F 69622, Villeurbanne, France</footnote>

<footnote><sup>f</sup> Institute of Hydrogeology and Environmental Geology, Chinese Academy of Geological Sciences, Shijiazhuang, 050061, China</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: cornelia.rumpel@inrae.fr (C. Rumpel).</footnote>
determined up to now.

Traditionally, earthworm species have been classified into (i) epigeic species, comprising small, heavily pigmented, short-lived worms that live in and consume litter at the soil surface, (ii) anecic species, including larger worms with a dark antero-dorsal pigmentation that feed on litter and transport it into the soil; and (iii) endogeic species, composed of intermediate-sized worms that live within the soil and feed on soil organic matter (SOM) (Bottinelli et al., 2020; Bouché, 1977). These ecological groups have been taken as proxies for their functionality, although this was not their purpose when this classification was developed (Capowiez et al., 2024a). Indeed, many studies emphasized the limitations of the ecological categories for predicting earthworm effects on ecosystem processes (Babiy et al., 2021; Curry and Schmidt, 2007; van Groenigen et al., 2019; Vos et al., 2019), as it does not account for within-group functional diversity (Hedde et al., 2022).

The trait-based approach has emerged as a powerful alternative for understanding how organisms influence their environment and contribute to ecosystem processes (Marichal et al., 2017; McCary and Schmitz, 2021; Pham et al., 2023, 2024). Unlike taxonomy-based methods, which focus on species identification, this approach emphasizes functional diversity by linking measurable traits of individual organism to ecological functions and environmental responses (Brousseau et al., 2018; Joimel et al., 2024; Pey et al., 2014; Violle et al., 2007). Applied to earthworm communities, the trait-based approach enables the quantification of key processes, such as organic matter decomposition, translocation, and stabilization (Bonfanti et al., 2024), while improving predictions of community responses to environmental changes, including soil contamination (Hedde et al., 2013), land-use alterations (Pelosi et al., 2016), and climate change (Bonfanti et al., 2022). By focusing on specific traits, such as those influencing soil structure, water infiltration, and cast properties, this approach may provide a mechanistic understanding of the relationships between earthworm communities and soil functioning (Bonfanti et al., 2024; Joimel et al., 2024; Mcgill et al., 2006; Pham et al., 2023, 2024). It thus addresses the limitations of traditional classifications by capturing within-group variability and offering a more nuanced perspective on earthworm contributions to ecosystem processes. We hypothesized that this approach may also be adequate to address the impact of earthworms on SOM dynamics and in particular, the physicochemical properties of their casts.

In this study, we thus investigated the importance of earthworm traits in determining cast properties related to the biogeochemical cycling of carbon and nitrogen. Our objectives were to (1) assess the impact of earthworm species belonging to four ecological categories, soil types and their interaction on cast properties, and (2) explore the link between earthworm traits and cast properties, i.e., to identify functional traits. We selected 15 morpho-anatomical, 3 physiological, and 3 behavioral traits of nine temperate earthworm species spanning four ecological categories (Bottinelli et al., 2020; Bouché, 1972, 1977). Physiological traits, related to an organism's internal functions such as metabolism and nutrient assimilation (Calow, 2012; Secor, 2001), and behavioral traits, including feeding preferences and cast production (Bolton and Phillipson, 1976; Curry and Schmidt, 2007), were considered particularly relevant for biogeochemical cycling. Cast production was performed under controlled laboratory conditions using two soil types with contrasting properties. We determined the physicochemical parameters, total and available carbon and nitrogen concentrations of these casts, and used statistical analyses to identify traits significantly impacting cast properties. Additionally, we examined the importance of earthworm species, soil type and their interaction for cast characteristics. We hypothesized that (1) the link between earthworm species and cast properties is soil type-dependent, and (2) physiological traits play a predominant role in determining cast properties.

## 2. Material and method

### 2.1. Earthworm species sampling and soil preparation

Adult individuals belonging to nine earthworm species of the *Lumbricidae* family were collected in Avignon (Vaucluse, southern part of France) in an experimental orchard of INRAE (43°54'55.8" N, 4°52'42.3" E) and additional individuals of two species (EH and LT) were purchased in a fishary store (Decathlon, Avignon, France). In the orchard, 150–200 adult individuals of each species were collected by hand sorting. We sampled earthworms belonging to four ecological categories defined by Bouché (1972, 1977) and Bottinelli et al. (2020). These included epigeics (*Lumbricus castaneus*, LC; *Eisenia hortensis*, EH, also referred to as *Dendrobaena hortensis* (Capowiez et al., 2024a), epi-anecics (*Lumbricus terrestris*, LT), anecics (*Aporrectodea nocturna*, AN; *Aporrectodea caliginosa meridionalis*, ACM) and endogeics (*Aporrectodea rosea*, AR; *Aporrectodea caliginosa typica*, APT; *Aporrectodea icterica*, AI; *Allolobophora chlorotica* (leucotypic morph), ACL). After sampling, earthworms were put into boxes filled with soil collected in the same orchard. All earthworm species were transported back to the laboratory and kept in a refrigerator at 6 °C before starting the experiments. For each species, 5 individuals corresponding to replicates were used. These individuals were chosen because of similar body mass with a standard deviation of 5 %.

