Available online at www.sciencedirect.com
European Journal of Soil Biology 43 (2007) S149eS156
http://www.elsevier.com/locate/ejsobi
Original article
Fate of soil bacteria and fungi in the gut of earthworms
Boris A. Byzov a,*, Nikita V. Khomyakov a, Sergei A. Kharin a,
Alexander V. Kurakov b
b
a
Department of Soil Biology, Faculty of Soil Science, Russian Federation
International Biotechnological Center, Moscow Lomonosov State University, Russian Federation
Available online 24 September 2007
Abstract
The responses of soil microorganisms to the action of gut fluids of three earthworm species, Aporrectodea caliginosa, Lumbricus
terrestris and Eisenia fetida were investigated. It was found that the midgut fluid taken form anterior part of the digestive tract could
suppress formation of colonies of bacteria, inhibit the germination of spores and reduce the radial growth rate of some fungal colonies. Heating the midgut fluid at 98 C for 10 min did not eliminate its suppressive activity. This suggests that a non-protein compound (s) is involved in this action of the midgut fluid. The suppressing effects were shown to be selective towards soil bacteria.
However, the responses of microbial cells to the midgut fluid did not correspond to the taxonomic affiliation of the microorganisms
tested. The mechanism of the suppressing effect might be a destruction of microbial cell membranes, as demonstrated for the gut of
soil millipedes. The selective activity of the gut fluid of earthworms could be a significant factor for the animal’s nutrition as well as
for regulating the steady state of the intestinal microbial community, and modification of microbial communities in soil.
Ó 2007 Elsevier Masson SAS. All rights reserved.
Keywords: Earthworms; Bacteria; Fungi; Gut fluid; Digestion; Suppressive and stimulating effects; Soil microbial communities
1. Introduction
Earthworms can directly regulate microbial populations by consuming large amount of soil. This leads to
elimination of some microorganisms and proliferation
of others in the digestive tract, drilosphere and faeces
of earthworms [12,4,5,11,1,18].
Microorganisms are an essential food component of
soil invertebrates including earthworms. The importance of fungi and bacteria as principal sources of
food is indicated by several phenomena. Soil animals
avoid consuming fresh leaf litter, which could be toxic
* Corresponding author. Fax: þ7 (495) 939 0989.
E-mail address: [email protected] (B.A. Byzov).
[10]; the animals themselves have no intrinsic capacity
to digest cellulose [17,8] and they depend on microorganisms as sources of essential amino acids [14]. It
was proposed that earthworms derive more of its energy
and nutrients from gut specific microbiota than from
microbiota already present in the ingested soil [15].
Moreover, microbial biomass and its structural components were found to be assimilated more efficiently than
cellulose, which supports the hypothesis that microorganisms are an important dietary resource for soil
macro invertebrates [9].
Mechanisms of the effects of the gut passage through
earthworm on soil microorganisms are not known. Little
information exists about impact of gut fluid of earthworms on the viability of soil microorganisms and their
1164-5563/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejsobi.2007.08.012
S150
B.A. Byzov et al. / European Journal of Soil Biology 43 (2007) S149eS156
digestion in the gut. Few studies have shown suppressive
effects of the earthworm’s gut fluid on germination of
fungal spores [11] and viability of ciliates [13]. We propose that modification of soil microbial community
could be due to different responses of microorganisms
under the influence of gut fluid of earthworms (stimulation, suppression, death of cells or absent of such
responses).
The aim of our study was to explore the changes of
quantity of soil microorganisms under the gut passage
of earthworms and to characterize the reaction of pure
cultures of some bacteria and fungi affected by the
incubation in the gut fluid.
2. Materials and methods
2.1. Earthworms
The earthworms Aporrectodea caliginosa and Lumbricus terrestris were collected from ploughed horizon
(0e20 cm) of fertilized (NPK) soddyepodzolic arable
soil under crop rotation (red clover with mixture of
grasses) at the Ecological Soil Station of Moscow
Lomonosov State University (Solnechnogorsk district,
Moscow region, Russia). Eisenia fetida were collected
from manure compost at Chashnikovo farm (Solnechnogorsk district, Moscow region, Russia). The earthworms A. caliginosa and L. terrestris were maintained
in laboratory in the soddyepodzolic soil, and E. fetida
were maintained in the cow manure compost, before
experiments.
