Kevin Hoeffner
email: kevin.hoeffner@gmail.com

Cécile Monard

Daniel Cluzeau

Mathieu Santonja

Response of temperate anecic earthworm individual biomass to species interactions

Keywords: soil fauna, Aporrectodea, Lumbricus, earthworm mass gain, species assemblages, temperate grasslands Bi-assemblages Bi-assemblages SAn/EpA

Earthworms contribute to a wide range of ecosystem services provided by the soil.

, Aporrectodea nocturna (Evans, 1946), Aporrectodea giardi (Savigny, 1826). Twenty-one pairs of individuals were established following five assemblages: monospecific pairwise assemblages of epi-and strict-anecic earthworms (2 × 3 treatments), bispecific pairwise assemblages within epi-and within strict-anecic earthworms (2 × 3 treatments) and bispecific pairwise assemblages with one epi-and one strict-anecic earthworm (3 × 3 treatments). Treatments were maintained in mesocosms for 30 days under controlled conditions (food provided at the soil surface at the beginning of the experiment) and changes in the earthworm individual biomass were measured. Strict-anecic earthworms in monospecific or bispecific assemblages maintained their initial biomass. In contrast, epi-anecic earthworms exhibited an increase of 12.4% and 23.7% of their biomass in monospecific and bispecific assemblages, respectively. In bispecific assemblages combining one epi-and one strict-anecic earthworm, epianecic earthworms solely gained biomass leading to a total increase of a 6.9%. Surprisingly, the biomass' changes were not homogenous within the two sub-categories as the six earthworm species exhibited species-specific responses. The greatest increases in individual biomass were recorded for epi-anecic earthworms in the bispecific assemblages. This study provides further evidence for the distinction between the two anecic sub-categories, as it indicates that species interactions is positive only for epi-anecic earthworm biomass.

Introduction

Earthworms are widespread organisms and constitute the most important animal biomass in European soil under temperate climates [START_REF] Hole | Effects of animals on soil[END_REF][START_REF] Curry | Grassland invertebrates: ecology, influence on soil fertility and effects on plant growth[END_REF][START_REF] Bar-On | The biomass distribution on Earth[END_REF].

According to their physiology, morphology and behaviour, earthworms are classified into three ecological categories: epigeic, endogeic and anecic species [START_REF] Bouché | Lombriciens de France: écologie et systématique[END_REF][START_REF] Bouché | Strategies lombriciennes[END_REF]. Nevertheless, several studies highlighted that within anecic earthworms two ecological sub-categories can be identified based mainly on their feeding [START_REF] Ferlian | Carbon food resources of earthworms of different ecological groups as indicated by 13C compound-specific stable isotope analysis[END_REF][START_REF] Andriuzzi | Organic matter composition and the protist and nematode communities around anecic earthworm burrows[END_REF][START_REF] Larsen | Substantial nutritional contribution of bacterial amino acids to earthworms and enchytraeids: a case study from organic grasslands[END_REF] and burrowing [START_REF] Bastardie | X-ray tomographic and hydraulic characterization of burrowing by three earthworm species in repacked soil cores[END_REF] behaviours. Briefly, epi-anecic earthworms (Lumbricus sp.) feed preferentially on surface organic matter, mainly composed of leaf litter, which they can bury into their main permanent burrows. In contrast, strict-anecic earthworms (Aporrectodea sp.) feed on humified organic matter already incorporated into the soil with a slight proportion of leaf litter and they establish a denser burrow network than epi-anecic earthworms.

Consequently, ecosystem services provided by earthworms belonging to these two anecic subcategories are expected to be different.

In this context, improving our understanding of the assembly rules of anecic earthworm communities is important since (i) from one to six different anecic earthworm species can coexist in European soils under temperate climates [START_REF] Poier | Spatial distribution of earthworms and soil properties in an arable loess soil[END_REF][START_REF] Murchie | Responses of earthworm species to long-term applications of slurry[END_REF] and (ii) inoculations of anecic earthworms are frequently realised in order to enhance soil functions [START_REF] Butt | Earthworms in soil restoration: lessons learned from United Kingdom case studies of land reclamation[END_REF][START_REF] Forey | Inoculation of an ecosystem engineer (Earthworm: Lumbricus terrestris) during experimental grassland restoration: consequences for above and belowground soil compartments[END_REF]. Previous studies reported that interactions within anecic earthworms could either delay or increase their growth rate depending on the degree of niche overlap between species [START_REF] Butt | Interactions between selected earthworm species: a preliminary, laboratory-based study[END_REF]Lowe andButt, 1999, 2002;[START_REF] Uvarov | Inter-and intraspecific interactions in lumbricid earthworms: their role for earthworm performance and ecosystem functioning[END_REF]. However, our knowledge about these interactions within and between epi-and strict-anecic earthworms is still limited.

