Ecotoxicology and Environmental Safety 183 (2019) 109538

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Ecotoxicology and Environmental Safety

journal homepage: www.elsevier.com/locate/ecoenv

Species-specific effects of arsenic on the soil collembolan gut microbiota T

a,b a,b b,c a,*
Yi-Fei Wang , Min Qiao , Hong-Tao Wang , Dong Zhu
a
State Key Laboratory of Urban and Regional Ecology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing, 100085, China
b
University of Chinese Academy of Sciences, 19A Yuquan Road, Beijing, 100049, China
c
Key Laboratory of Urban Environment and Health, Institute of Urban Environment, Chinese Academy of Sciences, 1799 Jimei Road, Xiamen, 361021, China

A R T I C LE I N FO A B S T R A C T

Keywords: It is well established that arsenic (As) pollution has a severe threat to food security and soil non-target organisms,
Gut microbiota however, its influences on soil fauna gut microbiota are poorly understood. The gut microbiota of soil fauna play
Arsenic an important role in host health and nutrient cycling. Here, we used dietary exposure to investigate the effects of
Species-specific As on the mortality and gut microbiota of two model soil collembolans (Folsomia candida and Onychiurus yodai)
Microarthropods
and determine the accumulation of As in collembolan body tissues. The results showed that, although As ex-
Indicator bacteria
posure did not induce the mortality of the two species, dose dependence of As accumulation was indeed detected
in their body tissues. Oral As exposure (500 μg g−1 yeast) significantly altered the community structure
(P < 0.05) of F. candida gut microbiota and reduced its diversity (by more than 20%; P < 0.05) compared to
the control; however, no significant effects were observed in O. yodai gut microbiota. The two collembolan
species possess significantly different gut microbiota (P < 0.05), which may partly explain the differences of the
two collembolan gut microbiota response to As exposure. We further found that the genera Ochrobactrum,
Geobacter and Staphylococcus were sensitive to As exposure in F. candida (P < 0.05), but these bacteria were low
abundance and not altered in O. yodai. Moreover, the relative abundance of these bacteria was significantly
correlated with As bioaccumulation in F. candida body tissues (P < 0.05, R2 > 0.6). Higher As bioaccumulation
factor was also found in O. yodai body tissues compared to the F. candida. These results indicate that collembolan
gut microbiota present a species-specific response to As and may be a more sensitive indicator than the mortality
of collembolan.

1. Introduction microbiota is dynamically changing and susceptible to the host's en-

Collembolans are one of the most abundant microarthropods in the
soil ecosystem and have major influences on the processes of litter
decomposition and nutrient cycling (Bardgett et al., 1993; Hopkin,
1997; Czarnetzki and Tebbe, 2004). They are widely distributed from
the Arctic to tropical rain forests and are easily found in the soil in
numbers up to 100, 000 individuals m−2 (Rusek, 1998). They are
omnivores with a wide range of diets, such as bacteria, protozoa, plant
litter, soil detritus, and animal feces (Joosse and Buker, 1979; Ponge,
1991; Bardgett et al., 1993; Rusek, 1998). They harbour a large number
of symbiotic microbes in their gut lumen (Agamennone et al., 2015;
Bahrndorff et al., 2018; Zhu et al., 2018b, 2018c). Increasing evidences
demonstrate that gut microbiota are not just a component of the host
organisms, they regulate important biological processes such as food
digestion (Brune, 2014), essential vitamin supply (Dillon and Dillon,
2004), and immune response (Engel and Moran, 2013). In turn, the gut
vironment and diet. The soil ecosystem is suffering from effects of
various contaminants. Soil animals may be exposed to a variety of toxic
substances during food intake, but the knowledge on effects of con-
taminants on gut microbiota of non-target soil fauna is currently very
limited. To date, only two studies have described the effects of anti-
biotics (Zhu et al., 2018b) and microplastics (Zhu et al., 2018c) on
collembolan gut microbiota. Therefore, research on the influence of
environmental pollutants on collembolan gut microbiota should be
strengthened.
Arsenic (As) is a major toxic pollutant in the soil ecosystem. It is
commonly found in the geosphere and has greatly increased in en-
vironment because of anthropogenic activities (Zhu et al., 2014; Shi
et al., 2017), especially mining (Williams et al., 2009; van Coller-
Myburgh et al., 2015). Arsenic has different valence forms in the en-
vironment. In the oxic environment, As (V) is dominant, whereas As
(III) is major in the anoxic environment (Dixit and Hering, 2003; Wang

