Effects of Arsenic on Gut Microbiota and Its Biotransformation Genes in

Subscriber access provided by UNIV OF TEXAS DALLAS

Ecotoxicology and Human Environmental Health

Effects of Arsenic on Gut Microbiota and Its Biotransformation Genes in Earthworm Metaphire sieboldi Hong-Tao Wang, Dong Zhu, Gang Li, Fei Zheng, Jing Ding, Patrick J O’Connor, Yong-Guan Zhu, and Xi-Mei Xue Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b06695 • Publication Date (Web): 15 Mar 2019 Downloaded from http://pubs.acs.org on March 16, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 33

Environmental Science & Technology

1

Effects of Arsenic on Gut Microbiota and Its Biotransformation

2

Genes in Earthworm Metaphire sieboldi

3

Hong-Tao Wang,a,b Dong Zhu,a,b Gang Li, a Fei Zheng,a,b Jing Ding,c Patrick J

4

O’Connor,d Yong-Guan Zhu,a,b,c and Xi-Mei Xue*,a

5

a

6

Chinese Academy of Sciences, 1799 Jimei Road, Xiamen 361021, China

7

b

8

China

9

c

Key Laboratory of Urban Environment and Health, Institute of Urban Environment,

University of Chinese Academy of Sciences, 19A Yuquan Road, Beijing 100049,

State Key Laboratory of Urban and Regional Ecology, Research Center for

10

Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China

11

d

12

Australia

Centre for Global Food and Resources, University of Adelaide, Adelaide 5005,

13 14

*Address Correspondence to Xi-Mei Xue,

15

Address: Institute of Urban Environment, Chinese Academy of Sciences, 1799 Jimei

16

Road, Xiamen 361021, China

17

Phone number: +86(0)592 6190559

18

Fax number: +86(0)592 6190977

19

Email address: [email protected]

20 21 22 23 1

ACS Paragon Plus Environment


Page 2 of 33

24 25

ABSTRACT:

26

Arsenic biotransformation mediated by gut microbiota can affect arsenic

27

bioavailability and microbial community. Arsenic species, arsenic biotransformation

28

genes (ABGs) and the composition of gut microbial community were characterized

29

after the earthworm Metaphire sieboldi was cultured in soils spiked with different

30

arsenic concentrations. Arsenite (As(III)) was the major component in the earthworm

31

gut, while arsenate (As(V)) was predominant in the soil. A total of 16 ABGs were

32

quantified by high-throughput quantitative polymerase chain reaction (HT-qPCR).

33

Genes involved in arsenic redox and efflux were predominant in all samples, and the

34

abundance of ABGs involved in arsenic methylation and demethylation in the gut was

35

very low. These results reveal that the earthworm gut can be a reservoir of microbes

36

with the capability of reducing As(V) and extruding As(III), but with little

37

methylation of arsenic. Moreover, gut microbial communities were dominated by

38

Actinobacteria, Firmicutes and Proteobacteria at the phylum level, and were

39

considerably different from those in the surrounding soil. Our work demonstrates that

40

exposure to As(V) disturbs the gut microbiota of earthworms and provides some

41

insights into arsenic biotransformation in the earthworm gut.

42 43 44 45 46 2


Page 3 of 33


47

INTRODUCTION

48

Arsenic is widely distributed in the soil and can be bio-accumulated through the food

49

web in soil, plants and animals.1-3 Arsenic bioaccumulation can result in toxicity for

50

soil biota and may ultimately affect human health. Most of previous studies

51

concentrated on the bio-concentration and toxicity of environmental arsenic in the

52

earthworm. Using a laboratory vermicomposting system, Fischer and Koszorus4

53

studied the sublethal effects, lethal concentrations, accumulation and elimination of

54

arsenic, selenium, and mercury in Eisenia foetida. The effect of edaphic factors (pH,

55

depth in soil profile, and soil organic matter content) on the toxicity and accumulation

56

of arsenate (As(V)) were investigated in the earthworm Lumbricus terrestris.5

57

Furthermore, a few laboratory and field surveys investigated diverse arsenic species in

58

earthworm body tissue.6,

59

Dendrodrilus rubidus collected from the arsenic mine and an uncontaminated soil was

60

arsenobetaine.8 In addition, earthworm activity may influence the species, mobility,

61

and partitioning of metals and metalloids in soil and pore waters by changing soil pH,

62

soluble organic carbon, or microbial communities.9 However, these studies mostly

63

focused on arsenic toxicity on earthworm and arsenic species in earthworm body

64

tissues or gut, with little attention to the mechanism of arsenic detoxification or

65

biotransformation in the earthworm gut.

