SYSTEMATIC REVIEW

published: 07 June 2019

doi: 10.3389/fenvs.2019.00081

Earthworms Building Up Soil

Microbiota, a Review
Regina M. Medina-Sauza 1† , Marycruz Álvarez-Jiménez 2,3† , Alix Delhal 4 ,
Frédérique Reverchon 3*, Manuel Blouin 4 , José A. Guerrero-Analco 3 , Carlos R. Cerdán 2 ,
Roger Guevara 5 , Luc Villain 6 and Isabelle Barois 1*
1
Red de Ecología Funcional, Instituto de Ecología A.C., Xalapa, Mexico, 2 Facultad de Ciencias Agrícolas, Universidad
Veracruzana, Xalapa, Mexico, 3 Red de Estudios Moleculares Avanzados, Instituto de Ecología A.C., Xalapa, Mexico,
4
Agroécologie, AgroSup Dijon, CNRS, INRA, University Bourgogne Franche-Comté, Dijon, France, 5 Red de Biología
Evolutiva, Instituto de Ecología A.C., Xalapa, Mexico, 6 UMR- Interactions Plantes Microorganismes Environnement, Institut
de Recherche pour le Développement (IRD) and Centre de Coopération Internationale en Recherche Agronomique pour le
Développement (CIRAD), Montpellier, France

The positive effect of earthworms on soil processes and plant growth has been
Edited by: extensively documented. The capacity of earthworms to decompose organic matter
Juan José Jiménez, has been attributed to the microbial communities that inhabit their digestive track
Consejo Superior de Investigaciones
or the structures they build, which in turn contribute to make up the drilosphere, a
Científicas, Spain
hotspot for microbial activity. However, how earthworms modify the structure of soil
Reviewed by:
Alexei V. Tiunov, microbial communities and how these changes affect soil microbial processes is still
Severtsov Institute of Ecology and unclear. Do earthworms reduce microbial abundance and activity because they feed
Evolution (RAS), Russia
Claire Le Bayon, on microorganisms or do they select and stimulate specific microbial groups? We
Université de Neuchâtel, Switzerland hypothesise that “the effect of earthworms on nutrient cycling and plant growth is not
*Correspondence: only a direct effect but is mainly mediated indirectly, via modifications of the microbial
Frédérique Reverchon
community.” The objective of this review is to synthesize the existing literature concerning
[email protected]
Isabelle Barois the influence of earthworms on the structure and function of soil microbial communities,
[email protected] as well as to understand how earthworm-induced changes in the soil microbiota would in
† These authors have contributed turn impact soil processes, particularly those occurring in the rhizosphere and involved
equally to this work as co-first authors in plant growth and health. Recent reports have shown that specific bacterial groups
consistently increase in soils where earthworms are present, regardless of the earthworm
Specialty section:
This article was submitted to functional group. The extent of this increase seems to be dependent upon the type of
Soil Processes, substrate under study. Our synthesis also reveals that endogeic and anecic earthworms
a section of the journal
Frontiers in Environmental Science
regularly induce an increase in soil nutrients, whilst this positive effect is not as evident
Received: 29 January 2019
in the presence of epigeic earthworms. The effect of earthworms on nutrient cycling
Accepted: 21 May 2019 has been further investigated with microbial functional genes, although existing reports
Published: 07 June 2019
largely focus on nitrogen cycling. Earthworms seem to enhance denitrification, most likely
Citation:
through the increase in organic compounds due to organic matter decomposition. By
Medina-Sauza RM,
Álvarez-Jiménez M, Delhal A, enhancing soil nutrient availability, earthworms indirectly promote plant growth, which
Reverchon F, Blouin M, has also been attributed to the induction of signal molecules. However, no experiment
Guerrero-Analco JA, Cerdán CR,
Guevara R, Villain L and Barois I
to date has been able to prove a direct causal relationship between specific signal
(2019) Earthworms Building Up Soil molecules, earthworms and plant growth promotion. Finally, we propose a framework
Microbiota, a Review.
for earthworm-microbiota interactions and recommend further research.
Front. Environ. Sci. 7:81.
doi: 10.3389/fenvs.2019.00081 Keywords: soil nutrient hotspots, interactions, soil biotransformation, signal molecules, drilosphere, microbiome

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Medina-Sauza et al. Microbiata and Earthworms

INTRODUCTION and in a limited soil space (“hot spot”), at the microscale

of a biopore or aggregate (Kuzyakov and Blagodatskaya,
Earthworms are considered as ecosystem engineers that play 2015) which reverberates on a larger scale, at the drilosphere
an important role in shaping soil structure and cycling nutrients and soil levels (Brown et al., 2000; Hoang et al., 2016;
(Blouin et al., 2013). Earthworms promote litter decomposition, Lipiec et al., 2016).
nitrogen (N) mineralisation and water infiltration, as a result of Earthworms have a direct and important effect on the soil
their feeding and burrowing habits (Baker, 2007), and therefore microbiota through their nutrition. This effect may depend on
deeply affect soil properties (Hättenschwiler and Gasser, 2005). their food preference, selection, food ingestion rate, digestion
They also play a crucial role in the provision of soil ecosystem and assimilation, as mentioned by Curry and Schmidt (2007).
services (Lavelle et al., 2016). The soil volume directly influenced Earthworms can digest microorganisms (Brown, 1995; Chapuis-
by earthworms, known as the drilosphere (Bouché, 1977; Lavelle, Lardy et al., 2010) thereby decreasing microbial biomass,
2002), is an important functional region of the soil, made by the especially that of fungi (Shan et al., 2013). They may also select
earthworm community and the structures it creates: middens, or stimulate soil microbes (Khomyakov et al., 2007; Nechitaylo
burrows, tunnels, and casts. Earthworms are thus builders of et al., 2010) which help them digest the soil organic matter,
habitats for other organisms, which establishes them as physical since the earthworm gut often lacks the sufficient enzymes to
or allogenic engineers (Jones et al., 1994; Lavelle et al., 1997, do so (Lattaud et al., 1997, 1998; Fujii et al., 2012). This process
2016). Besides, these building activities constitute an input of may enrich the soil in certain bacterial taxa, for example in
organic matter to the soil and a pathway for the stabilization bacteria able to decompose the organic matter that earthworms
of soil organic carbon (Corg) through the formation of organo- feed on or in denitrifying bacteria able to survive in the reduced
mineral aggregates (Deeb et al., 2017). This enrichment in oxygen conditions of the earthworm gut (Drake and Horn, 2007;
organic matter mainly results from earthworm food choice Hong et al., 2011).
(Curry and Schmidt, 2007), its digestion and excretion of The physiology, morphology and behaviour of earthworms is
intestinal or cutaneous mucus that can be cementing (Shipitalo essential to understand their effect on soil functions (Figure 1,
and Le Bayon, 2004) or used as an energy source (Lavelle arrow 1). However, there is increasing evidence that the
et al., 1995). Therefore, in addition to shaping soil structure, effect of earthworms on soil functions may be mediated
earthworms also have an important impact on soil organic through soil microbial communities (Figure 1, arrow 2). It
matter dynamics and microorganisms in their gut, casts and is yet not clear how the different ecological groups may
drilosphere (Andriuzzi et al., 2016) and are also identified promote or select soil microorganisms and there are many
as biochemical (Lavelle et al., 2016) or autogenic ecosystem contradictory results concerning the effect of earthworms on
engineers (Lawton and Jones, 1995). soil microbial communities (Byzov et al., 2015). However, the
Earthworms are divided into three main functional groups drilosphere is generally acknowledged as being a soil hotspot
or ecological categories, which determines how they influence with a positive effect on ecosystem functions such as nutrient
the soil compartment and its microbial communities (Thakuria cycling and plant growth (Brown et al., 1999; Scheu, 2003;
et al., 2010): (1) epigeic earthworms live on the soil surface Van Groenigen et al., 2014).
and feed from the litter; (2) endogeic earthworms live in Considering that the involvement of microorganisms in
the soil and produce horizontal tunnels, while feeding on these functions is fundamental, it is therefore necessary to
mineral soil and partially decomposed material, being then consider microbial communities and how they are influenced
geophagous; (3) anecic earthworms produce permanent vertical by earthworms in order to understand and predict the effect of
burrows and feed on the litter that they drag into their earthworms on ecosystem functions. It is our hypothesis that
burrows to be pre-decomposed by microorganisms, while “the effect of earthworms on nutrient cycling and plant growth
depositing their casts at the burrow entrance (Bouché, 1977; is not only a direct effect but is mainly mediated indirectly, via
Lavelle, 1981; Lee, 1985). modifications of the microbial community” (Figure 1). Thus,
Earthworms are considered as key ecological mediators that the objectives of this review are two. The first is to determine
have the capacity to affect soil functions and microbial activities whether some patterns can be drawn from the existing literature
(Binet et al., 1998; Lavelle et al., 2016), by producing an regarding the effect of the different earthworm functional groups
energy-rich mucus that activates microorganisms through a (epigeic, endogeic, and anecic) on the abundance, structure
priming effect (Jenkinson, 1966) and signal molecules that have and diversity of soil microorganisms (bacteria, archae, and
hormone-like effects and influence plant gene expression (Puga- fungi) at the different sites (earthworm gut, casts, burrows, bulk
Freitas and Blouin, 2015). The mutualistic interaction existing soil, rhizophere, others). The analytical methods used are also
between earthworms and the soil microbiota has been named considered [Gram+/–, fingerprinting, phospholipid fatty acids
the “Sleeping Beauty Paradox” (Lavelle et al., 1995; Brown (PLFA), sequencing]. The second is to establish the impact of
et al., 2000), where dormant soil microorganisms, awaiting earthworms on microbial processes involved in nutrient cycling,
suitable environmental conditions are activated by the kiss of on the production of signal molecules and as a consequence,
the earthworm made of easily assimilable glycoproteins present on plant growth promotion. The selected literature was mainly
in the drilosphere in the form of intestinal or cutaneous chosen from studies that deal with earthworms and microbial
mucus as already mentioned. This triggers the acceleration of (microorganisms, microbiome) interactions and nutrient cycling
microbial processes for a short period of time (“hot moment”) (Nitrogen, Phosphorus) between 1980 and 2018; however, for

