# Behavioral and functional responses of Hormogaster samnitica to different organic amendments

Francesco Serafini, Margherita Santoni, Ottorino-Luca Pantani, Gaio Cesare

Pacini, Yvan Capowiez

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Francesco Serafini, Margherita Santoni, Ottorino-Luca Pantani, Gaio Cesare Pacini, Yvan Capowiez. Behavioral and functional responses of Hormogaster samnitica to different organic amendments. Applied Soil Ecology, 2026, 218, pp.106716. 10.1016/j.apsoil.2025.106716. hal-05444469

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# Behavioral and functional responses of *Hormogaster samnitica* to different organic amendments

Francesco Serafini<sup>a</sup>, Margherita Santoni<sup>b,*</sup>, Ottorino-Luca Pantani<sup>a</sup>, Gaio Cesare Pacini<sup>a</sup>, Yvan Capowiez<sup>c</sup>

## ARTICLE INFO

**Keywords:**

Agroecology

Agricultural residues

Earthworms

Burrowing behavior

Soil bioturbation

Tomography

## ABSTRACT

The decline of soil organic matter in Mediterranean agroecosystems, coupled with the underutilization of crop residues, calls for sustainable strategies that enhance soil health while valorizing local organic resources. This study investigates the behavioral and functional responses of *Hormogaster samnitica*, a large-bodied, endogeic earthworm, to four organic amendments derived from agricultural residues: vine pruning compost (PRUN), vine pruning compost with a commercial accelerator (COMM), vine pruning compost inoculated with Indigenous Microorganisms (INDG), and vermicompost from spent coffee grounds (VERM). The experiment was conducted in laboratory mesocosms using repacked soil columns. Earthworms were introduced at three body size classes ("big", "medium" and "small"), and their impact on soil porosity and infiltration was assessed via X-ray tomography and infiltration tests. Results showed that body size significantly affected earthworm activity, with larger individuals creating wider and deeper burrows and markedly increasing water infiltration. Among treatments, COMM significantly enhanced macroporosity in the amended soil layer and induced a marked shift in the vertical distribution of macropores. However, despite increased porosity, compost-based amendments generally reduced infiltration rates compared to the CTRL. VERM showed limited effects on both bioturbation and infiltration. A two-choice preference test revealed that *H. samnitica* exhibited weak substrate preferences, while the more generalist *Aporrectodea caliginosa* clearly favored compost-based treatments over VERM and CTRL. Overall, these findings highlight the importance of considering species-specific traits and amendment properties when evaluating the ecological impact of organic inputs, and support the use of compost as a bio-stimulant for soil fauna and structure enhancement in Mediterranean systems.

## 1. Introduction

Agricultural intensification and the separation between crop production and livestock farming have markedly reduced the return of organic materials to cultivated soils (Manlay et al., 2007). This transformation has played a major role in the steady decline of soil organic matter (SOM), a fundamental component of soil fertility and the long-term functioning of agroecosystems (Garrett et al., 2020). In the past, SOM levels were maintained through the regular application of farm-generated inputs such as manure and plant residues. With the reduction of mixed farming systems and a growing dependence on synthetic fertilizers, these traditional nutrient cycles have been increasingly disrupted, particularly in arable systems that no longer benefit from

livestock-based organic inputs (Canali et al., 2005; Feller et al., 2012). As a result, many Mediterranean soils now face a SOM deficit, with consequences that include reduced structural stability, lower water-holding capacity, diminished microbial activity, and a general decline in long-term productivity (Ferreira et al., 2022).

At the same time, modern agriculture generates vast quantities of plant-based residues that are often underutilized or poorly managed, representing a missed opportunity for SOM replenishment. Crop residues alone account for a substantial portion of this organic waste, with global annual production estimated at around 74 Tg of dry matter, including approximately 45 Tg derived from rice and wheat cultivation (Medina et al., 2015). Vineyard prunings, for instance, represent a considerable biomass flow in viticultural regions but are commonly

<footnote><sup>a</sup> Department of Agricultural, Food, Environmental and Forestry Sciences (DAGRI), University of Florence, Piazzale delle Cascine 18, 50144, Florence, Italy</footnote>

<footnote><sup>b</sup> Institute of BioEconomy (IBE), National Research Council (CNR), Via Madonna del Piano 10, 50019, Sesto Fiorentino, Florence, Italy</footnote>

<footnote><sup>c</sup> INRAE, EMMAH, UMR 1114 INRAE-Avignon University, Domaine Saint Paul, F-84914, Avignon cedex 09, France</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: margherita.santoni@ibe.cnr.it (M. Santoni).</footnote>
disposed of by burning or left to decay unmanaged. These practices not only waste a potentially valuable resource but also contribute to atmospheric pollution and greenhouse gas emissions (Lou and Nair, 2009). Addressing the dual challenge of SOM depletion and residue management therefore requires innovative strategies that are both economically effective and environmentally sound (Kanwal, 2021).