Before the beginning of cast production, we conducted a three-day fasting period to empty earthworm guts as described in Le Mer et al. (2022). Briefly, the worms were individually starved in the refrigerator at 6 °C using Petri dishes, filled with a wet paper towel, and covered with perforated aluminum foil. Two soil types (i.e.; Luvisol and Cambisol) were used to produce cast. The Luvisol was the soil in which earthworm had been collected. This soil was sampled in the same orchard at 0–30 cm depth and had the following characteristics: pH 8.3, organic carbon 15.4 g kg⁻¹, total nitrogen 1.8 g kg⁻¹, CaCO₃ 378 g kg⁻¹ and a loamy soil texture with 292 g kg⁻¹ clay, 372 g kg⁻¹ fine silt, 152 g kg⁻¹ coarse silt, 158 g kg⁻¹ fine sand and 26 g kg⁻¹ coarse sand. The Cambisol was collected from a field under agricultural use at the INRAE experimental site at Lusignan (Nouvelle Aquitaine, Western France; 46°25'12.91" N; 0°07'29.35" E) and was characterized by a pH of 5.9, an organic carbon content of 11.9 g kg⁻¹, total nitrogen of 1.2 g kg⁻¹, and a loamy soil texture with 167 g kg⁻¹ clay, 381 g kg⁻¹ fine silt, 285 g kg⁻¹ coarse silt, 63 g kg⁻¹ fine sand, 104 g kg⁻¹ coarse sand). Both soils were air-dried and sieved (<2 mm) before use in microcosms for cast production.

### 2.2. Preparation of microcosms and cast sampling

Cast production was conducted using microcosms. consisting of plastic boxes (10 cm × 10 cm x 10 cm) fitted with perforated lids to prevent earthworms from escaping. To ensure sufficient availability of fresh organic matter, 3 g of dry plant litter consisting of a mixture of leaves, roots, and stems from *Miscanthus giganteus*, crushed to 2 mm, were used as food source and mixed with 97 g of dry soil. To create optimal conditions for earthworm activity and cast production, we used a bulk density of 1 g/cm³ and adjusted moisture levels with distilled water to maintain to 80 % of the water holding capacity.

Earthworms were introduced into the respective microcosms at similar body mass with densities of 2 for anecic and epi-anecic, 4 for endogeic, and 8 for epigeic individuals. The experiment consisted of eleven treatments: (i) soil alone (C), (ii) soil with plant litter (C, L), and (iii) soil with plant litter plus individuals of one of the nine earthworm species. Each treatment was replicated five times, with the microcosms randomly placed in a dark chamber at 14 °C for four weeks. Fresh casts from the soil surface and control soil aggregates were collected daily during the first two weeks and every two days thereafter. They were manually sorted and cleaned from adhering organic debris before being air-dried. An aliquot of the dried casts and control soils was ground for chemical analysis.
## 2.3. Earthworm traits

We focused on 21 traits associated with earthworm feeding behavior, as well as food ingestion and digestion. The earthworm traits were categorized into four groups – morphological, anatomical, physiological, and behavioral (Table 1). We measured all traits except for the type of typhlosole. We considered that this trait varied little within the species. Consequently, the typhlosole type described by Bouché (1972) was used. The body mass and dorsal pigmentation were determined using living earthworms. The body length, body diameter, gizzard size, gizzard localization, pharynx size, crop size, buccal surface, morren gland, gut length were measured after submersion of the species in alcohol 70 % and dissection. The body diameter, body length and gut length were measured with a Vernier calliper.

The water and intestinal mucus content were measured based on the method described by Barois (1992). Briefly, after immersion for 2 s in boiling water, earthworms were dissected and the gut contents were removed and weighed. The water content was determined after desiccation of the samples over silica gel for 24 h, and expressed as a percentage of the moist gut content. The mucus content was extracted after dilution in hot water (60 °C), filtering through a 0.2 µm filter, and freeze-drying. After weighing, the result was expressed as a percentage of the dry gut content.

To determine the digestion time, earthworms were fasted in petri dishes for three days to empty their intestinal contents. Thereafter, they were placed in microcosms containing soil and litter and incubated at 14 °C for 1 h. After incubation, the worms were removed from the microcosms and transferred to crystallizing dishes lined with moistened white paper and covered with perforated Petri dish lids to allow air exchange and facilitate observation. The crystallizing dishes with the earthworms were then placed in an incubator equipped with a night-vision camera (SONY Corporation, Digital HD Video, Japan).

<caption>**Table 1**</caption>

Earthworm trait: categories, units and functions.

<table><thead><tr><th>Trait category</th><th>Trait</th><th>Unit</th><th>Function and/or indicator</th></tr></thead><tbody><tr><td rowspan="3">Morphology</td><td>Pigmentation</td><td>yes or no</td><td>surface activity and food preference (litter and/or soil).</td></tr><tr><td>Body size (body length, body diameter, body mass)</td><td>mm, g</td><td>overall strength and burrowing ability</td></tr><tr><td>Mouth surface area</td><td>mm<sup>2</sup></td><td>choice and quantity of food ingested</td></tr><tr><td rowspan="7">Anatomical</td><td>Pharynx size (length, weight)</td><td>mm, mg</td><td>grinding capacity</td></tr><tr><td>Crop (weight, volume)</td><td>mg, mm<sup>3</sup></td><td>storage capacity</td></tr><tr><td>Gizzard size (weight, volume)</td><td>mg, mm<sup>3</sup></td><td>grinding capacity</td></tr><tr><td>Gizzard localization</td><td>ante pore or postpore</td><td>food processing</td></tr><tr><td>Morren gland</td><td>mg</td><td>pH regulation and food digestion capacity</td></tr><tr><td>Type of typhlosole</td><td>pinnate, bifid, trifid, lameliform</td><td>food digestion capacity</td></tr><tr><td>Length of intestine</td><td>mm</td><td>food digestion capacity</td></tr><tr><td rowspan="2">Physiological</td><td>Intestinal Water content</td><td>%</td><td>food digestion capacity</td></tr><tr><td>Intestinal mucus content</td><td>%</td><td>food digestion capacity</td></tr><tr><td rowspan="4">Behavioral</td><td>Digestion time</td><td>hour</td><td>food preference (litter and/or soil)</td></tr><tr><td>Litter ingested</td><td>g/day/g of worm</td><td>litter conversion efficiency</td></tr><tr><td>Cast produced</td><td>–</td><td>litter conversion efficiency</td></tr><tr><td>Litter-to-cast ratio</td><td>–</td><td>litter conversion efficiency</td></tr></tbody></table>

Following 24 h of incubation, the earthworms were removed, and the videos were reviewed. The digestion time, expressed in hours for each individual, was defined as the time until the earthworm produced its first cast.