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Medina-Sauza et al. Microbiata and Earthworms

FIGURE 1 | Hypothesis: effect of earthworms on nutrient cycling and plant growth is not only a direct effect but it is mainly mediated indirectly by microorganisms. The
impact of earthworms is direct (1), or indirect, through the stimulation of microorganisms (2). By modifying microbial communities (2a), earthworms impact the way
bacteria are influencing ecosystem functions (2b). This figure aims at illustrating the fact that indirect effects are as important as direct ones.

specific topics we used the Web of Science (WOS) with precise The Epigeics
keywords (see Figures 4–6 legend). The consequences of the presence of epigeic earthworms on
Finally, we propose a framework for earthworm—soil soil microbial abundance are variable (Figure 2). The literature
microbiome interactions and recommend further research to be shows that they can provoke either a decrease or an increase
directed towards elucidating the microbial processes occurring in in microbial biomass. Less frequently, reports show that the
the drilosphere. number of microorganisms remains unaffected by their action.
Most studies found in the literature are performed under artificial
laboratory conditions and use epigeic earthworm species Eisenia
andrei and E. fetida, grown in different feedstocks (dungs,
THE IMPACT OF EARTHWORMS ON THE agriculture by-products and mixtures of organic matter and soil).
ABUNDANCE AND ACTIVITY OF SOIL These studies report that these species induce an increase of the
MICROORGANISMS microbial biomass in the transformed substrate which is made
up mainly of casts, although the magnitude of this effect varies
The effect of earthworms on soil microbial communities is through different time scales. The activity and the numbers of
critical as they are one of the most important fauna group in microorganisms have a peak at the beginning of the digestion
soils, in terms of number and biomass (Blouin et al., 2013). which lasts at the most a few hours in the gut (Brown et al.,
Besides, earthworms can have a very high rate of substrate or soil 2000) and a bit longer in the fresh casts, these sites being “hot
ingestion. Epigeic earthworms can ingest 3–50 mg (dry matter) spots and moments.” After some months (3–4) there is a decrease
of dung or any other kind of litter per gram of earthworm in microbial activity and numbers in the casts or vermicompost
per day and the geophagous worms 200–6,700 mg (dry matter) and then there is a stabilization of both (Yakushev et al., 2009)
of soil per gram of earthworm per day (Curry and Schmidt, Koubová et al. (2015) indicated that microbial biomass measured
2007). In this section, we will synthesize the available information by PLFA was 2-fold greater in the earthworm gut than in the
regarding how earthworms influence the abundance or activity of non-ingested substrate and that biomass was also higher in casts
soil microorganisms, depending on their functional groups. than in the surrounding substrate, although this increase was

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Medina-Sauza et al. Microbiata and Earthworms

substrate they live on (Figure 2). Through measurements of

total PLFA, Gómez-Brandón et al. (2012) and Aira et al. (2002,
2006) found less microbial biomass in E. andrei vermicompost
from grape bagasse and in E. fetida and Eudrilus eugeniae
vermicompost from pig manure than in substrates without
earthworms, although this effect seemed to depend upon the
earthworm density (Aira et al., 2002). Overall, literature findings
show that no clear effect of epigeic earthworms can be detected
in microbial biomass, nor on the growth rate of microbial
populations. Discrepancies may be attributed to the different
species or substrates under study, as well as the different
analytical methods implemented (Yakushev et al., 2009; Gómez-
Brandón et al., 2012; Koubová et al., 2015).

The Endogeics
Although endogeic earthworms have been less studied, results
show contrasting effects on microbial abundance, compared
with the epigeics (Figure 2). For endogeics, the majority of the
works that observe their impact on the microbiome are made
comparing soils with and without earthworms. There are less
studies on the “hotspots” (gut and fresh casts). In mesocosms
with soil from arable fields (3.8% of organic matter) and potato
FIGURE 2 | The effect of earthworms on microbial abundance depending on residues on the surface, the endogeic Aporrectodea caliginosa
their functional group. Thirty-one studies from 1986 to 2018 were considered.
did not impact soil microbial biomass which was obtained from
All the studies were carried out in controlled conditions with the exception of
one (Stromberger et al., 2012). Five articles were from tropical regions and all
the biovolume of bacterial numbers measured by confocal laser
the others from temperate regions. The microbial abundance was measured in scanning microscopy (Postma-Blaauw et al., 2006). In contrast,
five gut contents, 11 casts, 12 vermicomposts, 11 soils, four burrows, and Chang et al. (2016) found an increase in the bacterial biomass
one midden from the different studies. Some studies presented results from of a forest soil mixed with litter, measured through PLFA, in the
more than one earthworm species, ecological category and site measurement.
presence of Octolasion lacteum. Similar findings were reported
for a pot experiment by Braga et al. (2016), who detected
increases in the bacterial 16S rRNA gene abundance in soils with
less important than in the gut. Yakushev et al. (2009) showed the tropical earthworm Pontoscolex corethrurus, compared with
an increase in microbial biomass of 2.7 times in the casts of E. soils without earthworms. In another study, microbial activity
fetida and evidenced that microbial growth in a period of 9 h was increased 6-fold in the gut of P. corethrurus and 1.6-fold in
124 times higher in vermicompost than in compost. In a mixture its casts, compared with the surrounding vertisol; more CFU
of pig manure and soil, the microbial biomass was 1.3 times were also observed in the casts by plate counting (Barois and
higher in the presence of earthworms (Aira et al., 2007). Toyota Lavelle, 1986; Barois, 1987). Contrastingly, soil microbial biomass
and Kimura (2000) found that bacterial biomass, evaluated by has also been shown to decrease with increasing endogeic
counting colony forming units (CFU), increased from 3.2 × 104 earthworm biomass (Scheu et al., 2002). Chapuis-Lardy et al.
CFU g−1 in composted farmyard manure to 1.3 × 107 CFU g−1 (2010) found that fungal and bacterial biomass significantly
in vermicompost with E. fetida. decreased (∼2-fold) in casts from P. corethrurus when compared
Although other studies show that epigeic earthworms do not to the parent soil, although the fungal-to-bacterial ratio was
have any impact on microbial abundance in the soil, this effect not modified by the earthworm casting activity. The effect
seems to be dependent upon the kind of feedstock assessed. On of endogeic earthworms on soil microbial biomass may also
leaf compost from alder, willow and birch with a C/N = 19.2, depend on the soil depth: it decreases with soil depth
the presence of E. fetida/E. andrei induced a great increase in (Sheehan et al., 2008).
the number of microorganisms whereas vermicompost made
from cattle manure (C/N = 15.4) did not present any differences The Anecics
with its respective control compost without earthworms (C/N The anecic worms feed on the surface and build permanent
= 16.5) (Yakushev et al., 2009). Sheehan et al. (2008) using tunnels that allow them to mix the different soil horizons. They
mesocosms showed that, in addition to the influence of the food form middens on the soil surface at the entrance of their burrows
supply, the effect of epigeic earthworms on the abundance of that contain visible pieces of organic matter and casts, these as
microorganisms also depends on the soil layer under study and well are excreted into the soil profile.
reported a larger increase in microbial biomass in the upper The impact of anecics on the soil may reach the whole soil
layers (0–7 cm) than in the deeper ones (7–14 cm). profile and this is not the case with the other functional groups.
On the other hand, several studies demonstrated that epigeic Most reports show that anecic earthworms have a positive or
worms can decrease microbial biomass in their casts or in the neutral impact on microbial abundance in the soil (Figure 2),