Within this context, the principles of circular economy and agroecology offer promising pathways (FAO, 2018). Agroecological approaches emphasize the recycling of nutrients and organic matter, the reduction of external inputs, and the valorization of on-farm resources, aiming to build resilient and self-sustaining agroecosystems. Circular economy strategies, likewise, promote the transformation of waste streams into productive inputs, aligning resource use efficiency with environmental stewardship (Vásquez Neyra et al., 2025).

Earthworms play a pivotal role in shaping soil structure, porosity, and water dynamics. Through their burrowing and feeding activities, they create macropores, redistribute organic matter, and stimulate microbial activity, thereby enhancing soil function and fertility. As ecosystem engineers, earthworms contribute to several key soil processes, including aeration, infiltration, and aggregation (Bertrand et al., 2015). Their response to organic amendments is therefore an indicator of amendment quality and a determinant of amendment efficacy. These amendments are applied to increase or restore the soil fertility and earthworms are important bioagents that will contribute directly or indirectly to their decomposition through their burial ingestion and translocation within the soil. Thus, earthworms' behavioral and functional traits (e.g., burrowing, casting, avoidance) are strongly linked to the physical processes of bioturbation that support their ecological role (Capowiez et al., 2024; Lowe and Butt, 2002). While the behavior of widely studied endogeic or anecic species such as *Aporrectodea caliginosa* and *Lumbricus terrestris* has been extensively investigated (Capowiez et al., 2021), much less is known about the functional traits of endogeic species native to Mediterranean soils. Previous studies have shown that the response of *A. caliginosa* and *L. terrestris* has different responses mainly linked to the location of these amendments (Frazão et al., 2019): amendments on or close to the surface benefited the anecics and those mixed with the soil promoted the endogeics. Beyond this typical response, amendments are also able to modify the burrowing behavior of earthworms of both ecological categories (Ducasse et al., 2025; Froseth et al., 2014; Mombo et al., 2018).

*H. samnitica*, a large-bodied, deep-burrowing earthworm endemic to the Mediterranean area (Omodeo and Rota, 2008), remains virtually unstudied despite its ecological relevance (Marchán et al., 2018). Adult weight can reach 20 g. Unlike more mobile and generalist species, *H. samnitica* inhabits undisturbed soils and exhibits a conservative life-history strategy, characterized by low reproductive rates and long lifespan. Its limited dispersal ability and sensitivity to environmental conditions make it a potentially informative species for evaluating soil management practices in Mediterranean contexts. However, to date, no studies have systematically characterized its bioturbation behavior or assessed its responses to organic amendments under controlled conditions. Bouché (1972) described this species as endogeic since non pigmented, however this species is relatively close to *Hormogaster praetiosa* which is more pigmented and thus described as an anecic (Bouché, 1972).

The present study aims to fill these knowledge gaps by evaluating the short-term effects of different organic amendments on the behavior and functional impact of *H. samnitica*, thereby contributing to its functional characterization within the existing ecological categories. The first objective of the experiment was to characterize the bioturbation activity of *H. samnitica* individuals depending on their different body sizes (classified into small, medium, and big), to determine whether weight class influences key outcomes such as cast production, burrow formation, and soil infiltration. The second objective was to evaluate the effects of the four organic amendments on earthworm activity (as indicated by weight change and macroporosity produced) and to assess

how this activity influences key soil physical indicators, such as water infiltration. We also included a two-choice preference test to assess whether *H. samnitica* and a more generalist species, *A. caliginosa*, exhibit differential attraction to the various amendments. This behavioral assay aimed to reveal short-term preferences that may correlate with amendment quality and inform future field-scale applications.

Accordingly, we hypothesized that: (i) larger *H. samnitica* individuals would have a greater influence on soil structure and function than smaller one; (ii) the addition of organic amendments would stimulate earthworm activity and modify burrowing patterns compared to unamended soil; and (iii) earthworms would display clear substrate preferences for fresh organic amendments and less to vermicompost that was already processed by earthworms (*Eisenia fetida*).

Overall, this study provides a first characterization of *H. samnitica* role in the soil under controlled conditions and offers new insights into how different composting strategies influence earthworm activity and soil physical properties. By focusing on locally sourced amendments and a native species, the research contributes to the development of context-specific, sustainable soil management practices in Mediterranean farming systems. It also underscores the importance of integrating soil fauna into the evaluation of organic amendments, not only as bioindicators but also as key agents of soil restoration.