2.2. Microorganisms
The list of microorganisms used in the tests is shown
in Table 1. Bacteria and fungi were isolated from soil,
compost, or from the guts of earthworms on R2A agar
(Difco) (bacto yeast extract, 0.5 g; bacto proteose peptone no. 3, 0.5 g; bacto casamino acids, 0.5 g; bacto dextrose, 0.5 g; soluble starch, 0.5 g; sodium pyruvate,
0.3 g; potassium phosphate, dibasic, 0.3 g; magnesium
sulfate, 0.05 g; bacto agar, 15 g; pH 7.2, distilled water,
1 l). For selection, fungal growth was inhibited by nystatin (500 mg per ml) and bacterial growth was limited
by streptomycin sulfate (50 mg per ml). Isolation of microorganisms was done by the plate dilution method.
Three separate samples of fresh soil/compost, gut content, or fresh excrement (50 mg weight each) were
used. Each sample was placed in 500 ml of sterile tap
water (for bacteria) or into 10 ml for fungi for preparation of dilution and homogenization. For homogenization of samples and desorbing microorganisms from
mineral/organic particles, the homogenizer DIAX 900
(Heidolph, Germany) was used. Aliquots of 20e
400 ml of the dilution were spread on the surface of
the agar medium. For the isolation of fungi were used
30 Petri dishes (10 for each sample) and for bacteria,
nine Petri dishes (three for each sample) for each dilution. The Petri dishes were incubated at 18e20 C for
2e10 days.
The strains of bacteria and fungi were preliminarily
identified according to cultural, morphological, physiological and biochemical properties to genera or species level based on current manuals with following
Table 1
Microorganisms tested for responses to gut fluids
Bacteria
a-Proteobacteria
b-Proteobacteria
g-Proteobacteria
Actinobacteria
Bacilli
Unidentified species
Fungi
Ascomycota
Basidiomycota
a
Aminobacter sp. (strains 408-1,a 408-2, 411-1, 411-2), Brevundimonas diminuta 384-1, Sphingopyxis witflariensis 397-1
Alcaligenes faecalis 345-1, Bordetella sp. 341-1, Delftia acidovorans 335-1
Kluyvera ascorbata 303-1, Pseudomonas proteolytica 599, P. putida (strains 304-1, 348-1, 429-2), P. reactans
(strains 383-1, 387-2, 394-2, 400-2), Pseudomonas spp. (strains 309-2, 310-1, 329-1, 387-1, 399-2, 428-1, 607-1)
Agromyces cerinus 347-1, Arthrobacter oxydans (strains 304-1, 304-2), A. globiformis 333-1, Arthrobacter spp.
(strains 392-1, 430-1, 430-2); Kocuria palustris (strains 405-2, 416-2), Microbacterium sp. 423-1, Nocardioides
sp. 410-1, Rhodococcus opacus 404-2, Streptomyces spp. (strains 389-1, 406-2)
Bacillus licheniformis 414-2, Bacillus mojavensis 317-1, B. megaterium (strains 401-1, 413-1, 413-2), B. subtilis
(strains 385-2, 386-2), Paenibacillus sp. (strains 412-1, 412-2)
Strains A9, C12, D12, B11, D8, B12
Alternaria alternata 1C321, Aspergillus versicolor 1G213, A. terreus 3S422, A. niger 3C132, Cladosporium
cladosporioides 2S41, Gliocladium catenulatum 2S723, G. roseum 2S522, Fusarium oxysporum 2S433,
Paecilomyces lilacinus 2S513, Penicillium aurantiogriseum 5S623, P. chrysogenum 4E232, P. decumbens 3S211;
P. melenii 4S523, Trichoderma harzianum 1E211
Bjerkandera adusta 7G521
Strains obtained from the collection of the Department of Soil Biology, Faculty of Soil Science, Moscow Lomonosov State University.
