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Earthworm Cast Formation and Development: A Shift
From Plant Litter to Mineral Associated Organic Matter
Alix Vidal, Françoise Watteau, Laurent Remusat, Carsten Mueller,

Thanh-Thuy Nguyen Tu, Franz Buegger, Sylvie Derenne, Katell Quenea

To cite this version:

Alix Vidal, Françoise Watteau, Laurent Remusat, Carsten Mueller, Thanh-Thuy Nguyen Tu, et al..
Earthworm Cast Formation and Development: A Shift From Plant Litter to Mineral Associated
Organic Matter. Frontiers in Environmental Science, Frontiers, 2019, 7, _xFFFF_10.3389/fenvs.2019.00055_xFFFF_.
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ORIGINAL RESEARCH
published: 24 April 2019
doi: 10.3389/fenvs.2019.00055

Earthworm Cast Formation and
Development: A Shift From Plant
Litter to Mineral Associated Organic
Matter

Alix Vidal 1,2*, Francoise Watteau 3, Laurent Remusat 4, Carsten W. Mueller 2,
Thanh-Thuy Nguyen Tu 1, Franz Buegger 5, Sylvie Derenne 1 and Katell Quenea 1

1 UMR Milieux Environnementaux, Transferts et Interactions dans les Hydrosystèmes et les Sols (METIS), Sorbonne Université
CNRS-EPHE, Paris, France, 2 Lehrstuhl für Bodenkunde, TU München, Freising, Germany, 3 INRA, LSE, Université de
Lorraine, Nancy, France, 4 Muséum National d’Histoire Naturelle, Sorbonne Université, UMR CNRS 7590, IRD, Institut de
Minéralogie, de Physique des Matériaux et de Cosmochimie, IMPMC, Paris, France, 5 Institute of Biochemical Plant
Pathology, Helmholtz Zentrum München, German Research Center for Environmental Health, Neuherberg, Germany

Earthworms play a major role in litter decomposition, in processing soil organic matter
and driving soil structure formation. Earthworm casts represent hot spots for carbon
turnover and formation of biogeochemical
interfaces in soils. Due to the complex
microscale architecture of casts, understanding the mechanisms of cast formation and
development at a process relevant scale, i.e., within microaggregates and at the interface
between plant residues, microorganisms and mineral particles, remains challenging.
We used stable isotope enrichment to trace the fate of shoot and root litter in intact
earthworm cast samples. Surface casts produced by epi-anecic earthworms (Lumbricus
terrestris) were collected after 8 and 54 weeks of soil incubation in mesocosms, in the
presence of 13C-labeled Ryegrass shoot or root litter deposited onto the soil surface. To
study the alteration in the chemical composition from initial litter to particulate organic
matter (POM) and mineral-associated organic matter (MOM) in cast samples, we used
solid-state 13C Nuclear Magnetic Resonance spectroscopy (13C-CPMAS-NMR) and
isotopic ratio mass spectrometry (EA-IRMS). We used spectromicroscopic approach to
identify plant tissues and microorganisms involved in plant decomposition within casts. A
combination of transmission electron microscopy (TEM) and nano-scale secondary ion
mass spectrometry (NanoSIMS) was used to obtain the distribution of organic carbon
and δ13C within intact cast sample structures. We clearly demonstrate a different fate of
shoot- and root-derived organic carbon in earthworm casts, with a higher abundance of
less degraded root residues recovered as particulate organic matter on the short-term
(8 weeks) (73 mg·g−1 in Cast-Root vs. 44 mg·g−1 in Cast-Shoot). At the early stages
of litter decomposition, the chemical composition of the initial litter was the main factor
controlling the composition and distribution of soil organic matter within casts. At later
stages, we can demonstrate a clear reduction of structural and chemical differences in
root and shoot-derived organic products. After 1 year, MOM clearly dominated the casts
(more than 85% of the total OC in the MOM fraction). We were able to highlight the shift
from a system dominated by free plant residues to a system dominated by MOM during
cast formation and development.