Behavioral traits were determined on living individuals. In particular, the quantity of plant litter consumed by each earthworm species after seven days of incubation was recorded gravimetrically from the litter remaining on the soil surface after drying. The mass of airdried casts was determined for each treatment at the end of the incubation. The litter conversion efficiency was calculated as the litter-to-cast ratio from these two parameters.

## 2.4. Physicochemical properties of casts and control soils

Moisture content of the samples was determined after drying at 105 °C (Allen, 1989). pH was measured in soil: water (1:5) suspension with a pH meter (ISO 10390). The organic carbon (OC) and total nitrogen (N) concentrations were determined by dry combustion using an elemental analyzer (Thermo Flash HT, Thermo Fisher Scientific Inc., Waltham, USA). Before the analyses, carbonates present in the Luvisol were removed by acid fumigation (Harris et al., 2001). Dissolved organic carbon (DOC) was extracted with deionized water at room temperature at a soil-to-water ratio of 1:10 w/v. After extraction, the solutions were centrifuged at 3000 rpm for 15 min and filtered through a polyethersulfone syringe filter (pore size 0.45 µm). The DOC concentration was quantified using a Shimadzu TOC-L analyzer (TOC-L series, Shimadzu Corporation, Japan).

The NO₃⁻ and NH₄⁺ concentrations were determined following extraction with 12 ml of 0.01 M CaCl₂, from 3 g of dry mass equivalent. Briefly, samples were shaken at 140 rpm for 2 h at 10 °C. The suspensions were filtered (pore size 0.2 µm) and stored at -20 °C before measurement of NO₃⁻ and NH₄⁺ concentrations by using a Smartchem 200 photometer (KPM Analytics, Frépillon, France). Measurements were performed at the AME platform (Microbial Ecology UMR1418, Lyon).

## 2.5. Statistical analysis

Statistical analyses were performed using R software (R Core Team, 2023), with a significance level set at 0.05. Quantitative earthworm traits were analyzed using one-way ANOVA, followed by Tukey's multiple comparison tests, implemented with the "agricolae" package (De Mendiburu, 2020). Before conducting the ANOVA, the homoscedasticity and normality of the data were verified using Levene's and Shapiro-Wilk's tests. To evaluate the effects of earthworm species and soil type on cast properties, a Principal Component Analysis (PCA) and a two-way ANOVA were conducted. The scores from the PCA were analyzed using two-way ANOVA, followed by Tukey's post hoc test, to provide detailed insights into the effects of earthworm species and soil type on the physicochemical properties of casts. Additionally, a two-way ANOVA with interaction plots between soil type and earthworm species, combined with Tukey's post hoc test, was used to examine differences across all combinations of species and soil types. Furthermore, an intra- and inter-class PCA was performed to rank and assess the relative impact of each factor (earthworm species and soil type) on cast properties.

To investigate the link between earthworm traits and cast properties, we conducted a co-inertia analysis on earthworm traits and cast parameters using the "ade4" package (Dray and Dufour, 2007). The level of association between the two datasets was measured with the RV matrix correlation coefficient. Covariation between both datasets was declared significant at the 0.05 probability level based on the Monte Carlo test. Prior to the co-inertia analysis, both data matrices were normalized to remove the effect of soil type and earthworm body mass. The trait data matrix was normalized by dividing the quantitative data by the earthworm weight, while the cast data matrix was normalized by dividing the cast parameters by the control soil parameters. Furthermore, a heatmap was generated to visualize the correlations between earthworm traits
and cast characteristics using “heatmaply” package.

## 3. Results

### 3.1. Variability of morphological, anatomical, physiological, and behavioral traits in earthworm's species

The 21 traits characterizing the nine earthworm species are presented in Table 2. All quantitative traits were significantly influenced by species (p < 0.001). The lowest values of all parameters were observed for *L. castaneus* and the highest always for *L. terrestris*. Cast production was higher in the Cambisol than in the Luvisol. In contrast, litter incorporation was greater in the Luvisol (higher litter-to-cast ratio).

### 3.2. Impact of earthworm species and soil type on cast properties

The PCA summarizing the effect of earthworm species and soil types on physicochemical properties explained 80.8 % of the data variability (Fig. 1A). PC1 accounted for 54.3 % of the total inertia. and was positively correlated with OC, NO₃ concentration, moisture, DOC content and C/N ratio. This axis displayed a gradient of physicochemical

properties from *L. castaneus* and *L. terrestris* towards the control soils, extending through *E. hortensis*, and the anecic and endogeic species, regardless of soil type. PC1 was also significantly influenced by earthworm species, soil type, and their interaction (p < 0.0001; Fig. 1B). Along this axis, treatments differed significantly between the two soil types, except for *A. nocturna* and *A. caliginosa meridionalis*, suggesting a low sensitivity or relative indifference of these species to variations in edaphic conditions. PC2 which explained 26.5 % of the total variability, was positively correlated with pH and negatively correlated with the NH₄⁺ concentration. This axis was significantly influenced by earthworm species, soil type, and their interaction (p < 0.0001; Fig. 1C) and it distinguished the Luvisol samples from the Cambisol samples. However, the between-within class PCA analysis revealed that the inertia ratio attributed to earthworm species (57.9 %) was higher than that of soil type (24.1 %; Fig. 1A–Table 3). Moreover, two-way ANOVA revealed that earthworm species was a significant factor (p < 0.001) influencing all cast physicochemical properties, while soil type and its interaction with the species were significant for most properties (p < 0.01). Soil type did not significantly influence DOC and C/N ratio, and species:soil interactions were non-significant for C/N (Table 4).