B.A. Byzov et al. / European Journal of Soil Biology 43 (2007) S149eS156
S151
verification by PCR amplification, sequencing of amplicons and analysis of sequence data [7].
The relative abundance of fungal species in the certain experimental variant was calculated as the percentage of the isolates of each species from all fungal
isolates from this variant counted on the agar plates
(on 30 Petri dishes used for each variant, were isolated
and counted around 500 colonies).
white, respectively. Bacterial and fungal biomass were
estimated on the base of volumetric measurements
(mean volume of bacteria cells 0.1 mm3 and dry weight
2 1014 g, mean width of fungal hyphae 5 mm and dry
weight of 1 m of fungal hyphae 3.9 106 g [19]).
2.3. Earthworm gut fluid
Bacteria were grown at 18e20 C in a liquid glucosee
peptoneeyeast medium (GPY) (glucose, 1 g; peptone,
2 g; yeast extract, 1 g; casein hydrolyzate, 1 g;
KH2PO4, 0.5 g; K2HPO4, 0.5 g; Difco agar, 15 g; pH
7.2, tap H2O, 1 l) for 18e20 h. Fungi were grown on
the GPY agar for 7e10 days. Fungal spores were harvested with sterile tap water and counted under a light
microscope. Three different treatments were carried
out to investigate the effects of gut fluid on fungi and
bacteria. In Experiment 1, a volume of 1 ml of bacterial
suspension or fungal spores (ca. 105 bacterial cells/
spores ml1) was applied to the surface of a thin film
(1.5e2 mm) of the agar medium R2A poured on a
microscope glass slide. After absorbing the bacterial
suspension (20e30 s) a volume of 2 ml of the gut fluid
was added. The slides were incubated for 1 day. Then
the numbers of CFU were determined by counting the
forming microcolonies using a light microscope. In
Experiment 2, 3 ml of cell suspensions was mixed
with 6 ml of the gut fluid. The mixture was incubated
for 2 h at 18e20 C. Then the numbers of CFU were
determined using the dilution plate technique. The percentage of spores germinated was determined on agar
films. In Experiment 3, fungal hyphae were disintegrated using a vortex blender and the suspension of
hyphae fragments was mixed with the gut fluid, 1:1.
Then the numbers of CFU were determined and the
radial growth rates of the fungal colonies were measured after 1e2 min, 2 and 24 h of incubation using
the dilution plate technique. In the controls, we used
sterile tap water instead of gut fluid.
The earthworms were fed with sterile sand or kept on
moist filter paper for 3e5 days to clean up the digestive
tracts. To obtain gut fluid, the worms were deactivated
by immersing in boiling water for 1 s to make them unmovable. Then they were partially desiccated by placing
on a freezing stage at 16 C. The earthworm body was
frozen within 2e3 min. Then it was allowed to thaw.
During this time, the digestive tracts taken behind the
clitellum were divided into two equal parts: anterior
part (foregut and midgut) and posterior part (hindgut).
The gut materials were centrifuged at 12,000 rpm for
15 min in order to separate the gut fluid from gut tissue
and the majority of microbial cells. A volume of 100e
200 ml of the gut fluid was heated at 98 C for 10 min
on a water bath to inactivate proteins. The precipitates
were removed by centrifugation at 12,000 rpm for
15 min. The fluid obtained was stored at 18 C.
2.4. Estimation of microbial biomass and cell count
Microbial biomass and numbers of colony forming
units (CFUs) were estimated in food substrates (soil or
compost), in the gut content, and in the fresh excrement.