Keywords: carbon isotopic labeling, root and shoot litter, microorganisms, NanoSIMS, TEM, 13C-CPMAS-NMR

Edited by:
Maria Luz Cayuela,
Center for Edaphology and Applied
Biology of Segura (CSIC), Spain

Reviewed by:
Andrey S. Zaitsev,
University of Giessen, Germany
Philippe Cambier,
Institut National de la Recherche
Agronomique (INRA), France

*Correspondence:
Alix Vidal
alix.vidal@wzw.tum.de

Specialty section:
This article was submitted to
Soil Processes,
a section of the journal
Frontiers in Environmental Science

Received: 05 November 2018
Accepted: 05 April 2019
Published: 24 April 2019

Citation:
Vidal A, Watteau F, Remusat L,
Mueller CW, Nguyen Tu T-T,
Buegger F, Derenne S and Quenea K
(2019) Earthworm Cast Formation and
Development: A Shift From Plant Litter
to Mineral Associated Organic Matter.
Front. Environ. Sci. 7:55.
doi: 10.3389/fenvs.2019.00055

Frontiers in Environmental Science | www.frontiersin.org

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April 2019 | Volume 7 | Article 55

Vidal et al.

Earthworm Cast Formation and Development

INTRODUCTION

Plant residues represent the main contributor to soil organic
matter (SOM), followed by microorganism biomass. Among
plant residues, the distinction between above (leaves and shoots)
and below ground (dead roots and rhizodeposits) inputs is
crucial. It is now commonly recognized that roots decompose
at a slower rate than shoots and root-derived carbon represents
a larger pool of carbon in soils (Balesdent and Balabane, 1996;
Puget and Drinkwater, 2001; Lu et al., 2003; Angst et al., 2016).
However, whether the slower root decomposition depends on
chemical composition, physical or physico-chemical protection,
remains unclear, due to the initial location of root in the soil
(Rasse et al., 2005).

Biotic factors driving plant residue decomposition encompass
the litter quality, as well as the activity of soil fauna and
microorganisms (Oades, 1988; Cortez and Bouché, 1998). Soil
fauna fragments, transports and partly decomposes residues
(Lavelle et al., 1993), and microorganisms decompose and
transform organic compounds (Kuzyakov and Blagodatskaya,
2015). Earthworms, ants and termites are considered as the
main ecosystem engineers having a significant
impact on
their environments, under suitable living conditions (Lavelle,
2002; Hastings et al., 2007). In temperate regions, earthworms
account for the main invertebrate biomass in soils (Lee, 1985;
Edwards, 2004). These saprophageous invertebrates ingest both
organic (plant litter, SOM and microorganisms) and mineral
soil particles. During ingestion, residues are fragmented and
the preexisting soil microstructures destroyed. Organic elements
are mixed with mineral particles, complexed with mucus, partly
assimilated and mineralized, and mainly released at the soil
surface in the form of biogenic organo-mineral aggregates called
casts (Lee, 1985; Six et al., 2004). Within a few weeks, the
presence of earthworms increases the proportion of macro
and microaggregates that are more stable compared with non-
biogenic aggregates (Six et al., 2004; Bossuyt et al., 2005; Zangerlé
et al., 2011). The mutualistic relationship maintained between
earthworms and microorganisms enhance litter decomposition
during the gut transit and in casts (Brown et al., 2000). This
results in higher microbial activity within casts compared to
bulk soil at the scale of days and weeks (Frouz et al., 2011),
inducing hotspots of microbial activity (Decaëns, 2010; Kuzyakov
and Blagodatskaya, 2015; Athmann et al., 2017). However, casts
are among the most complex and dynamic structures in soil
(Lee, 1985) and were recognized as potentially favoring long-term
carbon protection (Martin, 1991; Bossuyt et al., 2005; Frouz et al.,
2009; Sánchez-de León et al., 2014). The occlusion of SOM within
microaggregates, which can be found in casts, tends to protect
organic carbon (OC) from decomposition (Chenu and Plante,
2006; Lützow et al., 2006; Dignac et al., 2017).