The present study aimed to determine the effects of anecic earthworm interactions on individual biomass as a specific proxy of earthworm fitness [START_REF] Butt | The effects of temperature on the intensive production of Lumbricus terrestris (oligochaeta, Lumbricidae)[END_REF][START_REF] Butt | The life cycle of the earthworm Lumbricus terrestris L. (Oligochaeta, Lumbricidae) in culture[END_REF]. We measured changes in earthworm individual biomass after 30 days of experimentation with leaf litter placed at the soil surface to mimic natural leaf litter deposition. First, we hypothesized a loss of biomass in mono-and bispecific mixtures within each ecological sub-category due to resource competition. Second, we hypothesized no change in individual biomass when combining one epiand one strict-anecic species due to their different feeding behavior (no niche overlap expected).

Materials and methods

We studied three strict-anecic earthworm species, Aporrectodea caliginosa meridionalis [START_REF] Bouché | Lombriciens de France: écologie et systématique[END_REF], Aporrectodea nocturna (Evans, 1946), Aporrectodea giardi (Savigny, 1826) and three epi-anecic earthworm species, Lumbricus rubellus rubellus (Hoffmeister, 1843), Lumbricus centralis [START_REF] Bouché | Lombriciens de France: écologie et systématique[END_REF], Lumbricus terrestris, (Linné, 1758). We collected the soil (5-20 cm depth), fresh leaf litter of Lolium perenne (Linné, 1753; a typical grassland species), and the six anecic earthworm species from temporary grasslands in the Long Term Ecological Research (LTER) site ''Zone Atelier Armorique'' (48°50' N, -1°58' W), Brittany, France. We determined soil and leaf litter characteristics according to the protocols described in [START_REF] Hoeffner | Feeding behaviour of epi-anecic earthworm species and their impacts on soil microbial communities[END_REF]. We identified the soil as a brown soil with 48.2% sand, 37.5% silt and 14.3% clay, characterized by 2.9% organic matter, a C:N ratio of 9.7 and a pH of 6.4. The leaf litter was characterized by a C:N ratio of 13.9, a phenolic concentration of 3.4%, a water holding capacity of 392.6% and a specific leaf area of 330.2 cm 2 .g -1 .

Two weeks before the beginning of the experiment, we selected adult earthworms from the six species, we grouped them in monospecific boxes containing the soil previously hand-sieved at 4-mm and fed them with air-dried leaves of L. perenne. Thirty-six hours before the start of the experiment, we placed each earthworm on a moist sponge in a plastic box to void its gut content (depuration).

We filled mesocosms (PVC cylinder, 30 cm high, 10 cm diameter) with 4.9 kg of fresh sieved soil, placed 3.5 g of air-dried leaves of L. perenne at the soil surface to mimic the natural leaf litter deposition and re-humidified them with deionized water. Then, we determined the individual biomass of adult earthworms (i.e. presence of a turgid clitellum, to ensure taxonomic identity) and placed them in the corresponding mesocosm. The six earthworm species presented an initial mean biomass gradient varying from 0.62 g for L. rubellus to 4.72 g for L. terrestris (Supplementary Fig. S1). We placed the mesocosms in a climatic chamber at 12 °C, with a relative humidity of 85% and a 12 h: 12 h light: dark regime. From the six anecic earthworm species, we established 21 pairs of individuals in five replicates following five assemblages (Supplementary Fig. S2): monospecific pairwise assemblages of strict-and epi-anecic earthworms (2 sub-ecological categories × 3 earthworm species treatments), bispecific pairwise assemblages within strict-and within epi-anecic earthworms (2 sub-ecological categories × 3 earthworm species treatments) and bispecific pairwise assemblages with one strict-and one epi-anecic earthworm (3 epi-anecic species × 3 strict anecic species treatments). We maintained soil moisture by spraying deionized water on the soil surface twice a week. Given the large number of mesocosms, we established replicates of each treatment one day apart leading to five blocks of 21 mesocosms.

After 30 days, leaf litter was visually still available at the soil surface and we collected earthworm individuals by destroying the mesocosms. We determined each individual earthworm biomass after depuration, as previously described. We calculated the percentage of change in earthworm individuals' biomass following the formula: ((final fresh biomassinitial fresh biomass) / initial fresh biomass) × 100%. For monospecific assemblages, we assigned the biomass of the lightest earthworm at the end of the experiment to the lightest one at the beginning of the experiment.