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Medina-Sauza et al. Microbiata and Earthworms

using soil and litter from lime (Tilia cordia), the anecic L. terrestris
induced more microbial biomass in soil retrieved from its burrow
walls, compared with the surrounding soil (Tiunov et al., 2001).
However, these results seem to depend on the litter source and
the soil Corg and N content, as the same experiment repeated
with beech litter soil (which has three times more Corg and N
than the lime litter soil) showed a smaller increase in burrow
microbial biomass. Stromberger et al. (2012) also evidenced
a larger abundance of microorganisms measured by PLFA in
the burrow walls of L. terrestris when compared with the bulk
soil (89.4 and 56.7 nmol g−1 soil respectively). Similar results
measured also with microbial PLFA were found by Sampedro
and Whalen (2007) in the gut of L. terrestris and by Aira et al.
(2009) in middens. Overall, most of the literature points towards
a positive effect of anecics on microbial biomass in their burrows,
middens, or casts, although a few reports observed a neutral effect
(Postma-Blaauw et al., 2006) or negative effect (Zhang et al., 2000;
Yu et al., 2008).
Although most studies focus on the effect of one earthworm
species or species from the same ecological category on microbial
biomass, it is important to remember that all three functional
groups coexist in natural conditions. Investigating the earthworm
impact on soil microorganisms should therefore integrate the
whole earthworm community. Few studies have taken this
point into account, with the exception of Postma-Blaauw et al.
(2006) who showed that, although L. terrestris (anecic) did
not have any effect on soil microorganisms, the combined
presence of L. rubellus (epigeic) and L. terrestris induced an
increase in microbial biomass. These authors also observed a
smaller increase in the microbial biomass when L. rubellus and
A. caliginosa (endogeic) were tested together, and a decrease
of the microbial biomass when all three functional groups
were combined (Figure 3). The importance of investigating the
combined effect from distinct earthworm functional groups was
further confirmed by Scheu et al. (2002) who determined, in
a mesocosm experiment, that when epigeics (three species)
FIGURE 3 | Partial residual plots of the interaction effects (*) between (A) L. and endogeics (three species) were put together, soil microbial
rubellus and A. caliginosa (g), (B) L. rubellus and L. terrestris (g) and (C) L. biomass was larger than that observed when each earthworm
rubellus, A. caliginosa and L. terrestris (g) on the bacterial biomass (µg C g−1 ),
as determined by backward regression analysis (modified from
group was studied independently. Finally, it must be considered
Postma-Blaauw et al., 2006). that assessing soil microbial biomass as a whole may hide the
spatial heterogeneity of the effect of earthworms. This could
increase microbial abundance only in hot spots and decrease it
in the bulk soil, with the resulting effect depending on the rate of
although this may be biased by the fact that most studies are soil ingestion, earthworm density, and presence of the functional
carried out in temperate regions, using Lumbricus terrestris as groups (Sheehan et al., 2008). Considering microbial abundance
a model species. As shown in Figure 2, very few reports are as a whole can also prevent the detection of the fact that some
available for anecics in tropical regions. specific microbial taxa may have been promoted while others
Devliegher and Verstraete (1997) showed that the number of hindered. Therefore, it is important to look at how earthworms
CFU in the soil surface layer (0–5 cm) was 60–320 times larger in modify microbial community structure.
the casts of L. terrestris, fed with lettuce, than in the surrounding
soil. In a deeper layer (5–22 cm), the magnitude of the increase
was lower but still significant, with 6–32 times more bacterial IMPACT OF EARTHWORMS ON THE
CFU in the drilosphere than in the soil. The influence of soil STRUCTURE OF SOIL MICROBIAL
depth was further corroborated by Sheehan et al. (2008), who COMMUNITIES
observed a differential impact of the anecic worms Lumbricus
friendi and Aporrectodea longa on microbial biomass depending By feeding on soil and influencing soil factors such as porosity,
on the soil horizon under study. In a microcosm experiment water content, mineral N (NO− +
3 , NH4 ) or organic matter

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content, earthworms modify soil habitats and their resident here may be partly explained by the different methods that
microbial communities. In this section, we will make available were employed in the study of microbial diversity. Whilst
information regarding the impact of earthworms on the structure several results were obtained by using Terminal Restriction
and diversity of soil microbial communities and determine Fragment Length Polymorphism (T-RFLP) (Egert et al., 2004;
whether the resulting changes are consistent among functional Hoeffner et al., 2018) or clone libraries of the bacterial 16S
groups of earthworms. rRNA gene (Furlong et al., 2002), other studies have used
next generation sequencing (NGS) to increase the resolution
of diversity estimates (Gopal et al., 2017). The overall effect
Earthworms Modify the Diversity of Soil of earthworms on the soil microbial community also depends
Microbial Communities on soil conditions, particularly nutrient content. Koubová et al.
The effect of earthworms on the richness and diversity of (2015) showed that the effect of the epigeic earthworm E. fetida
microbial communities can be neutral, negative or positive, on soil microbial community biomass and composition, assessed
depending on the earthworm species and on the “micro- through PLFA and culturable bacterial counts, was stronger in
habitat” considered, i.e., whether the study focuses on the nutrient-poor habitats, where the stimulation of bacterial growth
earthworm gut, casts, or on the surrounding soil. Neutral effects in the earthworm intestine was more noticeable.
of earthworms on soil bacterial communities have been reported
by de Menezes et al. (2018), who showed that the introduction
of the endogeic Aporrectodea trapezoides did not influence the
Earthworms Modify the Abundance of
number of bacterial OTUs (Operational Taxonomical Units) nor Specific Taxa Within the Microbial
the Chao1 richness estimator of the whole soil. On the other Community
hand, positive effects on bacterial richness and diversity were Soil passage through earthworm gut has been reported to
observed by Hoeffner et al. (2018) in the burrows created by consistently increase the abundance of specific bacterial groups
four epi-anecic species from the Lumbricus genus, compared within the microbial community, such as that of Flavobacterium
to the bulk soil. These authors, however, showed that fungal (Schönholzer et al., 2002), Actinobacteria (Furlong et al., 2002;
diversity remained unaffected by the earthworms. The impact Rattray et al., 2010; Aira et al., 2016; Gopal et al., 2017;
that earthworms may have on soil microbial diversity was also Ma et al., 2017), Firmicutes (Furlong et al., 2002; Rattray
investigated through the study of the vermicomposting process. et al., 2010; Singh et al., 2015; Gopal et al., 2017; Ma et al.,
The epigeic earthworms Eudrilus sp. or E. fetida increased 2017) and γ-Proteobacteria, in particular members of the
bacterial diversity in the substrate, at least during the first stages Pseudomonas genus; (Furlong et al., 2002; Aira et al., 2016;
of their vermicomposting (Vivas et al., 2009; Gopal et al., 2017), Ma et al., 2017). Earthworms generally promote the growth
which showed the importance of considering different time scales of fast-growing bacteria such as γ-Proteobacteria due to the
in the study of bacterial diversity enhancement. labile carbon substrates they produce (Braga et al., 2016) in
Contrary results were observed when considering earthworm their gut or from their skin, which leads to increases in the
gut and casts. Negative effects of earthworms on bacterial Proteobacteria:Acidobacteria ratio (Gong et al., 2018). Specific
richness were found in earthworm gut and casts by Koubová et al. functional groups have also been shown to be enhanced by the
(2015), who showed that bacterial species richness (estimated presence of earthworms, such as denitrifiers (Ihssen et al., 2003)
from culturable bacteria) decreased during the passage through or cellobiose utilizers (Karsten and Drake, 1995). Sampedro
the epigeic Eisenia’s gut. Soil ingestion by epigeic earthworms was and Whalen (2007) also found significant changes in microbial-
also reported to decrease microbial diversity, as observed in the derived PLFA profiles of soil and gut and described that gut
gut of Eudrilus sp. (Gopal et al., 2017) and in casts of L. rubellus passage significantly increased the concentration of biomarkers
(Furlong et al., 2002). This decrease in microbial diversity after indicative of aerobic bacteria, microeukaryotes, and fungi.
soil ingestion has been attributed to the increased dominance of The advent of high-throughput sequencing of 16S rRNA
several bacterial groups in the earthworm casts, more specifically gene amplicons has allowed us to confirm and refine these
to an enrichment in bacterial taxa able to degrade benzoic and results. The presence of endogeic earthworms (A. trapezoides,
aromatic compounds (Furlong et al., 2002; Gopal et al., 2017). Metaphire guillelmi, or P. corethrurus) is associated with
Further studies evidenced that the type of food that earthworms increases in Bacteroidetes (especially in Flavobacteriaceae
ingest seems to have little influence on the diversity of bacterial and Sphingobacteriales), β-Proteobacteria (especially in
communities in casts, as shown by Aira et al. (2016) in the Rhodocyclaceae), Firmicutes (especially in Paenibacillaceae),
epigeic E andrei. Verrucomicrobia and ammonia-oxidizing Nitrosovibrio in
Overall, these studies show that the influence of earthworms the soil (Bernard et al., 2012; de Menezes et al., 2018; Gong
on microbial communities varies between micro-habitats, et al., 2018). The observed enrichment in these bacterial taxa is
although Egert et al. (2004) only found slight differences between usually attributed to an increase in the mineralisation of organic
the community structure of bacteria and archea in the gut, the residues (Bernard et al., 2012). Bernard et al. (2012) and de
casts and the surrounding soil in the case of the anecic earthworm Menezes et al. (2018) also found a promotion of chitinolytic
L. terrestris. On the other hand, for the same species, Sampedro bacterial taxa by P. corethrurus and A. trapezoides, respectively,
and Whalen (2007) found that the microbiome of its gut was such as Chitinophagaceae, Cytophagaceae, Neisseriaceae, and
different from the bulk soil. The contrasting findings highlighted Microbacteriaceae. The release of chitin in the soil, either