Many relevant processes in earthworm casts happen at
a fine spatial scale,
i.e., within microaggregates and at the
interface between plant residues, microorganisms and mineral
particles. As the gut passage leads to a fine scale mixing of
mineral and organic soil constituents together with earthworm-
derived mucus and bacteria, the resulting casts show a highly
complex microscale architecture (Vidal et al., 2016b). To gain

a more fundamental understanding of the processes at the
biogeochemical interfaces at the relevant process scale within the
casts, the use of spectromicroscopic imaging techniques allowing
for high spatial resolution is necessary. The visualization of OM
within undisturbed cast microstructures using spectroscopic and
microscopic methods can improve our understanding of plant
tissue degradation and their association with mineral particles
and microorganisms, at these soil biology hot spots. Due to
methodological difficulties, this scale of study has often been
left out for large scale investigations (Hastings et al., 2007), and
studies depicting the role of earthworm casts in the formation
and transformation of SOM at the fine scale are scarce (Barois
et al., 1993; Pey et al., 2014; Vidal et al., 2016b).

Existing studies focused on revealing cast constituents at a
given date, without discerning possible differences with respect
to the processing of different substrate materials (Barois et al.,
1993; Pey et al., 2014; Vidal et al., 2016b). As main plant-derived
SOM constituents, roots and shoots represent a major source
for OM during the buildup of cast-rich soils. However, while
it is recognized that roots and shoots have contrasted fates is
soil, little is known on the ability of earthworms to ingest and
transform roots (Curry and Schmidt, 2007; Zangerlé et al., 2011;
Cameron et al., 2014), and its impact on root decomposition in
casts. The physico-chemical processing of plant residues during
the gut passage, coupled to the intense microbial activity and the
formation of organo-mineral associations in casts compared with
soil, questions the different decomposition processes depicted
for roots and shoots in soils. This could significantly influence
the carbon cycling and storage in soils, considering that casts
might account for at least half of the surface soil layer in natural
conditions (Ponomareva, 1950; Lee, 1985).

The present study aimed at highlighting the transfer of plant-
derived C and the stage of decomposition of incorporated
residues into casts over time. We hypothesized that the chemical
characteristics of shoot residues will drive their rapid degradation
compared to root residues, and that associations between organic
and mineral particles within cast will develop over time. Cast
samples, produced in the presence of 13C-labeled shoots and
roots, were collected 8 and 54 weeks after the beginning of a
mesocosm experiment. The change in the amount and isotopic
composition of OM from initial plant residues to particulate
organic matter (POM) and mineral-associated OM (MOM) was
determined by isotope ratio mass spectrometry (EA-IRMS).
Particulate OM and MOM were studied separately to differentiate
the free from partly occluded plant residues, respectively. The
alteration of the chemical composition of the POM and MOM
derived from the casts with time was determined using solid-state
13C cross polarization magic angle spinning nuclear magnetic
resonance spectroscopy (13C CPMAS NMR). So as to follow the
decomposition processes in intact cast samples at the microscale,
we combined elemental and isotopic information obtained with
nano-scale secondary ion mass spectrometry (NanoSIMS) with
high-resolution information on the arrangement of organic
and mineral constituents obtained with transmission electron
microscopy (TEM). In previous works, we focused on the method
development and technical requirements of the micro-scale
analyses (Vidal et al., 2016b) and demonstrated soil alteration due

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Earthworm Cast Formation and Development

TABLE 1 | Characteristics of initial soil, as well as Cast-Control, Cast-Root and Cast-Shoot samples over time, before fractionation in POM and MOM (numbers in
parentheses indicate the standard deviation, n = 3).