Specifically, Luvisol casts exhibited higher levels of OC, NO₃,

<table><caption>**Table 2**
Mean and standard error of traits determined for the nine species.</caption><thead><tr><th>Earthworms Trait</th><th>LC</th><th>EH</th><th>LT</th><th>ACM</th><th>AN</th><th>APT</th><th>ACL</th><th>AR</th><th>AI</th><th>p-value</th></tr></thead><tbody><tr><td>Length (mm)</td><td>42.6 ± 0.87<sup>f</sup></td><td>68.6 ± 0.74<sup>d</sup></td><td>143.8 ± 0.37<sup>a</sup></td><td>78 ± 1.64<sup>c</sup></td><td>128.8 ± 1.01<sup>b</sup></td><td>63.8 ± 1.11<sup>d</sup></td><td>53 ± 1.67<sup>e</sup></td><td>79.4 ± 1.6<sup>c</sup></td><td>58.2 ± 0.58<sup>e</sup></td><td>***</td></tr><tr><td>Diameter (mm)</td><td>2.84 ± 0.09<sup>ef</sup></td><td>5.02 ± 0.02<sup>c</sup></td><td>10.6 ± 0.24<sup>a</sup></td><td>3.88 ± 0.04<sup>d</sup></td><td>6.04 ± 0.04<sup>b</sup></td><td>3.02 ± 0.02<sup>e</sup></td><td>2.7 ± 0.12<sup>f</sup></td><td>2.9 ± 0.1<sup>ef</sup></td><td>3.02 ± 0.02<sup>e</sup></td><td>***</td></tr><tr><td>Body mass (g)</td><td>0.23 ± 0.00<sup>h</sup></td><td>0.89 ± 0.01<sup>c</sup></td><td>5.51 ± 0.10<sup>a</sup></td><td>0.715 ± 0.01<sup>d</sup></td><td>1.86 ± 0.02<sup>b</sup></td><td>0.36 ± 0.00<sup>f</sup></td><td>0.33 ± 0.00<sup>g</sup></td><td>0.32 ± 0.00<sup>g</sup></td><td>0.53 ± 0.00<sup>e</sup></td><td>***</td></tr><tr><td>Mouth area (mm<sup>2</sup>)</td><td>0.87 ± 0.04<sup>f</sup></td><td>3.8 ± 0.15<sup>c</sup></td><td>9.3 ± 0.29<sup>a</sup></td><td>1.25 ± 0.02<sup>d</sup></td><td>7.5 ± 0.0<sup>b</sup></td><td>1.03 ± 0.03<sup>e</sup></td><td>1.08 ± 0.04<sup>e</sup></td><td>1 ± 0<sup>ef</sup></td><td>1 ± 0<sup>ef</sup></td><td>***</td></tr><tr><td>Pharynx length (mm)</td><td>2.01 ± 0.15<sup>e</sup></td><td>3.24 ± 0.16<sup>c</sup></td><td>5.98 ± 0.36<sup>a</sup></td><td>3.15 ± 0.34<sup>cd</sup></td><td>4.49 ± 0.13<sup>b</sup></td><td>2.01 ± 0.26<sup>e</sup></td><td>2.11 ± 0.13<sup>de</sup></td><td>3.13 ± 0.06<sup>cd</sup></td><td>2.28 ± 0.24<sup>de</sup></td><td>***</td></tr><tr><td>Pharynx weight (mg)</td><td>3.87 ± 0.51<sup>f</sup></td><td>10.86 ± 0.87<sup>cd</sup></td><td>84.49 ± 1.37<sup>a</sup></td><td>12.86 ± 0.53<sup>c</sup></td><td>35.72 ± 0.41<sup>b</sup></td><td>4.21 ± 0.50<sup>f</sup></td><td>8.02 ± 0.72<sup>de</sup></td><td>7.10 ± 0.3<sup>ef</sup></td><td>8.30 ± 0.34<sup>de</sup></td><td>***</td></tr><tr><td>Crop weight (mg)</td><td>0.59 ± 0.10<sup>e</sup></td><td>6.28 ± 0.31<sup>b</sup></td><td>53.80 ± 1.46<sup>a</sup></td><td>5.61 ± 0.45<sup>b</sup></td><td>7.41 ± 0.17<sup>b</sup></td><td>2.36 ± 0.11<sup>cd</sup></td><td>1.99 ± 0.13<sup>d</sup></td><td>2.31 ± 0.14<sup>cd</sup></td><td>3.14 ± 0.32<sup>c</sup></td><td>***</td></tr><tr><td>Crop volume (mm<sup>3</sup>)</td><td>1.63 ± 0.23<sup>cd</sup></td><td>6.97 ± 0.99<sup>b</sup></td><td>74.16 ± 6.54<sup>a</sup></td><td>7.27 ± 0.66<sup>b</sup></td><td>8.32 ± 0.73<sup>b</sup></td><td>1.02 ± 0.25<sup>d</sup></td><td>2.79 ± 0.69<sup>c</sup></td><td>2.28 ± 0.11<sup>c</sup></td><td>5.68 ± 0.74<sup>b</sup></td><td>***</td></tr><tr><td>Gizzard weight (mg)</td><td>6.27 ± 0.25<sup>e</sup></td><td>16.57 ± 0.42<sup>c</sup></td><td>132.83 ± 1.14<sup>a</sup></td><td>14.68 ± 0.42<sup>c</sup></td><td>24.33 ± 0.30<sup>b</sup></td><td>6.32 ± 0.19<sup>e</sup></td><td>3.81 ± 0.13<sup>g</sup></td><td>5.02 ± 0.05<sup>f</sup></td><td>7.78 ± 0.38<sup>d</sup></td><td>***</td></tr><tr><td>Gizzard volume (mm<sup>3</sup>)</td><td>9.51 ± 1.36<sup>bcd</sup></td><td>9.94 ± 0.71<sup>bc</sup></td><td>160.84 ± 13.37<sup>a</sup></td><td>10.49 ± 1.31<sup>b</sup></td><td>10.66 ± 0.46<sup>b</sup></td><td>4.13 ± 0.76<sup>e</sup></td><td>3.69 ± 0.54<sup>e</sup></td><td>5.75 ± 0.30<sup>de</sup></td><td>5.94 ± 0.31<sup>ede</sup></td><td>***</td></tr><tr><td>Morren gland weight (mg)</td><td>0.90 ± 0.07<sup>ef</sup></td><td>0 ± 0<sup>g</sup></td><td>22.77 ± 0.61<sup>a</sup></td><td>2.46 ± 0.19<sup>c</sup></td><td>13.75 ± 0.23<sup>b</sup></td><td>1.23 ± 0.07<sup>de</sup></td><td>0.81 ± 0.10<sup>f</sup></td><td>1.71 ± 0.14<sup>d</sup></td><td>1.07 ± 0.06<sup>ef</sup></td><td>***</td></tr><tr><td>Gut