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through the production of chitinase by earthworms or through Acidobacteria, Planctomycetes, Bacteroidetes, Nitrospirae, and
the degradation of fungal hyphae during gut passage, may be Chloroflexi have the highest relative abundance in soils
responsible for this increase in chitinolytic bacteria. In general, where earthworms are present, regardless of the ecological
gut bacteria of anecic and endogeic earthworms seemed to be category they belong to. Although epigeic earthworms seem
determined, in descending order of importance, by earthworm to induce a higher microbial diversity than endogeic and
ecological group, habitat, and species (Thakuria et al., 2010). anecic earthworms, these latter may impact the soil bacterial
In the epigeic earthworms E. fetida and Perionyx excavatus, community in a more consistent manner, as shown by a
gut bacterial communities were shown to be dominated by lesser proportion of rare phyla (Figure 4). A network analysis
Proteobacteria, Actinobacteria, and Firmicutes, with several (Figure 5) confirmed that the above referred eight phyla form
differences according to the species. Verrucomicrobia and the core of the network while interacting or being promoted
Chloroflexi were abundant in the gut of E. fetida whilst they by most earthworm species. It also revealed that epigeic
were absent in that of P. excavatus. On the contrary, Spirochaetes earthworms promote more rare phyla of bacteria (seven phyla)
were abundant in P. excavatus but not in E. fetida (Singh et al., than do endogeic earthworms (two phyla). Altogether, these
2015). The earthworm intestinal tract constitutes an environment findings suggest that some bacterial taxa respond in a consistent
that is enriched in C, N and water content and impoverished in manner to the presence of earthworms and could constitute
oxygen when compared with the surrounding soil (Barois and good indicators for predicting the impact of earthworms on
Lavelle, 1986). It has therefore been consistently shown to favour soil ecosystems.
the occurrence of anaerobic or facultatively anaerobic bacteria
and archea (Barois et al., 1987; Horn et al., 2003; Koubová et al., THE IMPACT OF EARTHWORMS ON
2015). Bacterial genera such as Aeromonas, Bacillus, Clostridium,
Paenibacillus, Propionibacterium, or Staphylococcus were shown
NUTRIENT CYCLING THROUGH THE
to be abundant in the guts of epigeic Eisenia earthworms (Toyota MODIFICATION OF SOIL MICROBIAL
and Kimura, 2000; Shin et al., 2004; Koubová et al., 2015). König COMMUNITIES
(2006) reported that Bacillus and Paenibacillus, in particular, were
commonly detected in the gut of earthworms and were especially Earthworms are decomposers feeding on organic matter, thereby
relevant since they were able to degrade aromatic compounds releasing nutrients through digestion and excretion with direct
under oxygen limiting conditions. consequences on plant growth (Figure 1, arrow 1). They also
Although consistent patterns could be observed, the effect of have an important impact on microbial communities which in
earthworms on soil bacterial community composition seems to turn affects nutrient cycling and plant development through their
be mostly dependent upon the type of substrate under study interactions (Figure 1, arrows 2a and 2b). In a recent study, Braga
(de Menezes et al., 2018). This was demonstrated by Gopal et al. (2016) showed that the introduction of the endogeic P.
et al. (2017) who showed that bacterial community structure corethrurus in the soil significantly changed around 70 microbial
changed throughout the vermicomposting process, as nutrient functions in the bulk soil and in the rhizosphere, which were
dynamics were modified. Gong et al. (2018) reported a decrease mainly related to biosynthesis and plant-microbe symbiosis.
in the relative abundance of Cloroflexi and Fibrobacteres by The presence of earthworms also modified the ecological
the anecic M. guillelmi in rice fields where mulch was applied, interactions among microbial functions. As shown in the
whereas their dominance increased in rice fields where straw was previous section, earthworms stimulate certain microbial taxa,
incorporated. These authors also reported a shift in keystone taxa and by doing so increase the importance of keystone functions
within the soil microbial community, which was dependent upon (Braga et al., 2016). In this section, we will summarise the main
the applied organic amendment. These findings were consistent findings concerning the impact of earthworms on microbial
with those described by Koubová et al. (2015), who recorded functions, emphasising how information about earthworm-
distinct shifts in microbial taxa depending on the environment associated microbial communities needs to be integrated in
under study. Earthworm (Eisenia spp.) excreta were enriched order to improve knowledge of the influence of earthworms on
in Actinobacteria in compost pile (plant remains) whereas they nutrient cycling.
were enriched in Firmicutes in large scale vermiculture plant
(cattle manure and agricultural waste) and forest soil. On the Earthworms Increase Nutrient
other hand, increases in Gammaproteobacteria were detected in Mineralisation in the Soil
the gut of earthworms. Increases in Gammaproteobacteria were Earthworms, in particular endogeic geophagous earthworms,
also found by Fjøsne et al. (2018) in the soil when the epigeic are known to promote C and N mineralisation in the soil
earthworm Dendrobaena veneta was present. These authors (Lavelle et al., 1998; Araujo et al., 2004; Coq et al., 2007; Gopal
consistently observed increases in Kluyvera cryocrescens and et al., 2017), most likely through a priming effect affecting
Pseudomonas putida, independently from the initial composition decomposition rates of the soil organic matter (SOM) (Barois
of the soil microbial community. et al., 1987; Bernard et al., 2012). This positive priming effect
To synthesize the available information, we looked at how is expected to promote the recycling of nutrients, especially of
often microbial phyla were found in soils or substrates influenced organic N and P, in the SOM (Kuzyakov et al., 2000; Bertrand
by earthworms belonging to different ecological groups. et al., 2015). This has been shown for P. corethrurus in several
Figure 4 shows that Proteobacteria, Actinobacteria, Firmicutes, studies, summarised in the recent review by Taheri et al. (2018).

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FIGURE 4 | Relative abundance of microbial phyla reported in soils or substrates processed by earthworms of different functional groups. Data was obtained from 11
peer-reviewed publications retrieved after a search made using the words: “earthworms,” “soil microbial communities,” and “phyla” in the Web of Science from 2009
to 2018 (before that, no information was found with the keywords “microbial phyla”).