*
Corresponding author. Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, 18 Shuangqing Road, Haidian District, Beijing, 100085,
China.
E-mail address: dongzhu@rcees.ac.cn (D. Zhu).

https://doi.org/10.1016/j.ecoenv.2019.109538
Received 13 January 2019; Received in revised form 27 July 2019; Accepted 1 August 2019
0147-6513/ © 2019 Elsevier Inc. All rights reserved.
Y.-F. Wang, et al.
et al., 2014). Previous studies have demonstrated that As (III) is more
mobile and toxic than As (V), and the redox is mainly driven by mi-
croorganisms (Bhattacharya et al., 2007; Zhang et al., 2017). A large
amount of As compounds are used worldwide, up to 82,000 metric tons
per year in the past (Bhattacharya et al., 2007). Arsenic has been used
in industry (Han et al., 2003), agriculture, livestock, and other fields,
dispersing to soils, waters and air and causing global environmental
pollution. Previous studies have revealed a wide range of As con-
centrations in soils ranging from the μg kg−1 level to 250,000 mg kg−1
(Mahimairaja et al., 2005; Mikutta et al., 2014). The adverse effects of
As are not only detected in carcinogenesis for mammals but in soil in-
vertebrates, such as earthworms and collembolans (Crouau and Pinelli,
2008; Langdon et al., 2010). However, the effect of As on collembolan
gut microbiota has not been investigated.
Various studies have indicated that the response of organisms to the
same contaminant exposure commonly exists a difference, because of
unique life cycle or body structure. For example, in chronic tests, col-
lembolans Folsomia candida showed higher sensitivity to Hg (II) than
Proisotoma minuta (Buch et al., 2016). The reproductive sensitivity to
cadmium (Cd) of Folsomia candida is higher than that of Onychiurus
yodai (Nakamori et al., 2008) and Hypoaspis aculeifer (Zhu et al.,
2016b). It has also been recognized that individual species of organisms
have their own distinct gut microbiota, possibly due to the differences
in diet and habitat (Li et al., 2014a, 2017; Franzini et al., 2016).
Therefore, pollutants may have diverse influences on gut microbiota of
varying species, which requires further investigation.
The F. candida (Fountain and Hopkin, 2005) and O. yodai (Kang
et al., 2001; Greenslade and Vaughan, 2003; Ke et al., 2004) have been
used in many studies to evaluate the ecotoxicity of pollutants due to
their high abundance in soil, which are relatively easy to breed in la-
boratory. These two species of collembolans are non-pigmented and
eyeless but possess different living behavior. O. yodai is a euedaphic
species (soil-dwelling) and has no furca (Nakamori et al., 2008),
whereas F. candida is a hemiedaphic species (Schrader et al., 1997). The
hemiedaphic species are more active than euedaphic species, living in
the soil as well as on the surface, which lead to dissimilarities of diet
and habitat between the two collembolan species. Therefore, we hy-
pothesized that the two species collembolans have a distinct gut mi-
crobiota, and As resistance of the gut microbiota of two species may be
different. The aims of this study were to 1) characterize the effects of
oral As exposure on mortality and bioaccumulation in collembolans, 2)
compare the differences in gut microbiota between two collembolan
species, 3) investigate the response of gut microbiota in different col-
lembolan species to As exposure using 16s rRNA gene high-throughput
sequencing. These results will help us to understand the contribution of
gut microbiota to toxicant resistance in soil fauna.
2. Materials and methods
2.1. Collembolans and As exposure
The F. candida and O. yodai used in the present study were originally
obtained from Aarhus University in Denmark and cultivated land in
Jiangsu Province, east China, respectively. They have been cultured in
our laboratory for over 2 years. Stock cultures of collembolans were
kept in Petri dishes that contain charcoal/plaster of Paris (ratio 1:8 w/
w) mixture, at 20 ± 2 °C and 75% relative humidity with a 16 h:8 h
dark: light, (800 lux) light regime. Dried baker's yeast (Angel Yeast Co.,
Ltd China) and ultrapure water were replenished twice a week.
According to the standardized methods of the Organization for