length (mm)</td><td>38.3 ± 0.66<sup>g</sup></td><td>64.2 ± 0.73<sup>d</sup></td><td>168.6 ± 1.02<sup>b</sup></td><td>75.2 ± 1.77<sup>c</sup></td><td>189.63 ± 0.75<sup>b</sup></td><td>55.4 ± 2.03<sup>e</sup></td><td>45.1 ± 1.14<sup>f</sup></td><td>69 ± 1.00<sup>d</sup></td><td>53 ± 0.44<sup>e</sup></td><td>***</td></tr><tr><td>Mucus content (%)</td><td>11.63 ± 1.59<sup>a</sup></td><td>5.65 ± 1.33<sup>b</sup></td><td>2.31 ± 0.23<sup>c</sup></td><td>2.47 ± 0.48<sup>c</sup></td><td>2.04 ± 0.03<sup>c</sup></td><td>3.08 ± 0.37<sup>bc</sup></td><td>3.75 ± 0.56<sup>bc</sup></td><td>2.63 ± 0.31<sup>c</sup></td><td>2.28 ± 0.34<sup>c</sup></td><td>***</td></tr><tr><td>Water content (%)</td><td>73.61 ± 2.24<sup>a</sup></td><td>70.34 ± 4.12<sup>ab</sup></td><td>62.23 ± 2.44<sup>abc</sup></td><td>55.99 ± 2.02<sup>c</sup></td><td>58.14 ± 1.94<sup>c</sup></td><td>50.67 ± 2.49<sup>c</sup></td><td>51.47 ± 2.20<sup>c</sup></td><td>59.22 ± 1.87<sup>bc</sup></td><td>59.03 ± 2.55<sup>bc</sup></td><td>***</td></tr><tr><td>Digestion time (hour)</td><td>5.41 ± 0.28<sup>cd</sup></td><td>5.65 ± 0.38<sup>cd</sup></td><td>10.14 ± 0.30<sup>a</sup></td><td>9.01 ± 0.64<sup>ab</sup></td><td>10.67 ± 0.38<sup>a</sup></td><td>8.45 ± 1.24<sup>ab</sup></td><td>6.91 ± 0.33<sup>bc</sup></td><td>1.39 ± 0.24<sup>c</sup></td><td>3.65 ± 0.07<sup>de</sup></td><td>***</td></tr><tr><td>Pigmentation</td><td>yes</td><td>yes</td><td>yes</td><td>yes</td><td>yes</td><td>no</td><td>no</td><td>no</td><td>-</td><td></td></tr><tr><td>Gizzard localization</td><td>post-pore</td><td>ante-pore</td><td>post-pore</td><td>post-pore</td><td>post-pore</td><td>post-pore</td><td>post-pore</td><td>post-pore</td><td>post-pore</td><td>-</td></tr><tr><td>Type of typhlosole</td><td>pinnate</td><td>lamelliform</td><td>pinnate</td><td>pinnate</td><td>pinnate</td><td>trifid</td><td>bifid</td><td>bifid</td><td>lamelliform</td><td>-</td></tr><tr><td>CastProd_Luvisol (g/day/g of worm)</td><td>0.64 ± 0.04<sup>c</sup></td><td>0.68 ± 0.01<sup>bc</sup></td><td>0.12 ± 0.00<sup>e</sup></td><td>0.59 ± 0.01<sup>c</sup></td><td>0.38 ± 0.01<sup>d</sup></td><td>1.07 ± 0.04<sup>a</sup></td><td>0.87 ± 0.08<sup>ab</sup></td><td>0.64 ± 0.02<sup>c</sup></td><td>0.63 ± 0.02<sup>c</sup></td><td>***</td></tr><tr><td>CastProd_Cambisol (g/day/g of worm)</td><td>1.47 ± 0.08<sup>bc</sup></td><td>1.26 ± 0.09<sup>bc</sup></td><td>0.22 ± 0.01<sup>e</sup></td><td>1.21 ± 0.03<sup>bcd</sup></td><td>0.95 ± 0.01<sup>d</sup></td><td>1.91 ± 0.05<sup>a</sup></td><td>2.23 ± 0.20<sup>a</sup></td><td>1.03 ± 0.06<sup>cd</sup></td><td>1.12 ± 0.04<sup>cd</sup></td><td>***</td></tr><tr><td>Litter_inguest_Cambisol (g/day/g of worm)</td><td>0.31 ± 0.01<sup>a</sup></td><td>0.08 ± 0.00<sup>ab</sup></td><td>0.025 ± 0.00<sup>c</sup></td><td>0.08 ± 0.00<sup>b</sup></td><td>0.07 ± 0.00<sup>b</sup></td><td>0.07 ± 0.01<sup>b</sup></td><td>0.08 ± 0.00<sup>b</sup></td><td>0.09 ± 0.00<sup>b</sup></td><td>0.07 ± 0.00<sup>b</sup></td><td>***</td></tr><tr><td>Litter_inguest_Luvisol (g/day/g of worm)</td><td>0.32 ± 0.01<sup>a</sup></td><td>0.08 ± 0.00<sup>de</sup></td><td>0.02 ± 0.00<sup>f</sup></td><td>0.08 ± 0.01<sup>de</sup></td><td>0.07 ± 0.00<sup>e</sup></td><td>0.13 ± 0.01<sup>b</sup></td><td>0.10 ± 0.00<sup>bc</sup></td><td>0.09 ± 0.00<sup>cd</sup></td><td>0.075 ± 0.00<sup>de</sup></td><td>***</td></tr><tr><td>Litter-to-cast_ratio_Cambisol</td><td>0.21 ± 0.01<sup>a</sup></td><td>0.06 ± 0.00<sup>c</sup></td><td>0.12 ± 0.00<sup>b</sup></td><td>0.06 ± 0.00<sup>c</sup></td><td>0.07 ± 0.00<sup>c</sup></td><td>0.04 ± 0.00<sup>d</sup></td><td>0.04 ± 0.00<sup>d</sup></td><td>0.07 ± 0.00<sup>c</sup></td><td>0.06 ± 0.00<sup>c</sup></td><td>***</td></tr><tr><td>Litter-to-cast_ratio_Luvisol</td><td>0.49 ± 0.02<sup>a</sup></td><td>0.12 ± 0.00<sup>d</sup></td><td>0.21 ± 0.01<sup>b</sup></td><td>0.14 ± 0.01<sup>cd</sup></td><td>0.18 ± 0.01<sup>bc</sup></td><td>0.12 ± 0.01<sup>d</sup></td><td>0.13 ± 0.01<sup>cd</sup></td><td>0.14 ± 0.00<sup>cd</sup></td><td>0.12 ± 0.00<sup>d</sup></td><td>***</td></tr></tbody></table>