Two to three-fold increases in mineralised C have also been growth rates and specialised catabolic capabilities (Bernard et al.,
observed in casts of the endogeic A. caliginosa, compared with the 2012), which are thought to be responsible for the observed
surrounding soil, which is attributed to the priming effect caused increase in SOM mineralisation by earthworms, was defined
by earthworm ingestion and digestion (Abail et al., 2017). Epigeic by Lavelle et al. (1995) as the “Sleeping Beauty” paradox. As
earthworms such as E. fetida and P. excavatus have also been described previously, the promotion of fast-growing bacteria (γ-
reported to enhance the decomposition rates of organic matter Proteobacteria for example) may be driven by the N-rich gut
(Singh et al., 2015). mucus, changes in soil physico-chemical characteristics or to
The increase in SOM mineralisation in earthworm casts, the degradation of fungal biomass during gut transit, through
compared with the surrounding soil, is associated with an which earthworms can produce labile C substrates (Brown, 1995;
enrichment in labile compounds and with a subsequent increase Brown et al., 2000; Braga et al., 2016). The induction of a
in microbial activity (Barois and Lavelle, 1986; Coq et al., priming effect by earthworm gut transit is further confirmed
2007; Abail et al., 2017), which could be attributed to the by observations showing that SOM mineralisation rates are
earthworm digestion itself and to the influence of the gut lower in old casts than in recent ones (Pulleman et al., 2005;
microbiome. The enhancement of r-strategist bacteria with fast Bertrand et al., 2015).

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FIGURE 5 | Network representation of microbial phyla (circles) in soils or substrates processed by earthworms (squares) of different functional groups (green =
endogeic and orange = epigeic). The size of the figures represents the relative frequency of reports for each taxon whilst the width of the links is the relative frequency
of each pair of interactions (n = 11). Aptr, Aporrectodea trapezoides; Deve, Dendrobaena veneta; Eifo, Eisenia fetida; Eusp, Eudrilus sp.; Megu, Metaphire guillelmi;
Peex, Perionyx excavatus; Poco, Pontoscolex corethrurus. The data is the same as that used in Figure 4.

Although the effect of earthworms on soil N dynamics may This was further confirmed by Athmann et al. (2017) who
vary depending upon the species considered (Clause et al., 2014; evidenced a positive effect of root and earthworm (L. terrestris)
Groffman et al., 2015), increases in mineral N in earthworm biopores, compared with the bulk soil, on the activity of
casts from the different functional groups have been consistently several enzymes involved in the C and N cycle, resulting in
observed (Decaëns et al., 1999; Aira et al., 2005; Clause et al., an increase in nutrient mobilisation. These findings point out
2014). Mineral N concentrations have been measured as 5- a positive interaction effect on nutrient mineralisation at the
folds in casts of P. corethrurus when compared with those of drilosphere and rhizosphere level, two hotspots of microbial
the surrounding soil (Lavelle et al., 1992). Increases of 31 and activity in the soil. As recently highlighted by Bray et al. (2019),
4% in soil NO− +
3 -N and NH4 -N, respectively, have also been there is a stimulatory effect of earthworms and other soil
observed in soils with the presence of A. caliginosa (McDaniel macrofauna on rhizosphere microbial communities and on the
et al., 2013). The epigeic E. fetida also enhanced organic N microbially-mediated processes, particularly on N mineralisation
mineralisation in the rhizosphere of Phormium tenax, a New and SOM formation.
Zealand lilaceous perennial (Zhong et al., 2017). The overall The enhancement of C and N mineralisation by the
positive effect of earthworms on C and N mineralisation in the earthworm-associated microbiota is mediated by an increasing
rhizosphere was shown by Wu et al. (2017) who demonstrated enzyme activity. Some of the bacterial taxa that may be
that P. corethrurus affected C and N processes and the soil promoted by earthworms, such as Pseudomonas spp., have been
microbial community in plots where living plants were present, associated with the production of enzymes involved in the
in contrast to plots where artificial plants were used as controls. degradation of complex organic molecules, which could favour

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BOX 1 | Earthworms interact with mycorrhizal fungi

Beside soil bacteria, fungi are key organisms in the dynamics of soil biogeochemistry and its ultimate effect on plant growth. In contrast to with bacteria, much less
information is available regarding their interactions with earthworms and the outcome of these interactions. Most of the attention, if not all in the fungi-earthworm
interactions have been focused on arbuscular mycorrhizal fungi. In a similar way to the interaction with bacteria, the interactions between earthworms and mycorrhizal
fungi, particularly arbuscular mycorrhizal fungi, have been found to modify the soil chemistry (Zhang et al., 2016, 2018) and soil nutrient availability (Milleret et al.,
2009; Xiang and Li, 2014) and, critically important, the uptake of nutrients by plants (Milleret et al., 2009; Li et al., 2012, 2013a,b; Aghababaei et al., 2014) and the
composition and abundance of the fungal community (Gormsen et al., 2004; Dempsey et al., 2013; Cao et al., 2015a,b,c, 2016, 2018; Zhang et al., 2016). Although
the understanding of the interactions between earthworms and mycorrhizal fungi has not been the primary focus of most published works, there is a considerable
amount of data that permits us to gain some insights on these interactions and their synergistic effects on plant performance (Wurst et al., 2004; Yu et al., 2005;
Zaller et al., 2011; Li et al., 2013b).
The scientific interest in the interactions between earthworms and mycorrhizal fungi dates back almost 30 years and tackled the fundamental question of how the
trophic activity of earthworms affects the availability of infective units of mycorrhizal fungi. While all studies focused on the abundance of spores of mycorrhizal fungi
in earthworms’ casts found a concentration effect, the density of spores in the casts was on average 66% higher than in surrounding non-earthworm processed soil
(Gange, 1993; Harinikumar and Bagyaraj, 1994; Lee et al., 1996) and remained viable for up to a year (Reddell and Spain, 1991). Another investigation found no
effect of earthworms in dispersing effectively the infective units of mycorrhizal fungi (Pattinson et al., 1997). However, it must be noted that the only investigation on
the dispersion of mycorrhizal infective units by earthworms used a different species of earthworm (the endogeic A. trapezoides) compared to the many studies where
concentration of the spores in the casts from different species were measured (the anecic L. terrestris and the endogeic P. corethrurus, Ochochaetona phillotti, and
Lampito mauritii). As considerable variation has been reported among species of earthworms in their ability to concentrate infective units of mycorrhizal fungi, the
lack of support for the capacity of earthworms to disperse the infective units of mycorrhizal fungi must be taken with caution until further investigation including the
three functional groups of earthworms (anecic, endogeic, and epigeic) is carried out.
Over 60% of the reviewed cases reported that earthworm activities enhanced root colonization by mycorrhizal fungi while 25% reported a reduction in root
colonization. It is worth noting that while for anecic and epigeic earthworms the ratio of positive to negative and neutral effects on root colonization by mycorrhizal
fungi worked out to 3:1 and 4:1, respectively, for endogeic species this was inverted to a 1:2 ratio. The mechanisms that may differentially affect root colonization by
mycorrhizal fungi when interacting with anecic/epigeic, and endogeic earthworms are poorly understood. Drilling by earthworms may damage the hyphal networks and
fine roots of plants (Gange and Brown, 2002; McLean et al., 2006). Horizontal drilling of endogeic species may cause a more extensive disruption of the extraradical
mycelium compared to the vertical burrowing of anecic species and this may affect the capacity of the fungi to colonize the roots. Cast deposits on the surface of
the soil carried out by epigeic and anecic species may favour the dispersion of mycorrhizal infective units and this in turn may favour the colonization of roots. The
unique investigation tackling the question of earthworms as dispersion agents of mycorrhizal infective units showed negative results and concordantly was carried
out with endogeic species, which are frequently reported to decrease root colonization by mycorrhizal fungi. A likely explanation is that fungi are often considered
as earthworm food (Curry and Schmidt, 2007; Shan et al., 2013). No data exists regarding the effectiveness of anecic and epigeic species in dispersing infective
propagules of mycorrhizal fungi. Therefore, a comparative study of earthworms with different ecologies as dispersing agents of mycorrhizal infective units is needed.
Additionally, earthworms may favour root colonization by mycorrhizal fungi indirectly by promoting particular groups of soil microorganisms that may cooperate with
mycorrhizal fungi (Zhang et al., 2016). Whether earthworms of different behaviours could favour the proliferation of particular microbial groups that in turn facilitate the
interaction between mycorrhizal fungi and plant roots is a totally unexplored area, although some efforts report correlative changes of Gram positive bacteria together
with mycorrhizal fungi (Dempsey et al., 2013).
A major drawback in the investigation of the interaction between earthworms and mycorrhizal fungi is that only a handful of species of fungi have been used
in the experimentation (Rhizophagus intraradices, Rhizophagus irregularis, Funneliformis mosseae, Glomus geosporum, Glomus caledoniun, Glomus etunicatum,
Claroideoglomus claroideum, and Acaulospora sp.), all from the Glomeraceae. This means that our understanding of these interactions is rather partial and efforts
need to be made to understand the dynamics of these interactions with a wider range of species of fungi from different families as it is known that there are marked
differences in colonization rates, growth of extra-radical mycelium and capabilities to move soil nutrients to their host plants. Surprisingly, we are aware of no study
documenting the role of earthworms as potential drivers of the composition and structure of mycorrhizal communities. Simple pot and mesocosm experiments
with known initial composition of added mycorrhizal communities with and without earthworms will help to advance this field. This is important as a great deal of
efforts are being made to include earthworms and mycorrhizal fungi in sustainable agricultural practices and we need to understand their fundamental interactions
and outcomes.