To obtain fresh excrement the earthworms were
placed on moist sterile filter paper for 2e3 h. Dilution
plate technique was used to estimate number of CFU
of bacteria and fungi and microbial biomass estimated
by luminescent microscopy. Bacterial cells and fungal
hyphae were stained by acridine orange and calcofluor
2.5. The study of response of bacteria
and fungi as affected by the gut fluid
Table 2
Fungal and bacterial biomass in soil (compost), guts and fresh excrement of the earthworms Aporrectodea caliginosa, Lumbricus terrestris and
Eisenia fetida as determined by luminescent microscopy
Substrate
Biomass (mg g1 dry weight)
Aporrectodea caliginosa
Soil (compost)
Gut content
Excrement
Lumbricus terrestris
Eisenia fetida
Fungal mycelium
Bacteria (102)
Fungal mycelium
Bacteria (102)
Fungal mycelium
Bacteria (102)
1.9 0.3
1.2 0.2*
1.3 0.1*
4.8 0.8
5.1 1.3
6.8 1.4
1.8 0.2
1.3 0.1*
0.9 0.1*
4.6 0.2
5.0 2.2
4.6 0.8
3.4 0.5
2.2 0.2*
1.9 0.3*
12.2 2.6
8.8 3.4
11.4 5.1
All data are means of 3e5 replicates; standard deviation.
* Significant differences comparing to soil (compost), p < 0.01.
B.A. Byzov et al. / European Journal of Soil Biology 43 (2007) S149eS156
S152
Table 3
CFU numbers ( 105 g1 dry weight) of fungi in soil (compost), gut
contents, empty guts and the fresh excrement of the earthworms Aporrectodea caliginosa, Lumbricus terrestris, and Eisenia fetida
Substrate
Aporrectodea
caliginosa
Lumbricus
terrestris
Eisenia
fetida
Soil (compost)
Gut content
Empty gut
Excrement
1.4 0.5
0.8 0.5*
1.2 0.4
1.3 0.3
1.7 0.6
ND
0.7 0.0*
1.1 0.1
4.4 1.2
ND
6.4 2.9
6.2 0.1
All data are means of 3e5 replicates; standard deviation; ND, not
determined.
* Significant differences comparing to soil, p < 0.01.
3. Results
3.1. Changes of bacterial and fungal biomass
and CFU numbers resulting from passage
through the guts of earthworms
The bacterial biomass did not change significantly
during gut passage. The biomass of fungal hyphae decreased 1.5e2 times from soil (compost) to excrement
for all earthworm species (Table 2).
The numbers of fungal CFU in the guts of A. caliginosa and E. fetida that were cleaned up of soil particles
were comparable to those of soil and fresh excrement.
In the gut of L. terrestris, the number of fungal CFU
was lower. The fungal CFU counts did not changed significantly from soil (compost) to excrement (Table 3).
3.2. Changes of fungal species composition during
passage through the earthworm guts
Considerable changes of fungal community composition were observed as consequence of passage through
the gut of A. caliginosa. Many fungi that occurred in soil
material could not be isolated from freshly collected
excrements. Species of Penicillium, Gliocladium, Acremonium, Chaetomium globosum and others decreased
in numbers and relative abundance in the fungal community of the fresh excrements or were not detected
in it. The relative abundance of Mucor hiemalis, Geotrichum candidum, Syspastospora parasitica, Bjerkandera
adusta, Mycelia sterilia increased in the excrements
compared to the soil, but Alternaria alternata, Aspergillus versicolor, Paecilomyces lilacinus and Trichoderma
harzianum showed no significant change in counts
(Table 4).
3.3. Effects of the gut fluid on the viability of bacteria
The midgut fluid of A. caliginosa caused decreases of
CFU numbers of eight strains of soil bacteria 10 times or
more. Another group of bacterial strains showed a moderate sensitivity to the midgut fluid, some bacteria were
Table 4
Changes of relative abundance (%) of fungal species resulting from passage of soil through the gut of the earthworm Aporrectodea caliginosa
Genus/species
Soil
Fresh excrement
Acremonium murorum
Acremonium sp.
Alternaria alternata
Arthrobotrys sp.
Aspergillus fumigatus
A. niger
A. versicolor
Chaetomium globosum
Cladosporium cladosporioides
Fusarium (F. oxysporum, Fusarium sp.)