## 2. Material and methods

### 2.1. Earthworms collecting

*H. samnitica* individuals used in this experiment were collected by digging the soil with a shovel up to 30 cm depth and hand sorting the soil in January 2025 from the Montepaldi Long Term Experiment fields (Supplementary Fig. S1), the longest-standing trial on organic farming in the Mediterranean area, located in San Casciano Val di Pesa (FI, Italy, Long. 11°0'08'' E, Lat. 43°40'16'' N, 90 m a.s.l.) where this earthworm species is highly dominant with more than 80 % of the total population as reported in previous studies carried out on the site (Pantani et al., 2022). The surrounding landscape is characterized by vineyards typical of the Chianti Classico DOCG region, where vine prunings are an abundant local residue. This material was selected for the composting process as part of a circular economy approach. After collection, the earthworms were categorized into three weight classes: “big” (>4 g, up to 12.79 g), “medium” (2–4 g), and “small” (<2 g, down to 1.07 g). Big earthworms were either adult or subadults (i.e. with a developed puberculum).

*A. caliginosa* adults used for the preference test were collected by hand-sorting the soil in April 2025 from a humid pasture close to Avignon. Their mean weight was 0.41 g.

### 2.2. Organic amendment treatments

The experiment included four organic amendment treatments (PRUN, COMM, INDG and VERM) and one unamended control (CTRL). Three of the organic amendments were derived from vine prunings collected at Castello del Trebbio, mixed in a 1:1 ratio with alfalfa, shredded together and composted near to the fields of the Montepaldi Long Term Experiment. In detail, the PRUN treatment consisted of composted vine prunings without additives. The COMM treatment included vine prunings composed with 600 $g m^{-3}$ of a commercial composting accelerator. The INDG treatment involved composting vine prunings inoculated with Indigenous Microorganisms (IMO$_2$), following Korean Natural Farming techniques. The IMO$_2$ culture was prepared through two stages, IMO$_1$ (collection) and IMO$_2$ (propagation), and applied to the compost pile at a rate of 500 g diluted in 10 L of water. All compost piles were equipped with three internal data loggers and were regularly turned and moistened to maintain aerobic conditions and enhance microbial activity. The VERM treatment consisted of vermicompost produced from spent coffee grounds and was provided by
Funghi Espresso Società Agricola s.r.l. Due to these different origins, VERM has different properties compared to the 3 other amendments.

Physico-chemical properties of the four amendments are summarized in Table 1.

## 2.3. Mesocosms and experimental design

To evaluate the impact of the different products on earthworm activities, we realized mesocosms in PVC columns (30 cm depth and 16 cm diameter) with a mesh (2 mm) glued at the bottom. The mesocosms were built with soil taken from an orchard since more than 15 years in Montfavet, near Avignon (43°55' N, 4°48' E) in the SE of France. The main physicochemical characteristics of the soil used to fill the mesocosms are reported in Supplementary Table S1.

The soil was sieved to 2 mm. For each layer, we used 650 g (± 1 g) of soil with an average moisture content of 16 %, and pressed it using a hydraulic press between 160 and 170 kPa for 3 min to obtain an average density of 1.2 g cm⁻³. This operation was repeated 12 times to obtain 26 cm depth columns.

Products were added in the last five layers (0–10 cm depth) close to the surface. The quantity of each product was calculated based on an application rate equivalent to 32 t ha⁻¹ FW, i.e. with the same rate than when these products were applied on the field. The products were homogeneously mixed with the soil. Ten soil columns were prepared for each of the five treatments. For each treatment, five columns were inoculated with one *H. samnitica* individual belonging to the “big” weight class and five with one individual from the “medium” class. Five columns were established without amendments and without earthworms as control cores. Additionally, to gain insights into the ecology of the different life stages of this species, five columns of the CTRL treatment were set up using individuals from the “small” weight class. In total, the experiment included 60 soil columns. Each earthworm was weighed prior to its introduction into the corresponding mesocosm.

The columns were left for 1 month in an outside shelter (with temperatures ranging from 13 to 18 °C during the experiment). During this period, surface casts were collected by hands every two days, oven-dried, and weighed.