Economic Co-operation and Development (OECD) (Zhu et al., 2016a),
7-9-day-old age-synchronized collembolan juveniles were obtained to
start the test.
Yeast (C content: 47.8% ± 0.35%, N content: 7.36% ± 0.16%, As
concentration: 0.9 ± 0.1 μg g−1) was selected as food in this study. As-
spiked foods were obtained by mixing yeasts with sodium arsenate
2
Ecotoxicology and Environmental Safety 183 (2019) 109538
(AsNa3O4·12H2O, purity 99%, CAS 13510-46-8) in ultrapure water. No
As-spiked yeasts as control were also dissolved in the same volume of
ultrapure water. The mixtures were freeze-dried, ground, and stored at
−20 °C until use.
2.2. Experimental design
The test of oral As exposure for the two collembolans species was
conducted in the Petri dishes (9 cm diameter). The test contained three
As treatments (0 μg g−1 as Control, 50 μg g−1 and 500 μg g−1), ac-
cording to the known environmental levels detected in farmlands and
other extreme sites such as ore fields and animal excreta (Hwangbo
et al., 2010; Li et al., 2014b; Gupta et al., 2018). Each treatment in-
cluded four replications. Sixty synchronized collembolans were placed
in each Petri dish and the culture condition was the same as mentioned
above. As-spiked yeasts (15 mg) and ultrapure water were provided
twice a week, and moldy food was removed. After 14-days exposure, all
collembolans were counted to identify mortality in each Petri dish, then
they were collected to determine As concentration of body tissues and
to extract DNA from the gut.
2.3. Determination of As concentration
Two milliliters of HNO3: H2O2: HF mixture (4:2:1, v/v/v) were
added to polytetrafluoroethylene tubes containing about 15 mg of
yeast. Before sealing, the tubes were kept for 2 h at room temperature.
Then, the sealed tubes were placed in a steel vessel and digested 6 h at
105 °C in a drying oven.
To determine the As bioaccumulation in collembolan body tissues,
the method of Zhu et al. (2017) was used. In brief, we digested the
collembolans for 8 h at 100 °C with 1 ml ultrapure nitric acid (Merck
Millipore, 65%, Darmstadt, Germany) on a heating block. The digests
were diluted 50-fold with ultrapure water, and the As concentration
was measured by ICP-MS (Agilent 7500cx, Agilent, Santa Clara, CA).
Recovery rates of the certified reference material GBW10050(GSB-28)
(King Prawns) for As were 93–108% in this study.
2.4. DNA extraction and amplicon sequencing
Chloroform was used to kill collembolans quickly to restrain in-
gestion and excretion of the gut contents. To degrade the surface mi-
crobiota, the dead collembolans were immediately dipped into 2% so-
dium hypochlorite (NaOCl) solution for 10s and rinsed with sterile
water five times to minimize the negative influence on gut microbiota.
After pre-treatment, 15 collembolans were dissected using aseptic for-
ceps under sterile conditions.
DNA was extracted from dissected gut using the DNeasy Blood &
Tissue Kit (QIAGEN, Germany) following the manufacturer's instruc-
tions. 1.0% agarose gel electrophoresis and spectrophotometric analysis
(Nanodrop ND-1000, Thermo Fisher) were performed to check the
isolated DNA quality and concentration. The extracted DNA was stored
at −20 °C until amplification.
The primer 515F: GTGCCAGCMGCCGCGG and 806R labeled with
unique barcodes: GGACTACNVGGGTWTCTAA that target the V4 hy-
pervariable region of the bacterial 16S rRNA gene were used to amplify
the gut DNA samples. The cycling conditions were as follows: 5 min at
95 °C followed by 28 cycles of 30 s at 95 °C, 30 s at 58 °C, and 30 s at
72 °C. The purified PCR products were obtained as described previously
(Caporaso et al., 2010a; Chen et al., 2016), and we used the NanoDrop
spectrophotometer to determine their concentration. The high-
throughput sequencing of the library built by 24 barcoded samples of
equal quality was operated on the Illumina MiSeq 300 platform (Meiji
biological medicine co., LTD, Shanghai, China).
Y.-F. Wang, et al.
2.5. Bioinformatic analysis
To analyze the high-throughput sequencing data, we followed on-
line instructions for Quantitative Insights Into Microbial Ecology
(QIIME, version 1.8.0) (Caporaso et al., 2010b). Briefly, the paired-end
reads were merged after the adaptor, and primer sequences, ambiguous