7

The only organoarsenicals found in L. rubellus and

66

Many of the ecosystem processes attributed to soil fauna may in fact be mediated

67

by the microbiome of those fauna, which is an important part of soil microorganism

68

process and will exert influence on the host metabolism and health.10-12 For example, 3



Page 4 of 33

69

earthworm gut microbiota has been shown to be involved in organic matter

70

decomposition, denitrification, nutrient stabilization, and other biogeochemical

71

cycles.13-15 The earthworm gut is considered to be a transient habitat for the microbes

72

of aerated soils.14 During passage through the gut, the unique micro-environment of

73

the gut of different species of earthworms from different environments appears to

74

selectively stimulate a specific subset of ingested soil microbes.14, 15 This selection of

75

earthworm gut microbiota has been shown to be influenced in the presence of

76

environment contaminants such as triclosan,16 and arsenic.17 In addition, the use of the

77

earthworm as an bioindicator of arsenic contamination was assessed by analyzing the

78

effects of arsenic on earthworm toxicity.18 Only a recent study revealed that soil

79

arsenic contamination could altered microbiome of the earthworm.17 However,

80

comprehensive studies of the interactions between arsenic species and the gut

81

bacterial flora of earthworms have not been undertaken.

82

Microbe-mediated arsenic metabolism plays an important role in arsenic

83

biogeochemical cycle, different types of genes involving in arsenic metabolism

84

encode proteins regulating arsenic species and solubility in environment.19-21 For

85

example, arsenic redox genes encoding cytoplasmic As(V) reductase (ArsC),

86

respiratory As(V) reductase (ArrAB), and As(III) oxidase (AioAB) perform an impact

87

on the species transformation between As(V) and As(III),21 and then change arsenic

88

toxicity and bioavailability. Arsenic methylation or demethylation is catalyzed by an

89

As(III) S-adenosylmethionine methyltransferase (ArsM) or a nonheme iron-dependent

90

dioxygenase with C·As lyase activity (ArsI).22 Organisms can also mobilize arsenic 4


Page 5 of 33


91

via phosphate transporters, aquaglyceroporins, or As(III) efflux systems.23 Using

92

qPCR amplifications with five primer pairs, Zhang et al.24 showed that ABGs

93

involved in arsenic redox reactions and methylation were widely distributed in paddy

94

soils. Metagenomic analysis was applied to reveal the co-existence of different arsenic

95

resistance system of microbes in low-arsenic soil habitats.25 Arsenic species changes

96

in earthworm body tissues associated with the related genes of arsenic detoxification

97

and biotransformation in the earthworm gut which is an anaerobic environment, like

98

flooded paddy soil.15 However, there is still relatively little known about the

99

distribution, diversity and abundance of

arsenic biotransformation genes (ABGs) in

100

soil fauna, not to mention the relationship between arsenic species and ABGs in the

101

gut of soil fauna.

102

To understand the influence of arsenic contamination in soils on earthworm gut

103

microflora and the relationships between arsenic species and ABGs in the gut, this

104

study was designed to: 1) determine arsenic species in the earthworm gut and tissues

105

after exposure to arsenic contaminated soils; 2) characterize the diversity and

106

abundance of ABGs in the earthworm gut; 3) profile the microbial communities in the

107

soil and the earthworm gut; and 4) examine the difference in the diversity of gut

108

microbial communities after earthworms were exposed to different

109

concentrations.