## 2.4. Monitoring of the soil porosity by X-ray tomography and 3D reconstruction of burrows

The soil cores were analyzed by X-ray tomography with a medical scanner in the INRAE centre of Nancy. The settings of the scanner were 120 kV and 50 mA. The images were then transformed into 8-bit images using ImageJ and the following parameters: –1000 for WW and 2000 for WL. The porosity created by the earthworms was obtained by binarizing the images with a fixed threshold (greylevel = 200) since the peaks for porosity and soil matrix were well separated (Capowiez et al., 1998). The macropores were characterized by computing the diameter, the volume of porosity (total and in two layers) and the barycenter. Diameter was estimated by the median of the most circular pores in the 2D images (circularity, as defined by ImageJ, > 0.8). The volume of macroporosity was the sum of the burrows whose volume was greater than 30 voxels. The barycenter was computed as the vertical barycenter of all the burrows (taking their volume into account).

The macroporosity data were aggregated into two depth layers: the

upper layer (H1, 0–10 cm), corresponding to the first five scanned sections where organic amendments were applied, and the lower layer (H2, 10–26 cm), comprising the remaining seven sections. This stratification allowed the evaluation of amendment-induced effects on burrow formation near the surface as well as their propagation into deeper soil layers.

## 2.5. Water infiltration experiment and earthworm weight

Following the tomographic analysis, infiltration tests were conducted using a single-ring infiltration method, also known as the simplified ‘Beerkan’ approach (Capowiez et al., 2015). Each core was placed on a support that allowed free drainage, and a fixed water volume (140 mL, equivalent to a water layer of 0.7 cm) was poured onto the soil surface at time zero. The time required for the complete infiltration of this volume was recorded. When the first volume had infiltrated completely, a second fixed volume of water was added. This process was repeated sequentially for a total of eight identical water volumes. To estimate the final infiltration rate (expressed in mm min⁻¹), we performed a linear regression of cumulative infiltration versus elapsed time. It is important to highlight that this method employed primarily for comparative purposes and to simulate intense rainfall events.

At the end of the infiltration test, each mesocosm was carefully dismantled, and the surviving earthworms were recovered and weighed to assess weight changes during the experiment.

## 2.6. Preference test

A two-choice preference test was carried out using plastic boxes (26 × 28.4 × 8.5 cm) filled with 1 kg of sieved soil (a layer of 1 cm depth). Pairs of treatments were compared in all possible combinations (10 total), each replicated three times. Amendments were incorporated at a rate of 32 t ha⁻¹ FW in half of the box area (one treatment per side).

Two earthworm species were tested: *H. samnitica* (8 “small” individuals per box, chosen as they could move easily in the boxes and were available in sufficient numbers) and adults of *Aporrectodea caliginosa* (10 individuals per box). The boxes were kept in darkness at 18 °C for 60 h. At the end of the test, the soil was carefully examined to determine the number of individuals on each side.

## 2.7. Statistical analysis

All statistical analyses were performed in R version 4.5.0 (R Core Development Team, 2003), and data visualization was conducted using the *lattice* (Sarkar, 2008) and *ggplot2* (Wickham, 2009) packages. Data preprocessing and merging were handled through *dplyr* (Wickham et al., 2021) and *tidverse* (Wickham, 2023) functions. Linear models (LM) were employed to evaluate the effects of treatments and earthworm weight classes on continuous variables. The assumptions of normality and homoscedasticity were checked by visual inspection of Q–Q plots and residual vs. fitted plots, respectively. When heteroscedasticity was detected, data were log-transformed. Detailed ANOVA tables reporting the effects of treatment (TRT), earthworm weight class (CLASS), and their interaction on earthworm performance and soil physical parameters are provided in Supplementary Table S2.

Infiltration dynamics were assessed by estimating linear trends

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

Averages (± standard deviation) of the physico-chemical qualities of the organic amendments, determined according to internationally recognized standard procedures (International Organization for Standardization, 1995, 1998, 2005; U.S. Composting Council, 2002) (n = 3).

<table><thead><tr><th>Treatment</th><th>Density (kg L<sup>-1</sup>)</th><th>Moisture (%)</th><th>pH</th><th>EC (mS cm<sup>-1</sup>)</th><th>C (%)</th><th>N (%)</th><th>C/N</th></tr></thead><tbody><tr><td>PRUN</td><td>0.13 (0.01)</td><td>13.2 (0.25)</td><td>6.9 (0.05)</td><td>0.90 (0.36)</td><td>43.9 (0.67)</td><td>0.80 (0.03)</td><td>57.4 (3.39)</td></tr><tr><td>COMM</td><td>0.13 (0.01)</td><td>13.5 (0.40)</td><td>6.9 (0.02)</td><td>0.80 (0.02)</td><td>44.5 (0.24)</td><td>0.80 (0.02)</td><td>58.0 (1.61)</td></tr><tr><td>INDG</td><td>0.15 (0.01)</td><td>12.8 (0.06)</td><td>7.0 (0.01)</td><td>0.70 (0.02)</td><td>43.6 (1.34)</td><td>0.70 (0.05)</td><td>62.6 (3.03)</td></tr><tr><td>VERM</td><td>0.49 (0.01)</td><td>67.9 (0.78)</td><td>5.8 (0.05)</td><td>0.90 (0.06)</td><td>42.8 (1.53)</td><td>5.00 (0.19)</td><td>8.5 (0.03)</td></tr></tbody></table>
(slopes) of water infiltration over time, expressed in mm min⁻¹ (Capowiez et al., 2015). These trends were extracted using the *emtrends* function from the *emmeans* package (Lenth, 2017).