nucleotides and low-quality reads were removed. With the open-re-
ference method and uclust, operational taxonomic units (OTUs) were
gathered using 97% similarity. OTUs with only one sequence (single-
tons) were removed before downstream analysis. Representative OTUs
were aligned with PyNAST, and taxonomy was assigned with the Ri-
bosomal Database Project (RDP) classifier based on the Greengenes
13.8 16S rRNA gene database (Caporaso et al., 2010a; McDonald et al.,
2012). The sampling-based OTUs analysis was used to determine the
alpha diversity within different samples, which was indicated by
Shannon, Chao 1 and PD Whole tree indices. To identify the microbial
community structure of each sample, principal coordinates analysis
(PCoA) and HCLUST were calculated based on the Bray-Curtis distance
matrix. The Adonis test was used to examine the difference of microbial
community between different samples. The indicator species were de-
termined to reveal specific taxa in different treatment groups.
2.6. Statistical analysis
Data are expressed as mean ± standard deviation (SD). When the
data met the normal distribution and variance homogeneity tests, the
difference between samples was analyzed using one-way ANOVA, LSD
and t-tests using IBM SPSS version 22. And if the test did not meet the
test, the nonparametric Kruskal–Wallis test was used (Sizmur et al.,
2011; Adamowicz et al., 2018). A probability level of < 0.05 was used
as level for statistical significance. The Adonis test based on Bray−-
Curtis distances was used to identify significant differences in the col-
lembolan gut OTU composition between different treatments and was
performed using R version 3.5.0 with vegan 2.5-2 (Dixon, 2003). Be-
sides, the indicator species and the Shannon index of the gut microbiota
were calculated simultaneously (Dixon, 2003). The bubble chart was
conducted in Origin (2017), showing the indicator values and relative
abundances of the indicator bacteria in two collembolan species.
2.7. Accession numbers
The Sequence Read Archive (SRA) accession number for the se-
quences reported in this paper is PRJNA510244.
3. Results
3.1. Collembolan mortality and As bioaccumulation
In the control groups, the mortality of two collembolan species were
less than 20%, which met validity criteria of the OECD guideline (Fig.
S1). No significant difference in the mortality of the two collembolan
species was observed between all treatments (Fig. S1). With As con-
centrations increasing, As accumulated in body tissues increased sig-
nificantly in both species (Kruskal-Wallis test, all P < 0.05; Compared
with the control, O. yodai: 11 times in the 50 μg g−1 treatment and 88
times in the 500 μg g−1 treatment; F. candida: 15 times in the 50 μg g−1
treatment and 74 times in the 500 μg g−1 treatment). At exposure level
of 500 μg g−1 As, the As bioaccumulation in O. yodai body tissues was
significantly higher than that in F. candida (Fig. 1, t-test, P < 0.05), up
to 1.74 times. Higher As bioaccumulation factor (BF) was also found in
O. yodai body tissues (BF: 0.67 in the 50 μg g−1 treatment and 0.43 in
the 500 μg g−1 treatment) compared to the F. candida (BF: 0.38 in the
50 μg g−1 treatment and 0.18 in the 500 μg g−1 treatment).
3
Ecotoxicology and Environmental Safety 183 (2019) 109538
Fig.1. Bioaccumulation of As in collembolan bodies (μg g−1) after 28 days of As
exposure. Data are presented as mean ± standard deviation (n = 4). The two
asterisks indicate that there is a significant difference between the treatments at
0.01 level (t-test).
3.2. Collembolans gut microbiota composition
A total of 795,389 high-quality reads with an average of 33,142
reads per sample were obtained from the gut of the two species. The
sequences dataset was saturated for further analysis revealed by the
Shannon index curve (Fig. S2). A total of 1057 and 473 OTUs were
clustered at 97% similarity level in F. candida and O. yodai. At the
phylum level, an obvious bacterial composition difference was observed
between gut microbiota of the two species (Fig. 2a). The F. candida gut
microbiota was mostly composed of Proteobacteria (72.97%), Firmi-
cutes (12.09%) and Bacteroidetes (0.09%). The Tenericutes, Proteo-
bacteria and Bacteroidetes, together occupying an average of up to 95%