We performed statistical analyses with the R software 3.2.3 (R. [START_REF] Team | R: a language and environment for statistical computing[END_REF]. We evaluated significance in all cases at P < 0.05. Data met the conditions of normality and homoscedasticity. We excluded four soil mesocosms that contained dead earthworms (from random species and random assemblages) from the data analysis. First, we used a one-way ANOVA, followed by a Tukey HSD test for post hoc pairwise comparisons, to assess differences in initial biomass between the six earthworm species. Second, within the two ecological categories, we used two-way ANOVAs to test for the effects of species assemblage and block on earthworm biomass change. Third, we used two-way ANOVAs to test for the effects of species combinations and block on the biomass change of the 6 studied species. Finally, we used separated one-sample t-tests with adjusted P-values due to multiple comparisons to test whether (i) earthworm species and (ii) earthworm assemblages significantly lost or gained biomass during the experiment.

Results

During the experiment, four earthworms over the 210 died. Whatever the treatment, the surviving earthworms remained adults (with a turgid clitellum) until the end of the experiment.

Overall, strict anecic earthworms in mono-and bispecific pairwise assemblages within their subcategory maintained their initial biomass during the experiment (Fig. 1A). In contrast, the biomass of epi-anecic earthworms in mono-and bispecific pairwise assemblages within their sub-category increased (Fig. 1B), and this increase was twice higher in bispecific compared to monospecific assemblages (+23.7% vs. +12.4%, respectively, Fig. 1B). The biomass of earthworms in bispecific assemblages combining one epi-and one strict-anecic was 6.9% higher than at the beginning of the experiment, but this increase was solely due to the biomass gained by epi-anecic earthworms (+17.0%, Fig. 1B).

The biomass' changes were not homogenous within the two anecic sub-categories as the six earthworm species exhibited species-specific responses (Fig. 2). Contrary to the two other strict-anecic species, A. giardi lost biomass in monospecific assemblage (-9.2%) as well as in bispecific assemblages with A. nocturna (-7.3%), L. rubellus (-7.5%) and L. terrestris (-13.6%, Fig. 2). While the biomass of A. caliginosa meridionalis and A. nocturna did not vary significantly in both mono-and bispecific assemblages with an epi-anecic species, these two strict-anecic species gained biomass in presence of A. giardi (+6.8% and +9.0% for A. caliginosa meridionalis and A. nocturna, respectively, Fig. 2). Within the epi-anecic species, the biomass of L. centralis only increased in presence of A. caliginosa meridionalis (Fig. 2), while L. terrestris gained biomass whatever the pairwise assemblage considered (Fig. 2). Finally, the biomass of L. rubellus increased in monospecific assemblage (+12.0%) as well as in bispecific assemblages with L. centralis (+29.8), A. nocturna (+21.2%) and A. caliginosa meridionalis (+12.3%, Fig. 2).

Discussion

In contrast to our first hypothesis of a negative effect of species interaction on biomass change in pairwise assemblages within each anecic ecological sub-category [START_REF] Butt | Interactions between selected earthworm species: a preliminary, laboratory-based study[END_REF]Lowe andButt, 1999, 2002;[START_REF] Frazão | Soil structure formation and organic matter distribution as affected by earthworm species interactions and crop residue placement[END_REF], we did not observe any change in the biomass of strictanecic earthworms but a gain of biomass of epi-anecic earthworms. However, the organic resources and the experimental time used in previous studies differed to those applied in the present study (i.e. Lolium perenne and 30 days). For example, [START_REF] Lowe | Interspecific interactions between earthworms: a laboratory-based investigation[END_REF] terrestris burrows fed with L. perenne. These previous observations of positive effects of epianecic species on microbial communities could lead to enhanced leaf litter decomposition and then to higher organic matter assimilation by epi-anecic earthworms [START_REF] Hoeffner | Feeding behaviour of epi-anecic earthworm species and their impacts on soil microbial communities[END_REF]. To confirm that these observed effects are due to the ecological category of earthworms and not the genus they belong to, it would be interesting to study other genus within anecic earthworms such as Scherotheca, Octodrilus or Fitzingeria.

Overall, the effect of the interaction between one strict-and one epi-anecic earthworm was mainly additive confirming our second hypothesis of reduced niche overlap. Nevertheless, we observed some gain or loss of biomass depending on the earthworm assemblages. It may be assumed that other mechanisms, such as facilitation or competition for resource access and gallery formation, could explain these differences and some additional experiments are required.