110

MATERIALS AND METHODS

111

Soil and Earthworm Preparation. Soil samples, which were not contaminated with

112

arsenic, were collected in 2017 from disused farmland in Xiamen City, south-east

arsenic

5



Page 6 of 33

113

China. Detailed physical-chemical properties of the soil are described in Table S1 of

114

the Supporting Information (SI). The soil was ground in an agate mortar and sieved

115

through 2 mm nylon sieves after being separated from gravel particles and litter, and

116

then air-dried at room temperature. The stock solution of As(V) (448 mg L-1),

117

prepared by dissolving Na3AsO4·12H2O (Chemically pure, Chemical Reagent

118

Purchasing and Supply Station, Shanghai, China) in Milli-Q water (18.2 MΩ,

119

Millipore, UK), was mixed thoroughly with 900 g of the homogenized soil in

120

polyethylene plastic containers (25×15×12 cm) (Runpeng Plastic Co., Ltd., Jieyang,

121

Guangdong, China) to yield a final concentration of control (7), 70, 140 and 280 mg

122

As(V) kg−1 dry soil. The spiked soils had a water content of 30% and were activated

123

for 14 days under laboratory conditions (20 oC, 12 h light: 12 h dark cycle).

124

Adult earthworms (M. sieboldi) of the same age, with well-developed clitellum,

125

purchased from an earthworm company in Nanjing City, were acclimated for 14 days

126

in the untreated soil under laboratory conditions prior to the experiment. Oatmeal

127

(Nanguo Food Industry Co., Ltd., Haikou, Hainan, China) as food source was added

128

to the soil. Earthworm species was confirmed by sequencing the cytochrome oxidase I

129

(COI)

130

(5’-GGTCAACAAATCATAAAGATATTGG-3’)

131

(5’-TAAACTTCAGGGTGACCAAAAAATCA-3’)).17, 26, 27 The sequences obtained

132

were submitted to the National Center for Biotechnology Information (NCBI) via the

133

Basic Local Alignment Search Tool (BLAST) to identify the species of earthworm.

134

M. sieboldi was rinsed with Milli-Q water to remove adhering soils. Ten earthworms

barcode

gene

(primers: and

LCO-1490 HCO-2198

6


Page 7 of 33


135

with similar magnitude and wet mass were transferred to the spiked soils.

136

Arsenic-free oatmeal was mixed into the soil as a food source at the initial stage of the

137

experiment. The culture containers were covered with a lid, in which holes were

138

punched to maintain ventilation, and containers were maintained in an artificial

139

incubator (Saifu Laboratory Instruments Co., Ltd., Ningbo, Zhejiang, China) (20 oC,

140

70% relative humidity) with a 12:12 h light/dark cycle. The experiment consisted of a

141

control (no added As(V)) and three treatments with three replicates of each treatment,

142

giving a total of 12 containers. During the culture, dead earthworms were observed

143

and removed. The soil water content was maintained at 30% through providing

144

Milli-Q water at regular intervals.

145

DNA Extraction, High-Throughput Sequencing and Bioinformatic Analysis. The

146

earthworms, exposed to As(V) for 28 days according to US Environmental Protection

147

Agency (USEPA) (1988)28 and Organization for Economic Co-operation and

148

Development (OECD)

149

with absolute ethyl alcohol (Analytical pure, Sinopharm Chemical Reagent Co., Ltd,

150

Ningbo, Zhejiang, China). Worms were gently shaken and rinsed in sterilized water

151

five times to remove surface microbiota. The earthworm gut was obtained using

152

sterile forceps under sterile conditions.

(No.222) protocols,29 were collected and immediately killed

153

Earthworm gut and 0.5 g soil from each treatment (a total of 24 samples including

154

12 gut and 12 soil, i.e., control soil and gut (SC/GC); 70 mg As(V) kg-1 soil and gut

155

(S70/G70); 140 mg As(V) kg-1 soil and gut (S140/G140); 280 mg As(V) kg-1 soil and

156

gut (S280/G280)) were used to extract DNA using a FastDNA Spin Kit for soil (MP 7



Page 8 of 33

157

Biomedicals, Santa Ana, California, USA) according to the manufacturer's

158

instructions. In the end, 100 μL and 70 μL of DNA elution solution was used to

159

dissolve the soil and earthworm gut DNA, respectively. The extracted DNA was

160

stored at -20 oC after concentration and the quality of DNA were measured by

161

Nanodrop ND-1000 (Thermo Fisher, USA) and agarose gel electrophoresis.

162

The V4-V5 region of 16S rRNA was amplified and sequenced on the Illumina

163

platform (Majorbio, China) as previously described.30 Briefly, a 392-bp fragment of

164

16S

165

GTGCCAGCMGCCGCGGTAA-3’)

166

(5’-CCGTCAATCMTTTRAGTTT-3’), with R907 modified to contain a unique 8 nt

167

barcodes at the 5’ terminus to distinguish different source samples in the same pool.