reads, were dominant in O. yodai gut microbiota with a relative abun-
dance of 85.54%, 0.09% and 0.05%, respectively. Compared with the
O. yodai, F. candida had significantly higher proportions of Proteo-
bacteria and Firmicutes by 6.77 and 62.6 times respectively
(P < 0.05), while Tenericutes was strikingly lower by 85.5%
(P < 0.05). At genus level, the Bacillus, Lutispora, and Sedimentibacter,
belonging to the phylum Firmicutes, and Ochrobactrum, Wolbachia,
Dechloromonas, Geobacter, and Thermomonas belonging to Proteo-
bacteria were significantly enriched in F. candida versus O. yodai
(Fig. 2b, P < 0.05). Correspondingly, phylum Tenericutes with large
number of unclassified genera significantly enriched in O. yodai than F.
candida (Fig. 2b, P < 0.05), at least 2.78 times.
In the Venn diagram, the numbers of OTUs were presented by the
total numbers of detected OTUs in the same treatment (Fig. S3). For
both species, 80 OTUs were shared between gut microbiota of control
groups (Fig. S3a). In all control and As exposure groups, 408 (Fig. S3b)
and 271 (Fig. S3c) shared OTUs were observed for F. candida and O.
yodai, respectively. The number of shared OTUs for the two species in
groups exposed to 50 μg g−1 As (Fig. S3d, 146) was higher than that in
the control (80) by about a 0.8-fold enhancement and increased by
0.47-fold compared to that in 500 μg g−1 As exposure treatment (Fig.
S3d, 99). The segregation of the two species based on their gut mi-
crobiota was presented by PCoA (Fig. 3a, Adonis test, P < 0.05).
3.3. Altered gut bacterial composition under As-exposure
There was no significant difference in the proportions of dominant
microbiota at phylum level in gut microbiota of both species after As
exposure. For F. candida, the Bacillus significantly increased by 1.24
Y.-F. Wang, et al.
Fig.2. The average relative abundance of gut microbiota for the two species of collembolans (a) at phylum level and (b) at genus level. OTUs with average relative
abundance lower than 1% are categorized into “Other” (mean, n = 4). The asterisk represents a significant difference between F. candida and O. yodai (t -test). One
asterisk denotes P < 0.05 and two asterisks denote P < 0.01.
times after exposure to 50 μg g−1 As (Fig. 4, P < 0.05) compared with
the control group. Moreover, exposure to 500 μg g−1 As significantly
decreased the abundance of Geobacter, Staphylococcus, Symbiobacterium
and Thermomonas, by 0.70, 0.97, 0.80 and 0.71 times, respectively
(P < 0.05). In contrast, the abundance of Ochrobactrum significantly
increased by 5.44-fold enhancement after 500 μg g−1 As exposure
(P < 0.05). For O. yodai, exposure to As did not cause significant dif-
ference in microbial abundance at genus level.
3.4. Altered alpha and beta diversity of gut microbial community under As
exposure
The OTU, Chao 1, Shannon, and PD whole tree indices of F. candida
gut microbiota were significantly lower in the 500 μg g−1 As treatment
group by decreasing 0.28, 0.29, 0.20 and 0.30 times respectively
(Table 1, P < 0.05). However, these indices were not significantly
difference between the control and 500 μg g−1 As treatment groups in
4
Ecotoxicology and Environmental Safety 183 (2019) 109538
O. yodai. Moreover, PCoA based on Bray-Curtis distance also showed a
distinct separation of gut microbiota between 500 μg g−1 As exposure
and other groups for F. candida (Fig. 3b, P < 0.05). In contrast, the As-
exposure level did not alter the gut microbial community structure of O.
yodai (Fig. 3c). The HCLUST also showed the gut samples of the O. yodai
were clustered together (Fig. 3d).
3.5. Altered indicator species of gut microbial community under As
exposure
The Geobacter, Wolbachia and Staphylococcus were three dominant
genus in the F. candida gut microbiota of control treatment (Fig. 5a).
Exposure to 500 μg g−1 As altered the indicator species in the F. candida
gut microbiota, from Wolbachia to Ochrobactrum (IndVal = 0.801). As
bioaccumulation in collembolan tissues was significantly negatively
correlated with the relative abundance of genus Geobacter
(R = −0.601, P < 0.05) and Staphylococcus (R = −0.902, P < 0.05),
Y.-F. Wang, et al. Ecotoxicology and Environmental Safety 183 (2019) 109538