G. roseum
G. penicilloides
Mucor hiemalis
Paecilomyces lilacinus
Penicillium (P. aurantiogriseum, P. canescens, P. corylophilum, P. chrysogenum, P. janczewskii,
P. pinophilium, P. purpurogenum, Penicillium sp. 1, Penicillium sp. 2)
Trichoderma harzianum
Verticillium lateritium
Geotrichum candidum, Syspastospora parasitica, Bjerkandera adusta, Mycelia sterilia
0.5a
8.0
1.0*
0.5
2.0
1.0
1.0*
1.5*
0.5
0.5
3.2
0.8
2.0
7.0*
51.0
n.f.
1.0
1.0
n.f.
n.f.
n.f.
1.0
0.5
n.f.
n.f.
n.f.
n.f.
12.0
6.0
33.5
6.0*
0.5
13.0
8.0
n.f.
37.0
* No significant differences, all others data significantly different, p < 0.01.
n.f., not found.
a
Coefficient of variation of data 20e30%.
B.A. Byzov et al. / European Journal of Soil Biology 43 (2007) S149eS156
S153
140
120
CFU, % of control
100
80
60
40
0
Rhodococcus opacus 404-2
Pseudomonas reactans 383-1
Amininobacter sp. 411-1
Bacillus licheniformis 414-2
Paenibacillus sp. 412-2
Bacillus subtilis 386-2
Nocardioides sp. 410-1
Aminobacter sp. 408-2
Arthrobacter globiformis 333-1
Pseudomonas sp. 329-1
Bordetella sp.341-1
Arthrobacter 392-1
Arthrobacter oxydans 304-2
Bacillus subtilis 385-2
Agromyces cerinus 347-1
Delftia acidovorans 335-1
Streptomyces sp. 389-1
Pseudomonas sp. 607-1
Streptomyces sp. 389-1
Pseudomonas proteolytica 599-1
Pseudomonas sp. KL28 309-2
Bacillus megaterium 413-2
Kocuria palustris 405-2
Pseudomonas sp. 387-1
Brevundimonas diminuta 384-1
Pseudomonas sp. 399-2
Pseudomonas reactans 394-2
Pseudomonas putida 348-1
Streptomyces sp. 406-2
Kocuria palustris 405-2
Pseudomonas reactans 400-2
Bacillus mojavensis 317-1
Paenibacillus sp. 412-2
Aminobacter sp. 411-2
Aminobacter sp. 408-1
Sphingopyxis witflariensis 397-1
Kluyvera ascorbata 303-1
Arthrobacteer sp. 430-1
Pseudomonas reactans 387-2
Bacillus megaterium 413-1
Bacillus megaterium 401-1
Microbacterium sp. 423-1
20
Bacteria
Fig. 1. The effects of the midgut fluid of the earthworm Aporrectodea caliginosa on CFU numbers of soil bacteria (percentage of control are
given) (microcolonies numbers counted on agar films under light microscope). n ¼ 3e5.
shown to be resistant to the digestive fluid, and their
CFU number even slightly increased (Figs. 1 and 2).
Hence, effects of the midgut fluid appeared to be selective towards soil bacteria. However, we did not find any
correspondence between the effects observed and the
taxonomic affiliation of the tested strains. The same species can be sensitive or resistant (see, for example, Bacillus megaterium 413-1, 401-1, 413-2 and Pseudomonas
reactans 387-2, 383-1).
Heating of the midgut fluid of E. fetida at 98 C did
not eliminate its suppressive activity for Flavobacterium
sp. and Promicromonospora sp. although the effects
were less pronounced (Table 5).
The hindgut fluid showed no suppressive activity;
moreover, some bacterial strains produced more colonies under the influence of the fluid (Table 6).
3.4. Effects of the gut fluid on the viability
and germination of fungal spores and fungal hyphae
The midgut fluid of A. caliginosa suppressed
growth of some fungal species. The percentage of
B.A. Byzov et al. / European Journal of Soil Biology 43 (2007) S149eS156
S154
Table 6
The effects of hindgut fluid of the earthworm Aporrectodea caliginosa
on the numbers (CFU 103/ml1) of soil bacteria
> x 105
Bacteria
Control
No effects
Alcaligenes sp. 345-1
Alcaligenes sp. 382-1
Arthrobacter globiformis 333-1
Arthrobacter sp. 392-1
Bordetella sp. 341-1
Delftia acidovorans 335-1
Kluyvera ascorbata 303-1
Pseudomonas sp. 309-2
1500
1500
468
156
175
268
146
98
1480
1470
451
162
140
248
158
106
1
Growth stimulated
Pseudomonas putida 348-1
Pseudomonas sp. 329-1
Pseudomonas sp. 387-1
550*
560*
60*
1340*
1500*
111*
0
* Significant differences, p < 0.01.