For the preference tests, a binomial generalized linear model (GLM) was used to model the number of worms choosing each side as a function of treatment. Results were expressed as estimated probabilities on the response scale, and post hoc comparisons were conducted using Tukey-adjusted grouping letters (Lenth, 2017; McCullagh, 2019).

## 3. Results

### 3.1. Effects of the earthworms of different weight classes

All subsequent analyses presented in this section refer exclusively to the unamended control (CTRL) mesocosms, in which no organic amendment was applied. This allowed us to isolate the effect of *H. samnitica* body weight on soil structural properties and biological activity, independently of amendment-induced variability.

Cast production was observed exclusively during the first 48 h following the introduction of *H. samnitica* individuals into the soil columns. No surface activity or additional cast deposition occurred during the remainder of the one-month incubation period. Casts were recorded only in some mesocosms containing “big” (>4 g) and “medium” (2–4 g) individuals, while no surface casts were observed in the presence of “small” earthworms (<2 g).

Earthworm weight change at the end of the incubation varied among weight classes (Table 2, Supplementary Fig. S2). Individuals in the “big” and “medium” groups showed consistent weight gain (+33.0 % and +17.0 %, respectively), whereas the “small” class exhibited only marginal gain (+3.73 %) with a significant reduction in weight gain compared to the reference group “big” (*p* = 0.0125).

The structural characteristics of the macropore system varied across earthworm weight classes (Table 2, Supplementary Figs. S3 and S4). Individuals in the “big” class created significantly wider burrows (6.84 mm), while the “small” class exhibited markedly narrower macropores (3.8 mm). The difference in diameter between “small” and “big” individuals was statistically significant (*p* = 0.008), whereas the reduction observed in the “medium” class (5.94 mm) was not significant.

No significant differences among earthworms weight classes were detected for macropore volume in the upper H1 layer. In contrast, macropore volume in the deeper H2 layer was significantly affected by weight class. Linear model results showed that the “small” class had a significantly lower macropore volume than the “big” class (*p* = 0.042).

No significant differences were found among classes for total macroporosity (ranging from 38.7 cm³ to 18.4 cm³) or for the barycenter of macroporosity, suggesting that despite differences in burrow dimensions, the vertical distribution of macropores was not strongly influenced by earthworm size.

Beyond weight class based comparisons, a positive linear relationship was observed between individual earthworm weight (both before and after incubation) and the total burrow volume (Supplementary Fig. S5), suggesting that heavier and bigger individuals create more extensive burrow systems.

Water infiltration rates varied markedly among earthworm weight classes (Table 2, Supplementary Fig. S6). Individuals in the “big” and “medium” classes exhibited similarly high infiltration rates (both 5.04 mm min⁻¹), while the “small” class showed a dramatically reduced rate (0.199 mm min⁻¹). The rate measured in mesocosms containing “big” individuals was significantly higher than that associated with the “small” class (*p* < 0.0001), highlighting the greater capacity of larger individuals to influence soil hydraulic properties.

### 3.2. Effects of the different organic amendments

The final weight of *H. samnitica* individuals varied across the different organic amendment treatments (Fig. 1). Among the tested treatments, only the COMM treatment led to a statistically significant increase in earthworm weight relative to the control (CTRL), with an estimated effect of +22.8 %. All other amendments (PRUN, INDG, and VERM) did not differ significantly from the control.

The structure and distribution of macropores were markedly influenced by the type of organic amendment applied. In the upper soil layer (H1, 0–10 cm), where amendments were incorporated, a significant treatment effect on macroporosity was observed (Fig. 2A). The COMM treatment led to the highest increase in macropore volume (+6.2 cm³) compared to the control (CTRL), followed by INDG (+3.5 cm³) and PRUN (+3.1 cm³). These three treatments all differed significantly from the CTRL, while the VERM treatment did not induce a statistically significant change in volume of macroporosity in the H1 layer.