Fig. 3. (a) Principal coordinate analysis (PCoA) plots based on Bray-Curtis distances comparing gut microbiota composition in the two species of collembolans. The
numbers in parentheses indicate the variation between the samples that can be explained by the first two axes. Collembolan species are shown by shapes, and colors
represent As treatments. (b) PCoA plots based on Bray-Curtis distances comparing sample gut microbial composition in different As treatments for F. candida. (c)
PCoA plots based on Bray-Curtis distances comparing sample gut microbial composition in different As treatments for O. yodai. (d) HCLUST based on Bray-Curtis
distances showing the difference between samples.

Table 1
OTU number and Alpha diversity of gut microbiota for the two collembolan
species.
Control 50 μg g−1 As 500 μg g−1 As

F. candida OTU 1057 1058 761

Chao1 3530.64 3409.79 2505.07***
Shannon 5.80 6.06 4.62***
PD whole tree 92.32 94.14 64.24***
O. yodai OTU 473 616 497
Chao1 1530.66 2144.95*** 1789.49***
Shannon 2.21 2.70*** 2.20
PD whole tree 30.41 46.25*** 33.50

The asterisk represents significant differences between the As-exposed and

control groups. One asterisk denotes P < 0.05, two asterisks denote P < 0.01
and three denote P < 0.001.

whereas positively correlated with that of genus Ochrobactrum

Fig. 4. Species-specific effects of As-exposure on the gut microbiota, as illu-
(R = 0.783, P < 0.05). However, indicator bacteria of O.yodai were
strated by the heatmap constructed with the min-max normalized relative
abundance of genera in two collembolan species (mean, n = 4). Genera not altered by As exposure. Tenericutes with a large number of un-
with > 1% abundance are presented in the heatmap. The asterisk represents classified genera was the dominant phylum in O. yodai.
significant differences between the As-exposed and control groups. One asterisk
denotes P < 0.05 and two asterisks denote P < 0.01.
4. Discussion