SOM decomposition (Bertrand et al., 2015; Fjøsne et al., 2018). (Domínguez et al., 2004). Some authors, however, did not detect
Enzymes produced by the earthworm-associated microbiota are any earthworm effect on potentially mineralisable N (Fonte
also responsible for the reported increases in soil NO− 3 -N and and Six, 2010) or, on the contrary, evidenced a decrease in N
NH+ 4 -N in the presence of earthworms. For example, the activity mineralisation by earthworms (Groffman et al., 2015), most likely
of the soil enzyme β-N-acetylglucosaminidase has been shown due to an increase in microbial immobilisation that caused total
to be promoted in presence of P. corethrurus, which resulted soil N to decrease by 90 g N m−2 in presence of the epigeic
in “NH+ 4 -N hotspots” that might be accessed by arbuscular L. rubellus. A possible explanation which has been proposed by
mycorrhizal fungi (Box 1), hence providing benefits for plant several authors is that N mineralised by earthworms and their
growth (He et al., 2018). Increases in phenol oxidase and associated microorganisms might be used more readily by plants,
glucosidase activity by earthworms and other macrovertebrates thereby masking an increase in soil available N concentrations
were also observed in the rhizosphere of Festuca arundinacea (Pashanasi et al., 1996; González and Zou, 1999; Wu et al., 2017).
(Bray et al., 2019), and were attributed to the ingestion of fine Similarly, the amount of readily available phosphorus (P) has
roots and the stimulation of microbial activity during gut passage. been shown to be affected by earthworms, levels of available
The reduction of microbial immobilisation has been suggested P being higher in casts (Jiménez et al., 2003; Kuczak et al.,
as another driver of enhanced N mineralisation by earthworms, 2006; Vos et al., 2014; Ros et al., 2017) or in biopores formed
which may ultimately lead to an increase in NO− 3 -N leaching by L. terrestris (Athmann et al., 2017) than in the bulk soil.

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FIGURE 6 | Frequency of studies reporting: (A) overall changes in soil nutrients when processed by earthworms of different functional groups, (B) increments in
carbon, nitrogen and phosphorus in the soil or substrates when processed by earthworms of different functional groups, (C) overall changes in the soil microbial
community when the soil was processed by earthworms of different functional groups and (D) concurrence of data revealing changes in the soil microbial community
and in the soil nutrients. Percentage values are relative to each bar. (+) and (–) indicate when the frequency of studies is higher or lower respectively than that
expected under the null hypothesis: the chances of detecting nutrient increments are independent of the functional group of the earthworms. Data was obtained from
40 peer-reviewed publications retrieved after a search made using the words: “earthworms,” “soil microbial communities,” “nitrogen,” “phosphorus,” and “nutrient
cycling” in the Web of Science from 2003 to 2018. In Figure 6B only 70% of the studies were taken into account as only increases in nutrients were considered.

Concentrations of water-extractable P in casts of the anecic ingestion or in earthworm casts, although the magnitude of
earthworm L. terrestris have been reported to be 30–1000 times the increase in available P may differ depending on earthworm
larger than those found in bulk soil (Ros et al., 2017). These functional groups (Wan and Wong, 2004; Bernard et al., 2012;
earthworm-induced “P hotspots” depend upon the earthworm Vos et al., 2014).
species and have been shown to be larger for the epigeic L. Our synthesis of literature over the past 15 years revealed
rubellus than for the anecic L. terrestris or the endogeic A. that endogeic and anecic earthworms induced an increase
caliginosa (Vos et al., 2014). The influence of earthworms on in soil nutrients in around 70% of the consulted studies;
available P is particularly relevant in the rhizosphere, where epigeic earthworms, however, only induced an increase in soil
earthworms can interact with arbuscular mycorrhizal fungi to nutrients in 43% of the reported studies (Figure 6A). When
enhance P solubility and transfer to the plant (Milleret et al., 2009; analysing the effects of the different earthworm functional
Cao et al., 2015a) (Box 1). Soil available P has been reported to groups on particular nutrients (C, N, and P), more differences
increase in the presence of the endogeic earthworm P. corethrurus emerged. Epigeic earthworms were reported to increase P
(Lopez-Hernandez et al., 1993; Chapuis-Lardy et al., 1998; Patron levels in the soil or substrate under study more frequently
et al., 1999), or of epigeic E. fetida (Cao et al., 2015a), which than expected under the null hypothesis, whilst endogeics
has been linked to the enhanced microbial activity during soil were associated with N increases in the soil more frequently

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than the other two groups of earthworms (Figure 6B). It shown to reduce N2 O emissions when associated with rhizobial
is noteworthy that the reported increases in soil nutrients bacteria, most likely due to the aerobic conditions created by
by earthworms were rarely related to changes in the soil burrowing, which are detrimental to denitrification (Kim et al.,
microbial community (Figures 6C,D); moreover, when changes 2017). These contrasting findings may be due to the different
in microbial communities induced by earthworms were experimental settings that were implemented to study the effect
investigated, most studies (82%) did not report associated of earthworms on N2 O emissions. Lubbers et al. (2013) for
changes in soil nutrient contents (Figure 6D). Considering example, conducted a meta-analysis showing that earthworms
the functional groups of the bacterial phyla promoted by increase GHG emissions, in which most referenced studies are
earthworms (mainly Proteobacteria, Actinobacteria, Firmicutes, based on very short and simplified experimental set ups, in
and Acidobacteria) it is expected that the observed changes in which there are no plants to uptake the mineralised N, which
nutrient availability associated to earthworms are at least in part could indeed favour the emission of N2 O. Complexification
caused by the metabolic activity of bacteria rather than by direct of experimental set ups towards an integration of complex
effects of the earthworms. This calls for more studies integrating interactions between plants, macrofauna and microorganisms is
earthworm effects on soil microbial communities at a taxonomic therefore required in order to elucidate whether the presence of
and functional level, to unravel the link between microbial earthworms increase or decrease GHG emission in the long term.
diversity and ecosystem functions. Other microbial processes have also been reported to be
positively affected by earthworms. Functional genes associated
with carbohydrate and lipid metabolisms, biosynthetical
Earthworms Affect Microbial Functional pathways, translation, reduction-oxidation and cell proliferation
Genes Involved in Nutrient Cycling processes were more abundant in the soil when P. corethrurus
The influence of earthworms on nutrient cycling is not restricted was present (Braga et al., 2016). The introduction of P.
to their impact on SOM mineralisation through an induced corethrurus also promoted microbial functions associated with
priming effect. Several studies have also demonstrated a direct plant-microbe symbiosis in the rhizosphere of sugarcane, such
effect on the expression of bacterial genes involved in the N cycle. as plant cell colonization by N-fixing bacteria or plant growth
Soil N transformations, and thus soil fertility, have often been regulation (Braga et al., 2016). Finally, despite the reported
investigated through the study of microbial functional genes, effect of earthworms on P mobilisation, no studies have yet
which emphasise their importance as functional genetic markers investigated, to the best of our knowledge, how earthworms may
(Hosseini Bai et al., 2015; Ribbons et al., 2018). alter microbial functional genes associated with the P cycle.
Generally, the presence of earthworms has been associated
with an increase in denitrification. The presence of the endogeic
P. corethrurus was shown to increase the abundance of bacterial THE IMPACT OF EARTHWORMS ON
functional genes related to denitrification (nirK and nosZ) in SIGNAL MOLECULES PROMOTING PLANT
the soil and in the rhizosphere (Chapuis-Lardy et al., 2010; GROWTH
Braga et al., 2016). Similar findings were found by Nebert et al.
(2011) for the epigeic L. rubellus. The expression of the nosZ The positive effects of earthworms on plant growth and yield
gene, which encodes for the nitrous oxide (N2 O) reductase, are known to be related to improved soil physico-chemical
is directly linked with the amount of N2 O, an important variables, as earthworms facilitate the penetration of roots in
greenhouse gas (GHG) of which earthworms are thought to the soil, the absorption of nutrients and the exchange of gases
be promotors (de Menezes et al., 2018). An increase in the (Figure 1, arrow 1). Recently, these positive effects have also
abundance of the nosZ gene in the presence of earthworms may been attributed to the soil microbiota (Figure 1, arrows 2a
indicate the presence of larger denitrifying bacterial communities and 2b), through the activation of microorganisms producing
(Reverchon et al., 2015), which are known to be influenced by the signal molecules.
quantity and composition of organic compounds resulting from Despite all the literature documenting the co-occurrence
the decomposition of organic residues (Kandeler et al., 2006). between changes in the N cycle by earthworms and their
Earthworms and their associated microbiota, by promoting the positive effect on plant growth (Van Groenigen et al., 2014),
decomposition of SOM, could therefore create soil conditions some studies suggest that an increased nutrient mineralisation
that are able to sustain more abundant denitrifier communities. is not sufficient to explain the effect of earthworms on plant
Horn et al. (2003, 2006) indicated that the earthworm gut is a growth by itself (Blouin et al., 2006; Laossi et al., 2010). There
microenvironment ideal for N2 O-producing bacteria and that are other concomitant mechanisms, especially the emission of
gut denitrifiers are probably soil-derived. However, increases in signal molecules (SM) in the presence of earthworms, which are
N2 O emissions and in the abundance of the gene nosZ seem to involved in the effect of earthworms on plant growth (Puga-
be species-dependent, as no effect of the endogeic A. caliginosa Freitas et al., 2012b) and help explaining the earthworms positive
was detected on denitrification genes (Nebert et al., 2011). This effects. SM are molecules with strong effects on plant physiology
is also consistent with results by Depkat-Jakob et al. (2010) who despite their presence at low concentration and are generally
found that nosZ-containing taxa were not uniformly stimulated associated with qualitative changes. For example, SM are the
in the guts of worms from different feeding guilds. On the main factors driving plant development and immunity (Taiz
other hand, the anecic earthworm Maoridrilus transalpinus was and Zeiger, 2010). In turn, these qualitative changes can induce