Initial soil

Cast-control

Cast-shoot

Cast-root

8 weeks

54 weeks

8 weeks

54 weeks

8 weeks

54 weeks

OC

N content

C/N
δ13C

Litter-derived C

mg.g−1
mg.g−1

‰

%

12.1

1.30

9

–

19.0 (2.2)

1.97 (0.2)

10 (0.5)

16.8 (2.5)

1.73 (0.2)

10 (0.3)

−28.1

−25.3 (1.3)

−28.6 (0.1)

–

–

34.9 (1.2)

3.33 (0.1)

11 (0.1)

938 (3.0)

58.1 (0.2)

20.6 (1.6)

2.03 (0.21)

10 (0.3)

168 (1.9)

11.8 (0.1)

55.0 (0.8)

3.77 (0.1)

15 (0.3)

673 (25)

51.8 (1.8)

22.2 (0.6)

2.10 (0.1)

11 (0.2)

127 (8.2)

11.5 (0.6)

to earthworm activity using bulk measurements and molecular
analyses (Vidal et al., 2016a, 2017). We now use the developed
methods and bulk analyses in addition to fractionation and
NMR analyses to demonstrate the fine scale mechanisms of litter
degradation through time. This approach reflects the increasing
cognition in environmental science for the need to combine
imaging with classical bulk measurements to gain a deeper
understanding of biogeochemical processes (Mueller et al., 2013;
Baveye et al., 2018).

MATERIALS AND METHODS

Experimental Setup
Three mesocosms were filled with ∼75 L of a loamy-sand soil
(clay, 19%; silt, 25%; sand, 56%) collected on permanent
grassland in North of France (Oise, France). The soil
characteristics are described in Table 1 and available in
Vidal et al. (2017) and Vidal (2016). Mesocosms were placed
in a greenhouse where soil humidity and temperature were
maintained at 23% and 13◦C, respectively. Six Lumbricus
terrestris earthworms were deposited onto each mesocosm.

Plants of

Italian Ryegrass

(Lolium multiflorum) were
artificially labeled in 13C at the PHYTOTEC platform of the
Alternative Energies and Atomic Energy Commission (CEA)
in Cadarache (France). Plants were grown under a controlled
and constant 13CO2 enriched atmosphere (2.6% 13CO2). The
mean δ13C values were 1,632 ‰ (±16) and 1,324 ‰ (±42) for
shoots and roots, respectively. Shoots and roots were separated,
dried and subsequently homogenized separately during 40 s with
a laboratory blender (Waring Commercial) in order to obtain
small fragments with millimeter size. We deposited 250 g of
shoots and roots (∼0.9 g OC.kg soil−1) on the soil surface of
the two mesocosms, respectively. Although the design does not
reflect the real condition of in situ root systems, both roots and
shoots were voluntarily deposited onto the soil surface, under
the same conditions, in order to consider the sole effect of the
chemical composition of litter (without any initial physical
contact of the roots with the soil particles) on its incorporation
and decomposition in earthworm casts. No litter was applied
on the third mesocosm, which served as control. After 8 and 54
weeks of experiment, around 10 earthworm cast fragments were
randomly collected on the soil surface of each mesocosm using a
spatula and combined to form a composite sample of around 50
grams for each time step. The present work aimed at improving

fundamental processes by combining
the understanding of
bulk chemical and imaging techniques, which together provide
a more complete view on small scale soil functioning. As we
combined all used techniques on one sample per treatment each,
no replication could be achieved due to time concern. The time
points of sampling were selected according to the contrasted
isotopic and molecular composition measured on bulk samples
in previous works (Vidal et al., 2016a, 2017). For example, the
shift from more than 50–12% of litter-derived carbon in casts
from 8 to 54 weeks (Table 1) showed a clear differentiation
into a first and second decomposition phase which led to the
two chosen sampling dates. Casts were distinguished from
the bulk soil due to their round shape and smooth texture
(Velasquez et al., 2007). After 8 weeks of experiment most
casts were fresh when collected, while after 54 weeks, casts
started to age and dry. A sub-sample of 5 grams, made of
around three cast fragments, was directly processed for TEM
and NanoSIMS analyses after sampling. The rest of the sample
was dried, ground and subsequently fractionated into POM
and MOM physical soil fractions. The obtained SOM fractions
were analyzed for OC, N and δ13C. The chemical composition
of the SOM fractions was analyzed using 13C CPMAS NMR
spectroscopy. For the cast collected in the mesocosms containing
roots, shoots and no litter, we will refer to Cast-Root, Cast-Shoot
and Cast-Control, respectively.