In this study, we found that As exposure does not induce mortality

in collembolans. Proteobacteria and Tenericutes were the dominant

5
Y.-F. Wang, et al.
phylum in the F. candida and O. yodai gut microbiota communities,
respectively. As we hypothesized, the two species collembolans have a
distinct gut microbiota. Moreover, As exposure altered structure of gut
microbiota and reduced its diversity in F. candida, but had no effect in
O. yodai. These results suggest that collembolan gut microbiota may be
a more sensitive indicator for As than mortality and this response is
species-specific.
4.1. Collembolan mortality and As bioaccumulation
To our knowledge this is the first study reporting effects of oral As
exposure on mortality in collembolans, and our results showed that the
oral As exposure (50 and 500 μg g−1) does not induce mortality in two
collembolans species. A study conducted in artificial OECD soils re-
vealed that EC50 for F. candida growth was 21.7 μg g−1 dry soil for As
(Crouau and Moia, 2006). However, Liu et al. (2018) showed that the
growth was more sensitive than the mortality for F. candida when
6
Ecotoxicology and Environmental Safety 183 (2019) 109538
Fig.5. (a) A bubble chart revealing the indicator
species in the gut microbiota of two collembolan
species. The indicator species of F. candida and O.
yodai gut microbiota are displayed at genera and
order level, respectively. The indicator value of spe-
cies is presented by circle color, and the circle size
represents the average relative abundance of in-
dicator species (mean, n = 4). The indicator value of
species represents the strength of association be-
tween indicator species and the treatment, with
larger values indicating higher relationship. The as-
terisk represents significant differences between the
As-exposed and control group. One asterisk denotes
P < 0.05 and two asterisks denote P < 0.01. (b)
The correlation between the relative abundance of
As-sensitive bacteria and arsenic bioaccumulation in
collembolan bodies for F. candida.
exposed to field-collected polluted soil (Liu et al., 2018), which is
consistent with the result of mortality of our study. Moreover, Fountain
and Hopkin (2001) found that EC50 growth values were lower in metal-
contaminated soil compared with food contaminated with metals
(Fountain and Hopkin, 2001). Compared with other organisms i.e fish
Oryzias latipes (EC50 growth value 14.6 mg l−1) and shrimp Corophium.
orientale (EC50 growth value 3.51 mg l−1) (Suhendrayatna et al., 2002;
Gaion et al., 2013), collembolans are more resistant to As contamina-
tion because they are able to shed the midgut epithelium at each
moulting. Furthermore, Nota et al. (2008) demonstrated that the ABC
transporters are involved in metabolic detoxification of collembolans,
which can expel harmful substance from cells (Nota et al., 2008). In the
present study, higher As bioaccumulation (246 μg g−1) and bioaccu-
mulation factor (0.67) were observed in O. yodai than in F. candida
body tissues. A recent study showed that 97.8 μg g−1 As in body tissues
will lead to the mortality of earthworm (Wang et al., 2019), which also
suggested collembolans have a high resistance to As. Various researches
Y.-F. Wang, et al.
have presented that gut microbiota participate in host metabolism,
especially in detoxification of xenobiotics (Guo et al., 2014; Bhat et al.,
2018). This suggested that F. candida was more sensitive to As than O.
yodai, and its gut microbiota may cause it to metabolize more As re-
sulting in lower bioaccumulation.
4.2. Effects of As exposure on gut microbiota
We found that exposure to As (50 and 500 μg g−1) affected F. can-
dida gut microbiota. Gut microbiota is not only a component of host
organisms, but also regulates important biological processes, such as
food digestion, essential vitamin supply (Dillon and Dillon, 2004), and
immune response (Engel and Moran, 2013). Thus we further used the
indicator species in different treatment groups to identify the specific
taxon that may involve in As metabolism. The relative abundance of
Geobacter and Staphylococcus were inversely related to the accumula-
tion of As in collembolan bodies which was associated with the re-
duction of arsenate (As (V)). Islam et al. (2004) showed that the family
Geobacteraceae was the dominant microbiota in As-rich West Bengali
sediments, involving the significant release of arsenite (As (III)) (Islam
et al., 2004). Ji and Silver (1992) revealed that the arsC gene in Sta-
phylococcus can reduce intracellular As (V) to As (III), then use efflux
process to export As (III) (Ji and Silver, 1992). Therefore, in the present
study because of the greater toxicity of As (III) than As (V), F. candida
tend to decrease the relative abundance of As-reducing microbiota in
the gut to mitigate the toxic effects of As. In contrast, our results in-
dicate the relative abundance of Ochrobactrum was positively correlated
with the accumulation of As in collembolan tissues. A previous study