4
3
Bacteria
Fig. 2. The effects of the midgut fluid of the earthworm Aporrectodea caliginosa on CFU numbers of soil bacteria under the incubation
for 2 h (macrocolonies counted on Petri dishes). n ¼ 3e5.
germinating spores decreased 1.5e10 times. Other
species of fungi were not suppressed considerably
and the germination of A. alternata was slightly increased (Table 7).
Growth of fragments of hyphae of T. harzianum
1E211 and P. decumbens 3S211 was suppressed within
1e2 min of incubation in the midgut fluid of A. caliginosa. The fragments of hyphae of B. adusta 7G521 survived under incubation in the midgut fluid for 24 h, but
the radial growth rate of its colonies decreased (Fig. 3).
Table 5
The effects of native and pre-heated midgut fluids of the earthworm
Eisenia fetida on the numbers (CFU 1010 ml1) of soil bacteria
Flavobacterium sp. and Promicromonospora sp.
Treatment
Flavobacterium sp.
Promicromonospora sp.
Water (control)
Native midgut fluid
Pre-heated midgut
fluid
10 3
0.3 0.1*
1.0 0.5*
10.5 6
0.35 0.2*
1.2 0.4*
All data are means of 3e5 replicates; standard deviation.
* Significant differences comparing to soil, p < 0.01.
Hindgut fluid
B12
Arthrobacter sp. 430-2
Pseudomonas sp. 310-1
D8
Arthrobacter oxydans 304-1
B11
Pseudomonas sp. 309-2
D12
Pseudomonas putida 428-1
C12
Bacillus mojavensis 317-1
A9
Pseudomonas putida 304-1
2
Pseudomonas putida 429-2
CFU x 104 ml-1
5
4. Discussion
The results of gut passage generally agree with the
commonly held view that the numbers of soil bacteria
do not change or even increase and those of fungi
decrease from food to excrement of earthworms
[12,6,16]. However, we have demonstrated that composition of microbial (fungal) species changed under passage through the gut of earthworms. There are species
with similar or higher abundance in the fresh excrements
than soil, but most fungal species have much lower abundances in the excrements. Therefore, the earthworm gut
Table 7
The effects of midgut fluid of the earthworm Aporrectodea caliginosa
on the germination of fungal spores (1e2 min of incubation in the
midgut fluid)
Strain
Germination (%)
Control
Midgut fluid
Germination suppressed
Paecilomyces lilacinus 2S513
Aspergillus terreus 3S422
A. niger 3C132
Penicillium aurantiogriseum 5S623
78.7 4.1*
53.0 4.8
75.1 5.0
58.9 5.2
4.0 1.3
7.0 2.2
7.5 1.5
39.2 3.0
No strong suppression
Gliocladium catenulatum 2S723
G. roseum 2S522
Aspergillus versicolor 1G213
Cladosporium cladosporioides 2S41
Trichoderma harzianum 1E211
Penicillium chrysogenum 4E232
P. melenii 4S523
64.6 2.8
75.0 3.3
83.1 3.3
81.2 4.0
87.0 8.0
69.3 2.5
92.1 4.7
58.1 2.6
62.5 2.0
64.7 3.6
74.0 6.0
82.2 3.3
71.0 5.0
84.6 6.6
Germination stimulated
Alternaria alternata 1C321
58.8 4.0
75.0 2.1
All data are means of 3e5 replicates; standard deviation.
* All data are significantly different, p < 0.01.