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quantitative changes (e.g., growth). It is important to notice, or their casts. Transcriptomic approaches could also help
that SM differ from nutrients which are constitutive of biomass, unravel the microbially-mediated impact of earthworms on plant
generally present at relatively high concentration and mainly growth (Puga-Freitas et al., 2012b). Furthermore, considering the
responsible for quantitative changes. growing information available regarding earthworm-associated
It is widely accepted that SM are not exclusively produced microbial communities, it is necessary to carry out more
by plants. They are also produced by almost all soil organisms, systematic research on the SM produced by microorganisms that
including soil fauna and microorganisms (Brito-Vega and are detected in earthworm digestive tracts, casts, and tunnels.
Espinosa-Victoria, 2009; Puga-Freitas and Blouin, 2015). Pseudomonas spp., for example, have been detected in the gut of
Multiple organic compounds are included in SM, such as sugars, E. fetida (Pathma and Sakthivel, 2013). Since Pseudomonas spp.
organic acids and vitamins; these compounds are often involved have frequently been shown to emit SM that may promote plant
in the initiation of signalling pathways leading to the production growth, for instance through an induction of plant resistance to
of phytohormones (auxins, gibberellins, cytokinins, ethylene, pathogens (Bloemberg and Lugtenberg, 2001; Pieterse et al., 2009;
and abscisic acid), as well as secondary metabolites or volatile González et al., 2017); the combining next-generation sequencing
compounds that activate the plant’s immune system or regulate with state of the art metabolomic tools may help understanding
its growth and development. Up to date, it is unclear if soil the joint effect of earthworm and PGPB on plant growth.
fauna is able to produce these SM by itself, or if it activates Regarding plant response to SM in the presence of
microorganisms that produce them. However, Puga-Freitas earthworms, many observations of the “hormone-like effect”
et al. (2012a) revealed that culturable microorganisms extracted have been made with reference to vermicompost, for example
from earthworm-worked soils where producing more indole increased growth and yield, development of flowers and fruits,
acetic acid (IAA) (+46%) as compared with a control soil and other processes related to tolerance to biotic and abiotic
without earthworms, which supports the second hypothesis of stresses (Table 1). In general, E. fetida, A. caliginosa and A. rosea
a stimulation of bacteria (probably Plant Growth Promoting are the earthworm species that have presented greater positive
Bacteria, or PGPB) by earthworms. effects in plants, which has been attributed to the presence
Humic acids, IAA, aminocyclopropane-1-carboxylate (ACC), of IAA, ACC, and humic acids produced by their associated
as well as molecules tentatively identified as auxins and ethylene bacteria. Humic acids are SM extracted from vermicompost
have been reported as SM produced in the presence of produced from cattle manure that also enhanced root growth
earthworms, using indirect methods such as colourimetry. In and the number of sites of lateral root emergence in maize
many cases, their presence has been deduced from observations seedlings (Zea mays); these molecules were also shown to be
on plants that are similar to results observed in the presence responsible for a stimulation of the plasma membrane H+-
of exogenous SM application. However, recent evidence shows ATPase activity (Canellas et al., 2002). Quaggiotti et al. (2004)
that SM have been unequivocally identified by a reverse phase reported an accumulation of H+-ATPase gene transcripts in the
ultra-high-resolution liquid chromatography (UPLC) system roots and an increase of nitrate transporter gene transcripts in
coupled to a triple quadrupole mass spectrometry analyser, the shoots of plants exposed to earthworm-producing humic
which allowed to determine the presence of jasmonic (JA), substances. Using a transcriptomic approach for the screening
salicylic (SA), and abscisic acid (ABA) in vermicompost of of gene expression in Arabidopsis thaliana, Puga-Freitas et al.
E. fetida (Hernández, 2019). Nevertheless, the involvement of (2012b) found an accumulation of transcripts of 57 genes, most
microorganisms in the secretion of these molecules was not of which are known to be induced by exogenous hormone
elucidated with the exception of Pathma and Sakthivel (2013) application or microbial elicitors. They also showed the reversion
who identified bacteria from E. fetida casts. Most of the studies of the dwarf phenotype of an A. thaliana mutant for IAA
investigating the chemical composition of SM in earthworm transport in the presence of earthworms, suggesting that
casts were conducted on epigeic earthworms, particularly on earthworms were compensating the low auxin level in root
E. fetida, while one single paper studied the endogeic species cells by producing auxin-like compounds in the soil, which
Aporrectodea caliginosa (synonym Nicodrilus caliginosus) and were able to penetrate plant roots (Puga-Freitas et al., 2012b).
Aporrectodea rosea (synonym Allolobophora rosea). Finally, all Transcriptomic studies and exploration of plant signalling
studies have solely been related to the casts of earthworms pathways using mutants could be developed for different stages
(Table 1). So far, there are no publications that confirm the of plant development to better characterize plant response to the
isolation and unequivocal quantification of compounds such as presence of earthworms.
auxins or gibberellins and the identity of the microorganisms A hypothesis has been put forward that the activity of
associated with these molecules. Only two studies, at a 17-year earthworms has a positive impact on plant growth through SM
interval (Canellas et al., 2002 and Hernández, 2019), provided an released in the soil. However, the literature is composed of
unambiguous identification of SM (humic acids with ABA, SA, many “chapters” (reported in Table 1) relying either on changes
and JA). in the soil microbial community, an increase or decrease of
In the light of the new era of technology for the analysis and SM or modifications in plant development or immunity. This
quantification of organic molecules, a new panorama opens to is mainly due to the numerous scientific expertise required in
understand “the universe of molecules’ diversity” of soil. More soil chemistry, microbiology, plant physiology, and soil ecology.
research is required to elucidate the most efficient extraction Therefore, nowadays there is no single study integrating all the
methods and identification of these molecules on earthworms chapters in a complete story.

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Frontiers in Environmental Science | www.frontiersin.org

Medina-Sauza et al.
TABLE 1 | Studies showing the effects of earthworms on plants under controlled conditions.