Note: LC: *L. castaneus*, EH: *E. hortensis*, LT: *L. terrestris*, AN: *A. nocturna*, ACM: *A. caliginosa meridionalis*, AR: *A. rosea*, APT: *A. caliginosa typica*, AI: *A. icterica*, ACL: *A. chlorotica*.
<figure><img src="image_3.png" /><figcaption>**Fig. 1.** (A) Principal Component Analysis (PCA) of earthworm species, soil types and cast physicochemical properties. Position of individuals on the plane defined by the first two axes (left figure) and correlation circle representing variables on the first two axes (right figure). Ellipses indicate the center of gravity of samples with 88 % of samples within the ellipse. Inertia ratio of the between-class PCA analysis to classify and test the explanatory effects of soil type, earthworm species, and their interaction (residuals). Two-way ANOVA followed by Tukey's post hoc test on (B) the PC1 scores and (C) the PC2 scores. Significance levels are indicated as follows: (***p < 0.001; **p < 0.01; *p < 0.05 and ns for p > 0.05). LC: *L. castaneus*, EH: *E. hortensis*, LT: *L. terrestris*, AN: *A. nocturna*, ACM: *A. caliginosa meridionalis*, AR: *A. rosea*, APT: *A. caliginosa typica*, AI: *A. icterica*, ACL: *A. chlorotica*, C: soil alone, C_L: soil with plant litter.</figcaption></figure>

moisture, and pH, while Cambisol casts showed higher NH₄⁺ concentrations (Supplementary, Fig. S2). The OC content of casts significantly depended on earthworm species and soil type (p < 0.001), ranging from 16–45 mg g⁻¹ in the Luvisol and 10–27 mg g⁻¹ in the Cambisol. The highest OC content was observed in the casts of *L. castaneus*, and the lowest in those of *A. rosea*. DOC also varied with species in the casts of both soil types, ranging from 170–499 mg kg⁻¹ in the Luvisol and 143–456 mg kg⁻¹ in the Cambisol. The C/N ratio ranged from 9 to 14

across both soil types, with the highest value in the casts of *L. castaneus* and the lowest in those of *A. rosea*. Available nitrogen and moisture contents were influenced by both earthworm species and soil type, with *L. castaneus* and *L. terrestris* producing casts with the highest values. The pH ranged from 7.2–8.3 in the Luvisol and 5.9–6.8 in the Cambisol. Compared to the control, it increased in casts produced with Cambisol and decreased in those produced with Luvisol, demonstrating the role of earthworm activity in pH regulation within different soil types.
<caption>**Table 3**</caption>

Total inertia and first eigenvalues of the between-within class PCA analysis to rank and test the explanatory effects of soil type and earthworm species on the modification of cast properties. Inertia ratio indicates the percentage of variability explained by the between or within classes effect. Residuals indicate the percentage of the inertia that was neither explained by a soil effect nor an earthworm effect alone.

<table><thead><tr><th></th><th>Total inertia</th><th>First eigenvalue</th><th>Inertia ratio (%)</th></tr></thead><tbody><tr><td>PCA general</td><td>7</td><td>3,81</td><td>-</td></tr><tr><td>Between soils PCA</td><td>1,69</td><td>1,69</td><td>24,1***</td></tr><tr><td>Within soils PCA</td><td>7</td><td>4,59</td><td>80,58</td></tr><tr><td>Between EW PCA</td><td>4,06</td><td>3,52</td><td>57,98***</td></tr><tr><td>Within EW PCA</td><td>7</td><td>3,01</td><td>63,3</td></tr><tr><td>Residuals</td><td>-</td><td>-</td><td>17,92</td></tr></tbody></table>

* 'significant at the 0.05 probability level' by 'p < 0.05'.