Separation of POM and MOM Fractions
In order to differentiate between plant residue dominated and
mineral-associated OM, dry and ground cast samples were
fractionated to separate POM and MOM. Briefly, 4 g of cast
sample were saturated with 50 mL sodium polytungstate solution
with a density of 1.8 (TC Tungsten compounds, Grub am
Forst, Germany). After settling overnight, the floating free POM
was collected using a vacuum pump, washed to remove excess
Sodium Polytungstate (conductivity < 3 µS) using pressure filtration (22 µm filter) and freeze-dried. The mineral fraction containing the MOM was washed to remove salts (conductivity < 50 µS), centrifuged (3,000 g, 30 min) and freeze-dried. The density fractionation resulted in a mean recovery of 94 ± 2.9% of the initial sample mass. In the present study, the POM fraction is considered as the particulate plant residues, which are extractable by floatation in a dense liquid, while the MOM fraction comprises the organo-mineral associations. Frontiers in Environmental Science | www.frontiersin.org 3 April 2019 | Volume 7 | Article 55 Vidal et al. Earthworm Cast Formation and Development Bulk Elemental and Isotopic Analyses All POM and MOM fractions were analyzed (Helmholtz Zentrum, Munich, Germany) for organic carbon, nitrogen and δ13C using IRMS (delta V Advantage, Thermo Fisher, Dreieich, Germany) coupled to an Elemental Analyzer (Euro EA, Eurovector, Milan, Italy). An acetanilide standard, calibrated against several suitable international isotope standards (IAEA; Vienna), was used for calibrating. Prior to organic carbon and δ13C analyses, MOM fraction samples were decarbonated adding 20 µl of HCl 2N to 1–5 mg samples for 10 h and drying overnight at 60◦C. Additional samples were prepared (10–40 mg) for nitrogen analyses. The labeled litter-derived carbon in POM and MOM fractions of earthworm casts was expressed according to equation 1: Ultrastructural Analyses by TEM The materials and methods used to prepare undisturbed samples for TEM and NanoSIMS analyses were identical to those described in Vidal et al. (2016b). In brief, osmium tetroxide was used to chemically fix cast samples (2 g for each sample) and initial litter parts. To avoid sample disruption, cast structures were physically preserved with agar (Watteau et al., 2006). Cast samples were cut into cubes of few mm3 (around 10 for each sample), dehydrated in graded acetone series, and embedded in epoxy resin (Epon 812). Ultrathin sections (80–100 nm) were sliced using a Leica Ultracut S ultramicrotome, stained with uranyl acetate and lead citrate and analyzed with a JEOL EMXII transmission electron microscope operating at 80 kV (LSE, Nancy, France). Litter-derived C(%) = [(δ13Csample − δ13Ccontrol)/(δ13Clitter − δ13Ccontrol)]×100 (1) Where δ13Csample is the δ13C value of the POM or MOM fraction samples isolated from casts incubated with labeled roots or shoots, δ13Ccontrol is the δ13C value of the POM or MOM fraction samples isolated from control casts incubated without litter, δ13Clitter is the δ13C values of the labeled roots or shoots. The percentage OC of the MOM or the POM fraction compared to the total OC contained in both POM and MOM isolated fractions (% OC bulk) was also calculated. Nuclear Magnetic Resonance Spectroscopy The 13C-CPMAS-NMR analyses were performed (Chair of Soil Science, TUM, Freising, Germany) on initial litter, as well as POM and MOM fractions of cast samples, using a Bruker AvanceIII 200 spectrometer (Bruker BioSpin GmbH, Karlsruhe, Germany). The NMR was operated at a 13C-resonance frequency of 50 MHz, with a spinning speed of 6.8 kHz and according to the carbon content, a recycle delay time of 2 or 0.4 s, for initial litter and other samples, respectively. From around 1,000 to up to 200,000 scans were accumulated for