In the lower layer (H2, 10–26 cm), no significant differences in macroporosity were detected among treatments (Fig. 2B).

When considering the total macropore volume across the entire soil column, the compost-based treatments COMM, PRUN, and INDG significantly increased the total macroporosity relative to the control (Fig. 2C). In contrast, VERM had no appreciable effect.

In addition to changes in volume, the vertical distribution of macropores was also affected (Fig. 2D). The barycenter of macroporosity in the COMM treatment was significantly shifted toward the surface (−23.4 mm), indicating a concentration of macropore formation in the uppermost part of the soil column and a more shallow burrow system. No significant shifts in barycenter position were observed for the other treatments.

The analysis of infiltration trends revealed that all compost-based

<caption>**Table 2**</caption>

Mean values (± standard deviation) of selected parameters measured in mesocosms containing individual earthworms of different weight classes. Different letters within rows indicate statistically significant differences between weight classes based on Tukey-adjusted post hoc comparisons (*p* < 0.05).

<table><thead><tr><th>Parameter</th><th>Big (&gt; 4 g)</th><th>Medium (2 - 4 g)</th><th>Small (&lt; 2 g)</th></tr></thead><tbody><tr><td>Weight change (%)</td><td>33.0 (11.7)<sup>b</sup></td><td>17.0 (18.9)<sup>ab</sup></td><td>3.73 (15.9)<sup>a</sup></td></tr><tr><td>Macropore diameter (mm)</td><td>6.84 (1.160)<sup>b</sup></td><td>5.94 (0.579)<sup>b</sup></td><td>3.80 (0.699)<sup>a</sup></td></tr><tr><td>Macropore volume H1 layer (cm<sup>3</sup>)</td><td>2.47 (1.91)<sup>a</sup></td><td>1.24 (0.80)<sup>a</sup></td><td>1.60 (0.84)<sup>a</sup></td></tr><tr><td>Macropore volume H2 layer (cm<sup>3</sup>)</td><td>2.48 (2.00)<sup>a</sup></td><td>1.32 (0.51)<sup>a</sup></td><td>0.75 (0.36)<sup>a</sup></td></tr><tr><td>Total macroporosity (cm<sup>3</sup>)</td><td>38.7 (25.1)<sup>a</sup></td><td>20.3 (4.69)<sup>a</sup></td><td>18.4 (5.46)<sup>a</sup></td></tr><tr><td>Macroporosity barycenter (mm)</td><td>90.2 (30.6)<sup>a</sup></td><td>102.0 (19.7)<sup>a</sup></td><td>86.2 (21.9)<sup>a</sup></td></tr><tr><td>Infiltration rate (mm min<sup>-1</sup>)</td><td>5.04 (0.754)<sup>b</sup></td><td>5.04 (1.11)<sup>b</sup></td><td>0.199 (0.101)<sup>a</sup></td></tr></tbody></table>

<figure><img src="image_2.png" /><figcaption>**Fig. 1.** Weight variation (%) of *H. samnitica* individuals across different organic amendment treatments. Asterisks indicate statistically significant differences from the control treatment (CTRL): *p* < 0.05 (*), *p* < 0.01 (**), *p* < 0.001 (**).</figcaption></figure>
<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /><figcaption>**Fig. 2.** Effects of organic amendments on macropore characteristics. (A) Macropore volume in the H1 layer (0–10 cm), (B) Macropore volume in the H2 layer (10–26 cm), (C) Total macropore volume across the entire column (0–26 cm), (D) Vertical distribution of macroporosity (barycenter). Asterisks indicate statistical significance of pairwise comparisons versus CTRL: \* p < 0.05, \*\* p < 0.01, \*\*\* p < 0.001.</figcaption></figure>

treatments showed significantly lower infiltration rates compared to the CTRL group (Fig. 3). Specifically, the slope of the infiltration curve in CTRL (5.04 mm min⁻¹) was significantly higher than in PRUN (p < 0.01), COMM (p < 0.001), and INDG (p < 0.001), indicating slower infiltration in these amended soils. No significant difference was found between CTRL and VERM (p = 0.85), suggesting that the infiltration performance of VERM-treated cores was comparable to the unamended control.