168

The amplicons were purified and combined into one pool.

rRNA

was

PCR

amplified

using

the

and

the

forward reverse

primer

F515

primer

(5’R907

169

The Illumina sequencing data were analyzed using Quantitative Insights Into

170

Microbial Ecology (QIIME, version1.8.0) following the instructions at Getting Help

171

with QIIME.31 The default method was used to pick the operational taxonomic units

172

(OTUs) which were defined at the 97% similarity level by UCLUST clustering32 after

173

the raw reads were filtered, and representative sequences of the OUTs obtained. A

174

phylogenetic tree was constructed using the Fast tree algorithm33 based on the

175

multiple sequence alignment generated by a PyNAST prior to downstream analysis.34

176

Phylogenetic Distance (PD) Whole tree, Chao1 estimator, rarefaction curves and

177

Shannon entropy were used to describe alpha diversity for each sample. Principal

178

coordinate analysis (PCoA) based on Bray-Curtis distance was performed to evaluate 8


Page 9 of 33


179

the profiles of microbial communities in different treatments. The sequence has been

180

deposited in the NCBI Sequence Read Archive under the accession number

181

PRJNA516551.

182

HT-qPCR of ABGs. The abundance of ABGs in the gut and soil was estimated using

183

the Wafergen SmartChip Real-Time PCR System (WaferGen Biosystems, Fremont,

184

CA) as described in the study35 with minor modifications. A total of 80 primer pairs

185

(Table S2) was designed to target 19 ABGs and a 16S rRNA gene. The 100 nL

186

HT-qPCR reaction contained LightCycler 480 SYBR Green I Master, primers,

187

nuclease-free PCR-grade water, bovine serum albumin, and DNA template. The

188

thermal conditions were 95 oC denaturation for 10 min, 40 cycles of 95 oC for 30 sec

189

(denaturation), 58 oC for 30 sec (annealing), and a final 6-min extension step at 72

190

oC.47

191

(1), a threshold cycle (CT) of 31 was utilized as the detection limit. When three

192

replicates for each DNA sample were all above the detection limit, ABGs were

193

considered to be detected. Moreover, in order to minimize bias from background

194

bacterial abundances and DNA extraction efficiencies, ABG copy number was

195

normalized to the number of ABG copies per bacterial cell using the equation (2). The

196

bacterial cell numbers in one sample were estimated by dividing 16S rRNA gene

197

quantities by 4.1 based on the Ribosomal RNA Operon Copy Number Database.36

The gene copy number of ABGs or 16S rRNA was calculated using the equation

198

Relative Gene Copy Number = 10(31−CT)/ (10/3)

199

Normalized ABG Abundance = 4.1× (Relative ABG Copy Number/Relative 16S

200

rRNA Gene Copy Number)

(1)

(2) 9



Page 10 of 33

201

Determination of Total Arsenic Concentration and Arsenic Species. The

202

freeze-dried earthworm body tissues and gut contents were ground in an agate mortar

203

with liquid nitrogen to a fine powder prior to digestion and arsenic extraction. The

204

soil (200 mg, weighed to a precision of 0.1 mg), earthworm body tissues (30 mg,

205

weighed to a precision of 0.1 mg), or gut contents (30 mg, weighed to a precision of

206

0.1 mg) for the analysis of total arsenic concentration was precisely weighed into 50

207

mL polypropylene digestion tubes. A HNO3 (Merck Millipore, 65%, Darmstadt,

208

Germany):HF (Thermo Fisher Scientific, 49%, USA) mixture (5+1 v/v, 6 mL) for soil

209

or a HNO3:H2O2 (Merck Millipore, 30%, Darmstadt, Germany) mixture (2+1 v/v, 6

210

mL) for earthworm body tissues or gut contents was added to the samples and allowed