identified the Ochrobactrum capable of oxidizing As (III) to As (V) by the
enzyme arsenite oxidase and the cytochrome c was encoded in the
operon (Branco et al., 2009). We also found that the relative abundance
of Ochrobactrum increased in the gut of F. candida with increasing As
exposure concentrations. At the same time, we confirmed that several
bacteria in F. candida gut are related with As resistance, nevertheless
additional evidence is needed to determine their roles in the resistance
of collembolans to As contamination.
Exposure of As in O. yodai did not alter its gut microbial community.
Whereas, in our study, we found significant differences of alpha di-
versity of gut microbiota in F. candida with 500 μg g−1 As, while in O.
yodai with 50 μg g−1 As. This may be because the two collembolans
have different initial gut microbiota, which may lead to the response of
different collembolan gut microbiota to As exposure presenting differ-
ence. For the F. candida, 500 μg g−1 As exposure significantly decreased
the alpha diversity of its gut microbiota, which may be related to toxic
effects of As on F. candida. A notably higher alpha diversity in the O.
yodai was observed in 50 μg g−1 As treated samples compared to the
control, suggesting that As of low-dose can stimulate the diversity of
collembolan gut microbiome (hormesis). This has been confirmed in
recent studies involving effects of nanoplastics on soil enchytraeid mi-
crobiome (Zhu et al., 2018a) and impacts of combination of oxyte-
tracycline and saponin on alpha-diversity of Zebrafish Larvae micro-
biome (Nadal et al., 2018). The similar phenomenon was also found in
the luminescence of Photobacterium phosphorem and Vibrio qinghaiensis
when they were exposed to Cu (II), Zn (II), Cd (II) and Cr (VI)(Shen
et al., 2009). The strong resistance to As in O. yodai may be attributed
by the enrichment of Tenericutes in its gut, which is consistent with the
high abundance of Tenericutes in As contaminated soils (Luo et al.,
2014).
4.3. Compositional differences in gut microbiota between two collembolans
species
Different collembolans have formed a unique ecological niche in
their guts due to the difference of living modes during the long period
of evolution, which led to the difference of gut microbiota in the dif-
ferent collembolan species. F. candida has a furca and is a hemiedaphic
7
Ecotoxicology and Environmental Safety 183 (2019) 109538
species, which was more active than O. yodai (no furca and a euedaphic
species). Thus F. candida may have a wider range of feeding in the
natural soil ecosystem, which led to a more complex gut microbiota
compared to the O. yodai. Although both collembolans were subjected
to the same treatment in vitro, this difference of gut microbiota can be
inherited via the unique niche in their guts. A previous study had de-
monstrated that diet and taxonomy influence insect gut bacterial
communities, using meta-analysis. They found the gut microbiota of
lower wood-feeding termites (Rhinotermitidae) clustered separately
from higher wood-feeding termites (Termitidae), similar to our study of
collembolans (Colman et al., 2012). The indicator bacteria Staphylo-
coccus and Geobacter sharply decreased after As exposure in the gut of F.
candida (P < 0.05), but these bacteria were low abundance and not
altered in the gut of O. yodai. Moreover, the As-tolerant bacteria, Te-
nericutes (Luo et al., 2014), showed up to 90% relative abundance in
the O. yodai gut bacterial community, but was low abundance in F.
candida. Therefore, these bacteria may play important roles for the
resistance of As in collembolan guts.
5. Conclusion
Results of this study suggest oral As exposure (50 and 500 μg g−1)
can cause As bioaccumulation in body tissues, but no effects of mor-
tality on F. candida and O. yodai. We demonstrated that the two species
of collembolans presented a distinct different gut microbiota due to
their unique feeding behavior. Arsenic exposure altered the gut mi-
crobiota in F. candida but no in O. yodai. We further found that the
genera Ochrobactrum, Geobacter and Staphylococcus were sensitive to As
exposure in F. candida, but these bacteria were low abundance and not
changed in O. yodai. These results indicate that there is a species-de-
pendent gut microbiota shift in As-exposed collembolans, suggesting
that gut microbiota may contribute to As resistance of collembolans.
These findings expand our understanding on effects of As on soil non-
target organism, and will direct our attention to the role of soil fauna
gut microbiota in the soil ecotoxicology. Further studies could be ap-
plied to test why the response of intestinal microbes to contaminants is
species-specific and whether the same phenomenon will occur in other
organisms and contaminants.
Acknowledgment
This work was financially supported by National Natural Science
Foundation of China (No.21377154, 41571130063).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.ecoenv.2019.109538.
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