<figure><img src="image_7.png" /><figcaption>**Fig. 3.** Water infiltration rate (mm min⁻¹) measured in soil columns under different organic amendment treatments. Asterisks denote statistically significant differences from the control based on pairwise comparisons: p < 0.05 (*), p < 0.01 (**), p < 0.001 (**").</figcaption></figure>

### 3.3. Preference test

The two-choice preference test revealed that *A. caliginosa* displayed a clear preference for COMM, followed by INDG and PRUN, all significantly more attractive than CTRL. In contrast, *H. samnitica* exhibited weaker treatment differentiation. Although PRUN showed the highest selection probability for *H. samnitica*, the overlapping grouping letters among all treatments indicate that no definitive preference pattern emerged (Fig. 4).

<figure><img src="image_8.png" /><figcaption>**Fig. 4.** Estimated probability of soil side choice by *A. caliginosa* and *H. samnitica* across different organic amendment treatments in the two-choice preference test. Different letters indicate significant differences between treatments (p < 0.05; Tukey's post hoc test).</figcaption></figure>
# 4. Discussion

## 4.1. Characterization of the burrowing behavior of *H. samnitica*

The first objective of this study was to characterize the bioturbation behavior of *H. samnitica* and to verify whether its subdivision into three weight classes could meaningfully explain differences in soil structural modifications and biological responses. Our findings confirm that individual size within this species plays a substantial role in determining its activity and ecological impact.

The absence of surface cast production beyond the first hours is in line with the endogeic nature of *H. samnitica*, whose activity remains confined to subsurface layers (Bouché, 1977; Capowiez et al., 2024). Interestingly, casts were only observed in the presence of “medium” and “big” further indicating that these casts were made when burrowing the very first burrows.

Weight variation over the incubation period further supports this interpretation. While both “big” and “medium” individuals showed weight gain, “small” individuals exhibited a significantly lower weight increase, suggesting a reduced adaptability or lower feeding efficiency in the mesocosm conditions (Lowe and Butt, 2005). This aligns with observations in other earthworm species, where juveniles often exhibit slower acclimation and reduced mobility in constrained environments (Grigoropoulou et al., 2009). It could also indicate that young *H. samnitica* would require soils with a higher organic matter content (or quality) to grow.

Structural analysis of the burrow system revealed a significant effect of body size on macropore diameter. The smaller class generated narrower burrows, with a marked difference compared to the “big” individuals, confirming that larger earthworms exert a greater mechanical influence on soil structure (Capowiez et al., 2015). Although total macroporosity and vertical distribution (barycenter) did not differ significantly among classes, the H2 layer macroporosity was significantly reduced in the “small” group. This suggests limited vertical exploration by smaller individuals, potentially due to lower burrowing capacity or energetic constraints as assumed for *L. terrestris* juveniles (Grigoropoulou et al., 2009).

Water infiltration patterns confirmed the functional relevance of these differences. The presence of “big” and “medium” earthworms preserved high infiltration rates, while the “small” class drastically reduced water movement, reflecting both their reduced macropore diameter and limited soil exploration. These results support previous findings indicating that earthworm-induced macropores, particularly deep, continuous ones, are critical for enhancing soil hydraulic conductivity (Pelíšek, 2018; Shipitalo and Butt, 1999).

Overall, this first characterization of *H. samnitica* under controlled conditions suggests that this species is purely endogeic and its impact on soil physical properties is size-dependent. Larger individuals appear better adapted to the experimental context and more effective in creating preferential flow paths and altering soil porosity.

## 4.2. Effects of organic amendments on *H. samnitica* bioturbation activity

The second objective of this study was to explore the effect of different organic amendments on *H. samnitica* behavior and soil modification capacity. Differences among treatments were most apparent in the upper soil layer (H1), where amendments were mixed with the soil, supporting the hypothesis that organic matter availability modulates burrowing behavior (Capowiez et al., 2021; Frazão et al., 2019). This is well known for endogeic species (Hugnes et al., 1996).

Compost-based treatments (COMM, PRUN, INDG) significantly enhanced macropore volume in the H1 layer (0–10 cm), likely reflecting an increased feeding or exploration activity in response to the presence of labile organic matter, as observed in other endogeic species which preferentially concentrate their burrowing activity in organic-rich layers (Capowiez et al., 2021; Le Couteulx et al., 2015). The vertical profile of

macroporosity was also altered, with the COMM treatment inducing a marked shift of the barycenter toward the surface. This confirms that earthworm bioturbation was spatially influenced by amendment placement and suggests a positive response of *H. samnitica* to this type of amendment.

Interestingly, VERM treatment failed to induce significant changes in either macroporosity or barycenter position, mirroring what was observed for small individuals. This may be due to the more advanced decomposition stage of vermicompost and its reduced palatability, as previously suggested for other species (Ducasse et al., 2023, 2025).