** 'significant at the 0.01 probability level' by 'p < 0.01'.

*** 'significant at the 0.001 probability level' by 'p < 0.001'.

<caption>**Table 4**</caption>

F-value and significance of the two-way ANOVA analysis of the effects of nine earthworm species and two soil types on the cast physicochemical properties. Significance levels are indicated by asterisks: (***p < 0.001; **p < 0.01; *p < 0.05 and ns for p > 0.05).

<table><thead><tr><th>Cast property</th><th>Species</th><th>Soil</th><th>Species*Soil</th></tr></thead><tbody><tr><td>DOC</td><td>164.74***</td><td>0.05<sup>ns</sup></td><td>4.23***</td></tr><tr><td>OC</td><td>67.91***</td><td>331.67***</td><td>6.89***</td></tr><tr><td>C/N</td><td>19.23***</td><td>1.43<sup>ns</sup></td><td>0.46<sup>ns</sup></td></tr><tr><td>NO<sub>3</sub><sup>-</sup></td><td>48.08***</td><td>112.38***</td><td>25.97***</td></tr><tr><td>NH<sub>4</sub><sup>+</sup></td><td>37.57***</td><td>503.59***</td><td>4.19***</td></tr><tr><td>Moisture</td><td>48.12***</td><td>9.09**</td><td>4.33***</td></tr><tr><td>pH</td><td>31.13***</td><td>1784.57***</td><td>6.30***</td></tr></tbody></table>

### 3.3. Covariation between earthworm traits and cast properties

The co-inertia analysis revealed that the two projected inertias accounted for 90.1 % of the total co-inertia, indicating yet significant covariation (RV = 0.34, p < 0.01, Monte Carlo test, Fig. 2). Along axis 1, which explained 83.5 % of variance, DOC and NO₃⁻ concentrations, moisture, OC and N contents, and the C/N ratio covaried positively with gizzard size (weight, volume), pinnate typhlosolis, intestinal mucus, pigmentation, litter ingested, the mouth area, and gizzard located before the pores. Conversely, these properties were negatively associated with bifid and trifid typhlosoles. On axis 2, which explained 6.6 % of the variance, NH₄⁺ concentration covaried negatively with lamelliform typhlosoles, while pH was negatively associated with litter-to-cast ratio. To further explore the relationships between earthworm traits and cast physicochemical properties, we generated a heatmap based on the co-inertia analysis. The dendrograms at the top of the heatmap revealed three main clusters (Fig. 3). The first (left) and third (right) demonstrated strong correlations with cast properties, while the second cluster (center) exhibited weaker correlations. Notably, this analysis confirmed the positive correlations between cast properties with gizzard size, pinnate typhlosole, intestinal mucus, pigmentation, litter ingested, litter-to-cast ratio, buccal surface, and gizzard located before the pores. Additionally, it revealed a positive correlation between NH₄⁺ concentration and gizzard size, as well as pinnate typhlosole. A positive correlation between pH and cast production was also identified.

## 4. Discussion

### 4.1. What determines cast characteristics: soil type or earthworm species?

In agreement with a large body of literature, our results indicated that the concentrations of OC, DOC, NH₄⁺, moisture, NO₃⁻, and the C/N ratio were consistently higher in earthworm casts compared to control aggregates in both soil types (Supplementary, Fig. S2). Earthworm

activity also induced changes in pH, with an increase observed in the Cambisol and a decrease in the Luvisol, suggesting a potential pH-regulating effect (Parkin and Berry, 1999). This may be attributed to proton release in the gut (Scharenbroch and Johnston, 2011) and calcium carbonate secretion by the morren gland (Robertson, 1936), both of which play important roles in maintaining the acid-base balance (Briones et al., 2008).

The observed soil-type-specific differences in most parameters are largely attributed to the inherent properties of the soils, which are known to influence cast characteristics (Babiy et al., 2021; Bityutskii and Kaidun, 2008; Clause et al., 2014; van Groenigen et al., 2019). Notably, some species, such as *A. caliginosa meridionalis* and *A. nocturna*, exhibited minimal influence of soil type on their cast properties (Fig. 1B). This could reflect strong adaptive capacity or behavioral flexibility in response to varying edaphic conditions (Gergs et al., 2022). For example, *A. caliginosa meridionalis*, with its ability to exploit both litter and soil resources, may buffer environmental variations, resulting in relatively consistent cast composition (Capowiez et al., 2024a; b). Similarly, *A. nocturna*, characterized by its larger digestive system and specific behavioral traits (Bottinelli et al., 2020; Bouché, 1972; Capowiez et al., 2024a), may tolerate differences in soil physicochemical properties more effectively, further supporting its low sensitivity to soil type (Capowiez et al., 2024b). Such low sensitivity to soil type is not exclusive to temperate species. Tropical and subtropical earthworms, such as *P. corethrurus*, are also exhibit broad environmental tolerance, attributed to their high organic matter assimilation efficiency (Lavelle et al., 1995). This enables them to persist in nutrient-poor soils and colonize new habitats effectively (Lavelle et al., 1987; Marichal et al., 2010). Our findings underscore the importance of species-specific traits in shaping cast characteristics, even under contrasting soil conditions (Singh et al., 2020). Indeed, the inertia ratio analysis indicated that the influence of earthworm species on cast properties (59 %) was significantly greater than that of soil type (24 %), highlighting the dominant role of earthworm species in determining cast characteristics.