B.A. Byzov et al. / European Journal of Soil Biology 43 (2007) S149eS156
0.2
Radial growth rate, mm hour-1
0.18
0.16
0.14
0.12
0.1
0.08
0.06
0.04
0.02
0
no growth
Control
no growth
1-2 minutes
no growth
2 hours
24hours
Fig. 3. The effects of the midgut fluid of the earthworm Aporrectodea caliginosa on the radial growth rate of micromycetes. n ¼ 3e5.
, Penicillium decumbens 3S211;
Trichoderma harzianum
1E211;
Bjerkandera adusta 7G521.
environment may act as a selective ‘‘filter’’ for soil
microorganisms.
Our work shows for the first time selective effects of
the gut fluid taken from the anterior part of gut of the
earthworm A. caliginosa to the survival of some bacteria and fungi. We have demonstrated different responses
of soil bacteria and fungi on the action of the gut fluid.
The sensitive bacteria and fungi responded rapidly
(within a few minutes of incubation) to the action of
the gut fluid, their CFU numbers decreasing many
times. Germination of spores and radial growth rate
were suppressed. Interestingly, in most cases among
the sensitive microbial populations there was a small
portion of cells that were found to be resistant to the
incubation (2 and 24 h) in the gut fluid. It might indicate
that cells have some resistance mechanism to the suppressive action of the gut fluid.
The suppressive activity of the gut fluid was not eliminated (although the activity was lower) when the fluid
was pre-heated at 98 C suggesting that a non-protein
compound (s) was involved in the suppressive action.
We speculate that the observed suppressive activity is
an important factor enabling the digestion of soil microorganisms by the earthworms. Some groups of species
were found to be resistant to the gut fluid. Some of those
species that survive can even proliferate more rapidly
S155
after incubation in the gut fluid. The hindgut showed
no suppressive activity towards microorganisms tested.
These lines of evidence collectively indicate that the
earthworm specific gut environment may act as specific
‘‘filter’’ as well as a ‘‘fermenter’’ for some soil bacteria
and fungi with those bacterial cells that survive passage
through the midgut can even multiply in the hindgut.
Spores of some fungi that survived in the midgut environment and started to germinate could further actively
grow in fresh excrement. It could be due to the possible
stimulating effect of the midgut fluid on the spores.
The observed selectivity of these digestive processes
remains unclear. We could not find any correspondence
of the responses of soil bacteria and fungi to the action
of the midgut fluid with taxonomic affiliation of the species tested: both sensitive and resistant populations
could be found in one genus and even within species
of bacteria or fungi (Figs. 1 and 2, Table 7). Hence, different responses of the cells to the action of the midgut
fluid could not be due to the different composition of
microbial cell walls (gram-negative and gram-positive
bacteria, ascomycetous and basidiomycetous fungi).
Such mosaic responses of microorganisms could be
explained by the membrane-tropic mechanism of the
suppressive action of the midgut fluid as previously
demonstrated for the fluid of millipedes [2,3].
Suppressive action of the gut environment of earthworms has been earlier demonstrated for fungal spores
[11] and soil ciliates [13]. It was shown that the midgut
fluid had the suppressive ability rather than the mechanical (abrasive) action of the digestive tract. Here we
present evidence that midgut fluid of earthworms also
possesses a selective suppressive activity against soil
microorganisms. To establish existence of species affiliation of the suppressive effect of gut fluid is necessary
to test several strains of the same species. In conclusion,
this action of gut fluid can be regarded as a key mechanism playing an important role in feeding of earthworms. In addition, the different fates of microbial
cells passing the digestive tract of earthworms are an
important factor in the formation of the soil microbial
community.
Acknowledgements
We are grateful to Dr. Peter N. Golyshin and Taras
Yu. Nechitajlo for the identification of the bacterial
strains. We wish to thank Professor Jonathan M. Anderson, Dr. Alexei V. Uvarov and Dr. Vu Nguyen Thanh
for editing the manuscript. This work was supported
by the grants from the Russian Foundation for Basic
Researches (projects no. 05-04-48676 and 06-04-48557).
S156
B.A. Byzov et al. / European Journal of Soil Biology 43 (2007) S149eS156
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