Earthworm Functional Area of Microorganism Signal molecule Identification Effect of the References
species group influence species released or related method molecule on plants

Eisenia fetida Epigeic Casts Not reported IAA and humic acids Gas Growth of corn Canellas
chromatography seedling root (Zea et al., 2002
coupled to mays)
masses
IAA Not reported Growth regulator Arancon
et al., 2006
Humic acids Increased the
development of flowers
and fruits in peppers
Casts Pseudomonas aeruginosa, P. monteilii, P. fluorescens, IAA Colorimetric Growth regulator Pathma and
Bacillus pumilis, B. subtilis, B. flexus, Microbacterium method Sakthivel,
schleiferi, Acinetobacter calcoaceticus, A. baumannii, A. junii, 2013
A. schindleri, Stenotrophomonas maltophilia and
Enterobacter cloacae
Bacillus megaterium, B. thuringiensis, B. amyloliquefaciens, Aminocyclopropeno- Regulator of ethylene
B. tequilensis, B. licheniformis, B. cereus, B. pumilus, B. 1-carboxilate (ACC level in plants for
aryabhattai, B. subtilis, B. flexus, B. aquimaris, B. marisflavi, ethylene precursor) optimal growth
Microbacterium takaoensis, Acinetobacter calcoaceticus, A.
14

baumannii, A. junii, A. schindleri, Arthrobacter nicotianae,

Rhodococcus ruber and Enterobacter cloacae
Casts Not reported ABA Identified Not reported Hernández,
unequivocally 2019
JA through
SA UPLC in a
directed study
Nicodrilus caliginosus* Endogeic Casts Not reported Auxin-like Root growth Root growth in Daucus Muscolo
and Allolobophora inhibition test carota et al., 1999
rosea**
Nicodrilus caliginosus* IAA and low Enzyme Stimulates the uptake Quaggiotti
and Allolobophora molecular size linked of nitrate by roots and et al., 2004
rosea** humic substances immuno- the accumulation of the
sorbent assay anion at the leaf level in
(ELISA) Zea mays
June 2019 | Volume 7 | Article 81

Aporrectodea Similar to Not reported Increased total Puga-Freitas

caliginosa phytohormone, such biomass and biomass et al., 2012b

Microbiata and Earthworms

as auxin and production of Lolium
ethylene perennial meristem

We highlight the compounds or signal molecules (SM) that, are responsible for the reported effects on plants.
*Synonym: Aporrectodea caliginosa. **Synonym: Aporrectodea rosea.
Medina-Sauza et al. Microbiata and Earthworms

CONCLUSIONS their direct interaction with plants (Box 1). On the other
hand, saprotrophic fungi, like many bacteria, are key drivers
Earthworms are known to play a critical role in ecological of soil biogeochemistry but their roles have been largely
processes, through their improvement of soil structure, neglected especially in their synergistic or antagonist interactions
nutrient cycling and plant growth. Evidence also shows that with earthworms.
earthworms contribute towards the structuring of soil microbial Earthworms promote the mineralisation of N and P and alter
communities, either directly through their ingestion or indirectly microbial functional genes which modifies soil functions. More
through a priming effect resulting from an increase of available information is needed to understand which microorganisms and
labile substances. However, few investigations have combined microbial genes are activated by earthworms, especially on the
data on earthworm –microorganism interactions with studies on P cycle.
soil nutrient cycling, especially on P cycling, or the production Finally, the consequences of these earthworm-induced
of signal molecules, which prevents us to fully understand changes in soil functioning on plant growth cannot be fully
the mechanisms underlying the effect of microbial hotspots understood without the study of SM, produced either by
in the drilosphere on soil functioning. Our hypothesis “the the earthworms or most likely by microorganisms created by
effect of earthworms on nutrient cycling and plant growth is earthworm activity. The involvement of specific microbial taxa
not only a direct effect but it is mainly mediated indirectly, via in the secretion of these molecules needs to be elucidated
modifications of the microbial community” is largely verified at and this requires a collaborative effort from disciplines such a
the small spatial and short temporal scale (gut, casts, burrows metabolomics, microbiology, transcriptomics, and biochemistry
and tunnels). in order to unequivocally identify SM in earthworms or in
Earthworms influence microbial biomass and activity in their casts.
the soil but contrasting results can be found in the literature To plagiarize Aristotle, earthworms are indeed the intestine
regarding the direction of this effect. This could be due of the Earth, with their specific microbiota, which brings
to the nature of the organic matter earthworms feed on, us to a large spatial and temporal scale. This intestine is
particularly in the case of epigeic species or the substrate complex to understand because of its dynamics associated
they live in. This could also be linked with experimental with the activity of earthworms, other soil organisms and
conditions, since the most variable effects on microbial plant roots. Nevertheless, we have to consider these complex
abundance were observed in laboratory studies and consistent effects of earthworms on microbial communities in order
increases of microbial abundance by endogeic species were to understand the effect of earthworms on nutrient cycling
observed in the field (Figure 2). However, the effect on and plant growth promoting SM and ultimately to predict
microbial communities is less or neutral when the feedstock or plant-soil interactions, especially if earthworms ingest hundreds
the soil they feed on is rich in assimilable organic matter or thousands of tons of substrates or soil per hectare and
independently of the functional group. Nonetheless, a per year.
recurrent result of our review is the relevance of considering
earthworm ecological category (epigeic, anecic, or endogeic)
to highlight some trends in the effect of earthworms on AUTHOR CONTRIBUTIONS
the structure and function of microbial communities.
RM-S and RG did the bibliometric data analysis. FR, AD,
Complexification of experimental design, with interactions
JG-A, MÁ-J, MB, RM-S, RG, and IB contributed with the
between earthworm ecological groups and the presence of
writing of different sections. LV and CC gave ideas for
plants should therefore be considered in mesocosm studies
the structure and illustrations of the article and reviewed
in order to better mimic natural conditions and avoid
the manuscript. MÁ-J and MB designed and produced the
experimental artifacts.
illustrations. MB gave constructive suggestions throughout the
Although there is still no clarity in understanding if
work to organise the manuscript. FR and IB coordinated
earthworms have their own intestinal microbiome or it
the work and integrated and edited the different sections of
comes from the soil, most of the information says it
this article.
comes from the soil. We can however stress on some
general patterns: taxa such as Flavobacterium, Actinobacteria,
Firmicutes, and γ-Proteobacteria are consistently reported to ACKNOWLEDGMENTS
be promoted by gut transit and could therefore constitute
good indicators for predicting the impact of earthworms We would like to thank the Consejo Nacional de Ciencia
on soil processes. The increasing use of Next Generation y Tecnología for their financial support (CONACYT, Project
Sequencing (NGS) technologies in the study of soil microbial 257819) and the scholarship granted to RM-S and MÁ-J. We
communities and their diversity will help to refine our are grateful to Martin de los Santos for his help in formatting
understanding of how earthworms may shape them. Information the document and to Judy Shirley for revising the language
is also critically needed regarding the role of earthworms of this manuscript. We would also like to acknowledge the
as potential drivers of the composition and structure of editors of this issue for giving us the opportunity to be
fungal communities, particularly mycorrhizal fungi because of part of it.

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Medina-Sauza et al. Microbiata and Earthworms

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6:e29293. doi: 10.1371/journal.pone.0029293 Conflict of Interest Statement: The authors declare that the research was
Zhang, B. G., Li, G. T., Shen, T. S., Wang, J. K., and Sun, Z. (2000). conducted in the absence of any commercial or financial relationships that could
Changes in microbial biomass C, N, and P and enzyme activities be construed as a potential conflict of interest.
in soil incubated with the earthworms Metaphire guillelmi or Eisenia
fetida. Soil Biol. Biochem. 32, 2055–2062. doi: 10.1016/S0038-0717(00) Copyright © 2019 Medina-Sauza, Álvarez-Jiménez, Delhal, Reverchon, Blouin,
00111-5 Guerrero-Analco, Cerdán, Guevara, Villain and Barois. This is an open-access article
Zhang, W., Cao, J., Zhang, S., and Wang, C. (2016). Effect of earthworms distributed under the terms of the Creative Commons Attribution License (CC BY).
and arbuscular mycorrhizal fungi on the microbial community and maize The use, distribution or reproduction in other forums is permitted, provided the
growth under salt stress. Appl. Soil Ecol. 107, 214–223. doi: 10.1016/j.apsoil. original author(s) and the copyright owner(s) are credited and that the original
2016.06.005 publication in this journal is cited, in accordance with accepted academic practice.
Zhang, W., Wang, C., Lu, T., and Zheng, Y. (2018). Cooperation between No use, distribution or reproduction is permitted which does not comply with these
arbuscular mycorrhizal fungi and earthworms promotes the physiological terms.

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