### 4.2. Influence of earthworm effect traits on cast physicochemical properties

Earthworm species-specific effects on cast properties, irrespective of soil type, revealed a gradient of physicochemical properties ranging from *L. castaneus* and *L. terrestris* to control soils, successively passing through *E. hortensis*, *A. nocturna*, *A. caliginosa meridionalis* and the four endogeic species (i.e., *A. caliginosa*, *A. chlorotica*, *A. rosea*, *A. icterica*). This result indicates that there are species specific traits modulating the soil effect. Indeed, the co-inertia analysis revealed a moderate yet significant covariation (RV = 0.34) between morpho-anatomical, physiological and behavioral traits of the earthworms and the physicochemical properties of their casts. We identified nine earthworm traits, including pigmentation, buccal surface, gizzard size, typhlosole complexity, intestinal mucus, litter ingestion, and litter-to-cast ratio, which were significantly correlated with cast properties. Since these traits depend on the earthworms' physiology and morphology, they may be considered as effect traits directly influencing soil properties (Dawson et al., 2021; Lavorel et al., 2013; Pey et al., 2014). For instance, gizzard size and typhlosole type have been shown to influence soil structural properties, under-scoring the importance of morpho-anatomical features in shaping soil processes (Marichal et al., 2017). Moreover, physiological traits, such as intestinal mucus affect microbial and enzymatic activity, leading to organic matter decomposition and nutrient release (Barois, 1992), thereby influencing the physicochemical composition of casts.

Traits such as pigmentation indirectly influence cast quality by reflecting earthworm behavior (Bouché, 1972). Indeed, pigmented earthworms are adapted to surface-dwelling behavior and tend to consume more litter than SOM, resulting in casts enriched in litter (Capowiez et al., 2024a). Similarly, the buccal surface influences the ingestion and processing of litter, enabling selective consumption of
<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Co-inertia analysis between earthworm traits and cast physicochemical characteristics. The first and second axes explained 83.5 % and 6.6 % of variance, respectively. (A) Projection of individuals on the two first co-inertia axes. (B) Projection of the earthworm traits on the two first co-inertia axes. (C) Projection of the cast physicochemical characteristics. RV = 0.34, p < 0.01, Monte Carlo test. Abbreviations: *Pig.pigme*: pigmentation (*Yes* or *No*), *D*: diameter, *L*: length, *GuL*: gut length, *PhW*: pharynx weight, *PhL*: pharynx length, *CrW*: crop weight, *CrV*: crop volume, *GiV*: gizzard volume, *GiW*: gizzard weight, *GiL*: gizzard, localization (post pore, ante pore), *CaP*: cast production, *MoA*: mouth area, *DgT*: digestion time, *MgW*: morren gland weight, *GuW*: gut water content, *Li/Ca*: litter-to-cast ratio, *Lil*: Litter ingested, *GuM*: gut mucus content, *Ty*: typhlosolis (trifid, bifid, pinnate, lamelliform).</figcaption></figure>

organic materials and thus modifying the chemical composition of the
casts (Edwards and Arancon, 2022). High litter ingestion and a high
litter-to-cast ratio enhance the conversion of litter into casts, increasing
OC, N, DOC and moisture levels (van Groenigen et al., 2019). These
traits likely influence cast properties by determining the quantity and
quality of (fresh) organic material contributing to casts. Therefore, they
may be considered as indirect effect traits. Feeding behavior emerges as
a pivotal driver in shaping the physicochemical properties of casts, while
morpho-anatomical and physiological traits further modulate their role
in soil biogeochemical cycling. These finding highlight the critical role
of several contrasting earthworm traits in influencing soil properties,
providing a framework to better understand earthworm contributions to

SOM dynamics. Further research is needed to explore how these
species-specific traits influence SOM turnover processes.

5. Conclusions

In this study, we investigated the relationship between earthworm
traits and the physicochemical properties of casts produced by nine
species in two contrasting soil types. Our results demonstrated that
earthworm species had greater influence on cast properties than the soil
type they inhabited. Strong relationships were identified between
earthworm morpho-anatomical, behavioral, and physiological traits and
cast properties. Key traits-such as buccal surface, gizzard size and
<figure><img src="image_5.png" /><figcaption>**Fig. 3.** Heatmap from co-inertia analysis showing correlation between earthworm traits and cast physicochemical characteristics. Abbreviations: Pig.pigme: pigmentation (Yes or No), D: diameter, L: length, GuL: gut length, PhW: pharynx weight, PhL: pharynx length, CrW: crop weight, CrV: crop volume, GiV: gizzard volume, GiW: gizzard weight, GiL: gizzard, localization (post pore, ante pore), CaP: cast production, MoA: mouth area, DgT: digestion time, MgW: morren gland weight, GuW: gut water content, Li/Ca: litter-to-cast ratio, Lil: Litter ingested, GuM: gut mucus content, Ty: typhlosolis (trifid, bifid, pinnate, lamelliform).</figcaption></figure>

location, typhlosole complexity, intestinal mucus and cast production may influence the physicochemical properties of casts directly, while pigmentation, amount of litter ingested, litter-to-cast ratio may have indirect effects because they are related to the earthworms' ecological behavior. Given their strong relationship with the carbon content and the availability of carbon and nutrients of casts, these traits might be employed to increase our understanding of the earthworms' role in soil biogeochemical cycling, particularly in SOM dynamics.

### CRediT authorship contribution statement

**Yacouba Zi:** Writing – original draft, Investigation, Formal analysis, Data curation.
**Nicolas Bottinelli:** Writing – review & editing, Validation, Supervision, Formal analysis, Conceptualization.
**Malalatiana Razafindrakoto:** Writing – review & editing, Supervision, Data curation.
**Yvan Capowiez:** Writing – review & editing, Validation.
**Alesandro Florio:** Writing – review & editing, Validation, Supervision, Data curation.
**Chao Song:** Writing – review & editing, Data curation.
**Cornelia Rumpel:** Writing – review & editing, Supervision, Resources, Project administration, Methodology, Investigation, Funding acquisition, Conceptualization.
**Marie-France Dignac:** Writing – review & editing, Supervision, Investigation.

### 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.

### Acknowledgments

This project was financially supported by the ANR project U2 worm (ANR-20-CE01-0015-01). YZ received funding from French ministry for education. We are grateful to Phillipe Biron, Axel Felback, Jonathan

Gervaux, Charline Creuze-Des-Chatelliers, Abigail Delort for technical support of this study.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.soilbio.2025.109809.

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