211

to stand at room temperature for 2 h before the tubes were covered with caps and

212

transferred to the microwave-accelerated system (CEM Microwave Technology Ltd.,

213

Buckingham, UK). The microwave-assisted digestion for earthworm body tissues or

214

gut contents in closed tubes was carried out as described previously.37 The

215

temperature ramping program in the microwave digested for soil samples was shown

216

as the following: 105 oC for 20 min, 180 oC for 10 min, 180 oC for 30 min. Upon

217

reaching room temperature, samples were diluted to 50 mL with Milli-Q water and

218

filtered through 0.45 μm syringe filters (PVDF, Millipore, USA). Arsenic

219

concentrations of soil, earthworm body tissues, and gut contents were determined by

220

ICP-MS (Agilent 7500 ce, Agilent technologies, USA) in collision cell mode to avoid

221

interference from argon chloride (40Ar35Cl) on arsenic (75As). The total arsenic

222

measurement was validated against the certified reference material GBW07403, 10


Page 11 of 33


223

GBW07406, and GBW10050 bought from the National Institute of Metrology of

224

China with a certified value for arsenic of 4.4 ± 0.6 mg kg-1, 220 ± 14 mg kg-1, and

225

2.5 mg kg-1 (reference value); we obtained 4.3 ± 0.4 mg kg-1, 216 ± 15 mg kg-1, and

226

2.4 ± 0.4 mg kg-1 (n = 4), respectively. The recovery rates of the CRMs ranged from

227

90.0% to 108.2%.

228

The following extractants for arsenic species were varied for each sample type. Soil

229

(200 mg, weighed to a precision of 0.1 mg) was extracted with 5 mL of 0.05 M

230

aqueous ammonium sulfate,38 and freeze-dried earthworm tissues or gut (30 mg,

231

weighed to a precision of 0.1 mg) was weighed into a polyethylene vessel with 5 mL

232

of MeOH (HPLC Grade, Thermo Fisher Scientific, USA): H2O mixture (1:1 v/v).7

233

Arsenic species were extracted on a rotary wheel at 150 rpm overnight. The mixture

234

was centrifuged (4754 g, 15 min) at 4 oC to separate the supernatant from the pellet.

235

Soil extract was filtered through a 0.22 µm filter and stored at -80 oC prior to analysis.

236

The extract containing methanol was evaporated to dryness under a nitrogen stream at

237

room temperature and re-dissolved in Milli-Q water. HPLC (Agilent 1200, Agilent

238

technologies, USA)-ICP-MS (Agilent 7700, Agilent technologies, USA) was used to

239

analyze arsenic species. The species analysis was carried out on a PRP-X100 anion

240

column (250 mm × 4.1 mm length, 10 μm particle size) and a pre-column (11.2 mm

241

length, 12-20 μm particle size) from Hamilton (Reno, NV, USA) with the mobile

242

phase comprised of 10 mM diammonium hydrogen phosphate and 10 mM ammonium

243

nitrate (pH = 9.25, adjusted with aqueous ammonia).39 In addition, four normal

244

arsenic species including As(III), monomethylarsonic acid (MAs(V)), dimethylarsinic 11



Page 12 of 33

245

acid (DMAs(V)) and As(V), four arsenosugars (glycerol arsenosugar (sugar 1),

246

phosphate arsenosugar (sugar 2), sulfonate arsenosugar (sugar 3), and sulfate

247

arsenosugar (sugar 4)) purified from Fucus serratus, and arsenobetaine from shrimp

248

were analyzed in the column. The injection volume was 20 μL and flow rate was 1.0

249

mL min-1. ICP-MS signals were recorded at m/z 75 (75As and 40Ar35Cl) at dwell times

250

of 300 ms, at m/z 77 (77Se and 40Ar37Cl) for possible chloride interferences and at m/z

251

74 (74Ge) for an internal standard at dwell times of 100 ms. Quantification was based

252

on peak areas against external calibration with standards (0, 0.1, 0.5, 1, 5, 10, 50, and

253

100 μg L-1) containing four arsenic species (As(III), As(V), MAs(V) and DMAs(V)).

254

The calibration standard (10 ppb) and blank were analyzed every 10 samples to assure

255

instrumental stability based on our previous study.39

256

Statistical Analysis. The data analysis including basic statistics (mean-value, variable

257

coefficient, standard deviation (SD), standard error (SE), the percentage of arsenic

258

species and ABGs value) and alpha-diversity (PD whole tree, Chao1, rarefaction

259

curves, and Shannon index) were performed in Origin 8.5 (OriginLab, USA). The

260

PCoA and Adonis test were carried out using R version 3.4.3. Analysis of variance

261

(ANOVA) and the Pearson correlation test were performed using the statistical

262

software SPSS V18.0 (IBM, USA). Microsoft Excel 2010 (Microsoft, USA) was used

263

to generate other tabulations and graphics.