initial litter and other samples, respectively. The spectra were processed with a line broadening from 0 to 50 Hz, followed by phase adjustment and base line correction. The chemical shift regions were obtained by dividing the NMR spectra as followed: 0–45 ppm (alkyl-C), 45– 110 ppm (O-N-alkyl-C), 110–160 ppm (aromatic-C) and 160– 220 ppm (carboxyl-C) (Kögel-Knabner et al., 1992). It has to be noted that the 160–220 ppm chemical region also include carbonyl-C, but that the carboxyl-C are by far dominant. The ratio between alkyl-C and O-N-alkyl-C was used as an indicator of organic matter degradation. A higher alkyl-C/O-N-alkyl-C ratio generally reflects a higher OM degradation, as alkyl carbon chains tend to be less degradable compared with carbohydrates and proteins (source of O-N-alkyl-C) (Baldock et al., 1997). Although these values cannot be considered as absolute ones (due to overlapping signals and potential differences in relaxation times between the different types of C), they can be used for comparison purposes. Nano-Scale Isotope Analyses by NanoSIMS Ultrathin twin sections of 100–200 nm were sliced from the same blocks prepared for TEM analyses, allowing the comparison between NanoSIMS and TEM images. Samples were gold coated and images were acquired using the NanoSIMS 50 (Cameca, France) located at Museum national d’Histoire naturelle in Paris, France. The sample surface was sputtered by a 1.5 pA Cs+ beam to obtain 24 ×24 µm images (256 × 256 pixels) of 12C−, 12C14N−, 13C14N−, and 28Si− secondary ions. The images were processed using the L’IMAGE R(cid:13) software (L. Nittler, Carnegie Institution, USA). Secondary ion images of 12C14N− and 28Si− were used to distinguish organic structures from mineral particles. The 13C isotopic images, named as δ13C in the following, were generated using the 13C14N−/12C14N− ratio relative to the PDB standard. The heterogeneity of the δ13C values observed on similar organic structures on NanoSIMS images can either reflect a methodological bias (variable contribution of C from epoxy resin) or a natural process (variable extent of C recycling), both leading to variable degree of isotopic dilution. Given these approximations, δ13C values obtained in the present study were considered as indicators of the occurrence of labeled OC, and not taken as representative of accurate isotopic enrichment values. Statistical Analyses A principal component analysis (PCA) was performed with the R statistical software (package “FactoMinerR”) on the 12 fraction samples (POM and MOM) using the 4 NMR chemical shift regions as variables. Root and shoot litter samples were implemented as illustrated samples in the PCA. The variables were normally distributed, as tested by the Shapiro-Wilk test. RESULTS C and N Elemental and Isotopic Composition, and Distribution in POM and MOM Fractions At 8 weeks, the mass proportion of POM fraction was higher (73 vs. 44 mg.g−1, in Cast-Root compared to Cast-Shoot respectively) (Table 2). Particulate OM and MOM (Cast-Shoot Frontiers in Environmental Science | www.frontiersin.org 4 April 2019 | Volume 7 | Article 55 Vidal et al. Earthworm Cast Formation and Development TABLE 2 | Organic carbon, nitrogen, δ13C and chemical characteristics of particulate organic matter (POM) and mineral associated organic matter (MOM) fractions isolated from earthworm casts. Cast-control Cast-shoot Cast-root 8 weeks 54 weeks 8 weeks 54 weeks 8 weeks 54 weeks POM Mass proportion of fraction Litter-derived C Alkyl-C/O-N-alkyl-C MOM Mass proportion of fraction OC Total OC %C of bulk N content C/N δ13C OC Total OC %C of bulk N content C/N δ13C Litter-derived C Alkyl-C/O-N-alkyl-C mg.g−1 mg.g−1 mg mg.g−1 ‰ % mg.g−1 mg.g−1 mg mg.g−1 ‰ % 14 95 5 9 7 – 15 −28 0.43 952 15 57 91 1 12 −28 – 0.42 11 105 11 5 7 15 −28 – 0.40 