Within strict-anecic earthworms, A. caliginosa meridionalis and A. nocturna in monospecific assemblages maintained their initial biomass while A. giardi lost biomass. These findings are in agreement with previous studies that also reported no change in A. caliginosa meridionalis biomass after 31 days when fed with Castanea sativa [START_REF] Cortez | Decomposition of mediterranean leaf litters by Nicodrilus meridionalis (Lumbricidae) in laboratory and field experiments[END_REF] and a 38% loss of biomass of A. giardi after 31 days when fed with Triticum aestivum [START_REF] Cortez | C and N transfer in soil with or without earthworms fed with 14C-and 15N-labelled wheat straw[END_REF]. Overall, epi-anecic earthworms in monospecific assemblages gain biomass [START_REF] Shipitalo | Effect of diet on the feeding and casting activity of Lumbricus terrestris and L. rubellus in laboratory culture[END_REF][START_REF] Binet | Experimental microcosm study of the role of Lumbricus terrestris (Oligochaeta : Lumbricidae) on nitrogen dynamics in cultivated soils[END_REF][START_REF] Hoeffner | Feeding behaviour of epi-anecic earthworm species and their impacts on soil microbial communities[END_REF]. For example, [START_REF] Shipitalo | Effect of diet on the feeding and casting activity of Lumbricus terrestris and L. rubellus in laboratory culture[END_REF] reported a 100% and a 35% increase in biomass of L. rubellus and L. terrestris when fed with Medicago sativa litter after 32 and 36 days of experimentation, respectively. Thus, the present study confirms the two distinct behaviours to process the leaf litter of strict-and epi-anecic earthworms and provides support to this sub-category distinction. In fact, feeding on leaf litter at the surface by epianecic earthworms [START_REF] Larsen | Substantial nutritional contribution of bacterial amino acids to earthworms and enchytraeids: a case study from organic grasslands[END_REF][START_REF] Hoeffner | Feeding behaviour of epi-anecic earthworm species and their impacts on soil microbial communities[END_REF][START_REF] Hoeffner | Epi-anecic rather than strict-anecic earthworms enhance soil enzymatic activities[END_REF] allowed them to increase their biomass with our experimental conditions. In contrast, strict-anecic earthworms by feeding mainly on soil organic matter had restricted access to organic matter (2.9% of soil content). Additionally, we also cannot exclude that soil organic matter consumption by strict-anecic earthworms was restricted due to the low soil volume of 1571 cm -3 in the experimental mesocosms.

Finally, the positive species interaction between epi-anecic species highlighted in our lab experiment could partly explain field species assemblages reported in previous studies [START_REF] Decaëns | Assembly rules within earthworm communities in North-Western France -a regional analysis[END_REF][START_REF] Murchie | Responses of earthworm species to long-term applications of slurry[END_REF]. For example, [START_REF] Murchie | Responses of earthworm species to long-term applications of slurry[END_REF] observed three epi-anecic species and only one strict-anecic species in a same temperate grassland suggesting facilitation within epi-anecic species. Furthermore, [START_REF] Decaëns | Assembly rules within earthworm communities in North-Western France -a regional analysis[END_REF] observed a higher co-occurrence rate between epi-anecic species than between strict anecic species in twenty temperate grasslands.

Figure legends

  used separated cattle solids during 84 days and Frazão et al. (2018) a mixture of Triticum aestivum and Raphanus sativus during 61 days. Postma-Blaauw et al. (2006) observed that the assemblage of L. rubellus with L.terrestris fed with Solanum tuberosum enhanced the bacterial biomass in their burrows. In addition,[START_REF] Hoeffner | Feeding behaviour of epi-anecic earthworm species and their impacts on soil microbial communities[END_REF] observed specific fungal communities within L. rubellus, L. centralis and L.

Fig. 1 .

 1 Fig. 1. Earthworm biomass changes according to anecic earthworm assemblages of (a) strict-and

Fig. 2 .

 2 Fig. 2. Biomass changes of earthworms in mono-and bispecific pairs of AM, AN, AG, LR, LC

  Figure 1 284

Acknowledgements

We thank the Institut National de la Recherche Agronomique (Arras, France) for mineral soil analyses. We also thank Clément De Graaf, Clément Bertran, Albin Fertil, Alan Schneider and Anaïs Rancon for their technical assistance in the laboratory. Kevin Hoeffner was supported by a PhD grant from the French Ministère de la Recherche.