Despite increased porosity in the upper layer, infiltration rates were reduced in compost-treated soils. This counterintuitive result may be explained by the formation of smaller, more tortuous macropores near the surface, perhaps less continuous and thus less insufficient to ensure rapid vertical flow. Alternatively, enhanced biological activity may have favored soil aggregate reorganization or partial pore clogging, especially in PRUN and COMM treatments.

## 4.3. Preference behavior and amendment attractiveness

The two-choice test results provide further insight into the behavioral responses of *H. samnitica* and *A. caliginosa* toward amended soils. Across the board, both species showed higher selection probabilities for PRUN, COMM, and INDG compared to CTRL, while VERM did not differ significantly from the CTRL.

Species-specific responses were particularly informative. *A. caliginosa* displayed a clear hierarchy of preferences, with COMM being the most attractive, consistent with its known ability to discriminate organic substrates based on OM quality and stage of decomposition (Le Couteulx et al., 2015). *A. caliginosa* is often classified as a specialist regarding its diet (Hsu et al., 2023). In contrast, *H. samnitica* showed less distinct preferences, suggesting a more conservative exploratory behavior. This weaker differentiation is coherent with its ecological strategy and limited mobility in short-duration assays.

The fact that vermicompost was not significantly preferred by either species reinforces the notion that substrate freshness and microbial composition, both more typical of composts, play a decisive role in short-term attractiveness (Ducasse et al., 2025). Furthermore, preference responses were consistent with the observed changes in macroporosity and infiltration, confirming that bioturbation intensity and behavioral choices are strongly interlinked and mediated by amendment characteristics.

## 4.4. Limitations and future directions

This study, while providing novel insights into the behavior of *H. samnitica*, has some limitations. First, only a limited number of amendment types and a single application rate were tested, whereas agricultural practices involve greater variability. In addition, the focus on individual earthworms did not capture potential density-dependent effects or interactions among different species. Future studies should therefore extend the duration and scale of observations, test a wider range of organic inputs and doses, and integrate multi-species approaches under realistic agronomic conditions to assess the long-term implications for soil structure, fertility, and crop performance.

# 5. Conclusions

*H. samnitica* exhibits size-dependent effects on soil structural properties, suggesting that individual biomass is a key determinant of its functional role in soil ecosystems. Larger individuals created wider and deeper macropores, and enhanced water infiltration that are critically important for soil functioning and hydrodynamics. Juveniles, as often for earthworms, live closer to the surface presumably in link with their less powerful muscles. These findings validate the subdivision into weight classes as ecologically meaningful, with smaller individuals
showing limited adaptability, lower activity, and minimal influence on soil porosity and, in turn, on infiltration.

The application of organic amendments further modulated earth-
worm behavior and bioturbation. Compost-based treatments, particu-
larly the compost inoculated with commercial composting accelerator
(COMM), significantly increased macropore formation in the amended
layer and altered its vertical distribution, confirming their capacity to
stimulate biological activity. In contrast, vermicompost (VERM) had
limited effects, consistent with its more advanced decomposition stage
and reduced attractiveness. These structural changes, however, did not
always translate into improved water infiltration, likely due to pore
geometry or clogging effects.

Preference tests revealed that both *H. samnitica* and *A. caliginosa*
were able to discriminate among substrates, favoring fresher, composted
materials over the control and vermicompost. This supports the hy-
pothesis that OM freshness and microbial composition are central to
short-term substrate attractiveness and bioturbation induction.

Altogether, these results underscore the potential of specific
amendments, particularly compost, to enhance soil structure and favor
earthworm activity. They also highlight the need to consider species-
specific responses and individual-level traits such as body size when
assessing soil fauna reactions and effects. While the short-term meso-
cosm approach proved effective in differentiating amendment impacts,
future studies should validate these findings under field conditions,
where more complex interactions and environmental variability occur.

CRediT authorship contribution statement

**Francesco Serafini:** Visualization, Methodology, Investigation, Formal analysis, Data curation, Conceptualization, Writing – original draft. **Margherita Santoni:** Supervision, Resources, Methodology, Writing – review & editing. **Ottorino-Luca Pantani:** Validation, Resources, Investigation, Writing – review & editing. **Gaio Cesare Pacini:** Supervision, Project administration, Writing – review & editing. **Yvan Capowiez:** Validation, Supervision, Methodology, Formal analysis, Conceptualization, Writing – review & editing, Writing – original draft.

Funding

This research did not receive any specific grant from funding
agencies in the public, commercial, or not-for-profit sectors.

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.

**Appendix A. Supplementary data**

Supplementary data to this article can be found online at https://doi.
org/10.1016/j.apsoil.2025.106716.

Data availability

Data will be made available on request.

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