900 11 39 89 1 13 −28 – 0.50 44 306 54 42 21 14 1,177 73 0.25 917 21 76 58 2 12 754 47 0.38 15 171 10 15 12 14 87 7 15 58 85 1 11 0.46 955 198 14 0.49 73 317 93 43 14 23 935 71 0.11 879 35 124 57 2 15 846 65 0.17 139 8 5 7 9 16 185 16 0.35 886 17 60 93 1 13 190 16 0.38 The total organic carbon (Total OC) values were calculated using POM or MOM fraction masses. The % OC of bulk corresponds to the percentage of OC in POM or MOM fractions compared to the sum of the OC in POM and MOM fractions. and Cast-Root) fractions contained around 40 and 60% of the total OC isolated, respectively (Table 2). Cast-Control POM and MOM fractions contained around 10 and 90% of the OC of bulk casts, respectively. In both Cast-Shoot and Cast-Root fraction samples, at least 50% of OC was litter-derived, with a higher percentage in Cast-Root MOM fraction (65%) compared to Cast- Shoot MOM fraction (47%). At 54 weeks, the mass proportion of MOM fraction slightly increased compared with 8 weeks and more than 85% of the total OC isolated was contained in the MOM fractions of both Cast-Shoot and Cast-Root. In both POM and MOM fractions, the litter-derived carbon dropped to 15%, with a minimum of 7% in the Cast-Shoot POM fraction. The C/N ratio decreased, compared with 8 weeks, of 30% and 13% in the Cast-Root POM and MOM fractions, respectively. Chemical Characterization of Cast POM and MOM Fractions Initial roots and shoots presented similar NMR spectra clearly dominated by carbohydrates (O-N-alkyl-C) (Figure 1). In initial roots, the relative abundance of alkyl-C was lower, while the relative abundance of aromatic-C was slightly higher than shoots (Figures 1B,C and Table S1). At 8 weeks, spectra for the POM fraction of Cast-Root and Cast-Shoot presented similar characteristics as the initial litter (Figures 1B,C), while those of MOM fraction spectra were broader (Figures 1E,F). At 54 weeks, a general broadening of spectra was observed for both POM and MOM fractions (Figure 1). A PCA was carried out to highlight the chemical characteristics of organic matter in POM and MOM isolated from earthworm casts after 8 and 54 weeks of experiment (Figure 2). The two factors (F1, F2) generated by the PCA explained 97% of the variance. F1 clearly separated Cast-Control samples and 54-week samples from Cast-Shoot and Cast-Root samples collected at 8 weeks (Figure 2B). Cast- Control samples were represented by a high relative abundance of aromatic-C and carboxyl-C, while 8 week Cast-Shoot and Cast-Root samples contained higher relative abundance of O- N-alkyl-C. At 8 weeks, the Cast-Root MOM fraction remained relatively close to the Cast-Root POM fraction and the initial root chemical characteristics. In contrast, Cast-Shoot MOM fraction at 8 weeks presented similar characteristics to the samples collected after 54 weeks. After 54 weeks, the OM in Cast- Shoot and Cast-Root samples tended to evolve toward Cast- Control chemical characteristics (Figure 2). Compared with 8 weeks, the relative abundance of O-N-alkyl-C decreased, while that of alkyl-C and aromatic-C increased (Figure 2B), resulting in a higher alkyl-C/O-N-alkyl-C ratio for both Cast-Root and Cast-Shoot (Table 2). the microscale spatial assembly of Cast-Root and Cast-Shoot at the Microscale Structures of intact cast samples were analyzed with TEM to obtain detailed information and NanoSIMS in order the biogeochemical of interfaces. At 8 weeks, plant incorporated in earthworm casts presented similar structures compared to the initial shoots and roots (Figures S1, S2), although they showed different degradation stages (Figure 3). For example, parenchyma cells were partially degraded in Cast-Shoot residues Frontiers in Environmental Science | www.frontiersin.org 5 April 2019 | Volume 7 | Article 55 Vidal et al. Earthworm Cast Formation and Development FIGURE 1 | 13C Cross polarization magic angle nuclear magnetic resonance (NMR) spectra of POM and MOM fractions isolated from earthworm casts. Cast-Control (A,D), initial shoot and root spectra (B,C, respectively), the POM fraction spectra for Cast-Shoot (B) and Cast-Root (C) after 8 and 54 weeks of experiment, as well as their corresponding MOM fraction spectra (E,F, respectively). (Figure 3A) compared with initial shoot tissues (Figure S1A), tissues were well preserved (Figure 3A). while woody images highlighted some Both Cast-Shoot and Cast-Root preserved plant tissues (Figures 3A,G), long and thin laces identified as parenchyma cell wall residues (Figures 3B,F,H), microaggregates and microorganisms (Figures 3C–E,H). Intact or barely degraded plant structures (Figures 3G,H). Various were microorganisms, mainly fungi and bacteria, were depicted within Cast-Shoot: fungi attacking cell walls of woody tissues (Figure 3D), bacteria colonizing parenchyma cells (Figure 3E), (Figure 3C). or microorganisms within microaggregates (Figures 3B,I, 4A,B) in Cast-Root prevalent A few microorganisms were also observed in Cast-Root (Figures 3G,H). Many features identified in TEM images were recognized in NanoSIMS images. For Cast-Shoot, Figures 4A,B were comparable to Figures 3C,B, respectively, with the clear occurrence of a labeled fungus (Figure 4A), amorphous OM and plant cell wall (Figure 4B). On these images, 28Si− maps reflect an important proportion of small size mineral particles (i.e., mainly clay size < 2 µm) on images and 12C14N− maps showed organic structures among these mineral particles. Cast-Shoot images showed the presence of partially degraded plant structures derived from the labeled plants and labeled Frontiers in Environmental Science | www.frontiersin.org 6 April 2019 | Volume 7 | Article 55 Vidal et al. Earthworm Cast Formation and Development FIGURE 2 | Chemical characterization of earthworm casts. PCA carried out with the 4 variables of the 6 cast samples, fractionated into POM and MOM. The variables correspond to the 4 chemical shift regions integrated from 13C CPMAS NMR. (A) distribution of variables on the correlation circle and (B) distribution of the cast samples, with root and shoot litter as illustrative samples. Weeks after the beginning of the experiment are indicated using numbers next to samples. microorganisms involved in plant decomposition (Figures 4A,B, respectively). Degraded plant structures and microorganisms were both integrated into organo-mineral aggregates. For Cast- Root, Figures 4C,D were comparable to Figures 3G,H. Labeled plant structures observed on NanoSIMS Cast-Root images presented a lower degree of degradation and reduced associations with mineral particles (Figures 4C,D), compared with Cast- Shoot images. (Figures 5A–C) and Cast-Root At 54 weeks, microaggregates (from 20 to 30 µm) with complex organo-mineral composition, were frequently observed (Figure 5G) on Cast-Shoot images. Highly degraded plant tissues, cell walls or amorphous organic residues were prevalent in Cast-Shoot. Residues of woody tissues were still recognizable and colonized by bacteria (Figure 5D). On Cast-Root images, some cell wall residues surrounded by mineral particles were identified (Figure 5E) and some cell intersections were still recognizable (Figure 5F). Microorganisms were prevalent on both Cast-Shoot and Cast-Root images. Bacteria were either intact, present under residual form (dead microorganisms leaving cell wall residues) (Figures 5B,D; Figures 5E,H) or spores (presenting dark core and coat) (Figures 5B,H). On NanoSIMS images, labeled areas were scarce (Figure 6) compared with samples observed after 8 weeks of experiment (Figure 4). Three types of labeled structures are identified on Cast-Shoot and Cast-Root