264

RESULTS

265

Body Weight, Mortality and Arsenic Bioaccumulation. The body weight of M.

266

sieboldi was remarkably lower in treatments with additions of 140 and 280 mg As(V) 12


Page 13 of 33


267

kg-1 than in the control, corresponding to a reduction of 35.5 and 41.6%, respectively

268

(ANOVA, P < 0.01) after 28 days (Table 1). Earthworm mortality was significantly

269

different between all treatments (ANOVA, P < 0.001), and exhibited a clear

270

dose-response relationship (Table 1). Earthworm mortality increased sharply with

271

exposure to arsenic at 280 mg kg-1 (86.7%) compared to the control (16.7%)

272

(ANOVA, P < 0.001). With the increase in arsenic concentration in soil, total arsenic

273

concentrations of M. sieboldi body tissues or gut contents increased significantly

274

(ANOVA, P < 0.05). The bioaccumulation factor (BAF) of earthworms was estimated

275

to be 1.0~1.4.

276

Arsenic Species and Abundance of ABGs in Soil and Gut. Figure S1 shows that

277

cationic species including arsenobetaine and sugar 1, usually co-eluted at the solvent

278

front with As(III), can be separated from As(III) by the anion exchange column under

279

the condition. The results indicated that inorganic arsenic (As(III) and As(V)) alone

280

was detectable in soil, earthworm body tissues, and gut contents (Table S3). The

281

average extraction rate was 12.6, 52.6 and 79.5% for soil, body tissues and gut

282

contents, respectively (Table S3). The concentration of extracted arsenic species was

283

up to 119.3 mg kg-1 in G140 consisting of 90.2 mg kg-1 As(III) and 29.1 mg kg-1As(V)

284

(Table S3). As(V) (>71.7%) was the dominant species found in soils, while the major

285

form of arsenic in earthworm body tissues was As(III) (>77.9%) (Figure 1).

286

Moreover, As(III) was the predominant arsenic species (>75.6%) detected in the gut

287

contents (Figure 1).

288

The ABGs were divided into four types based on their function, namely, As(III) 13



Page 14 of 33

289

oxidation, As(V) reduction, arsenic (de)methylation, and arsenic transport. A total of

290

16 ABGs were detected in the soil and gut samples (Figure S2), in which some genes

291

involved in As(III) oxidation (aoxC, aoxD, and arxA) were absent (Figure 2A). More

292

ABG species in soil were found than those in gut contents (Figure S2), for example,

293

aoxR, aoxS and arsI were found only in soil and were not detected in earthworm gut

294

(Figure 2A). The normalized copy numbers of ABGs ranged from 0.017 to 0.032

295

copies per cell, with S280 and G280 harboring the lowest and highest gene copies of

296

ABGs, respectively (Figure 2B). The arsI and arsM were rarely detected in gut and

297

soil. The predominant ABGs in gut and soil were involved in As(V) reduction and

298

arsenic transport (Figure 2B).

299

Differences in Microbial Communities between Earthworm Gut Contents and

300

the Surrounding Soil. Across all samples, 2 635 484 nonsingleton reads were

301

calculated, and counts of sequences per sample ranged from 41 999 to 173 030. 49

302

993 OTUs in gut samples and 67 807 OTUs in soil samples were obtained,

303

respectively. The Venn diagram shows that a total of 28 381 OTUs are shared by the

304

two sources, accounting for 56.8% and 41.9% of the total reads from the gut and soil

305

sources, respectively (Figure S3A). Actinobacteria (accounting for 57.5% of the total

306

reads), Firmicutes (20.7%), and Proteobacteria (13.5%) were the major phyla in the

307

gut, while the predominant phyla in soil were Bacteroidetes (26.1%), Proteobacteria

308

(27.0%), and Actinobacteria (24.6%) (Figure S4). Actinobacteria, Bacteroidetes, and

309

Firmicutes varied greatly in earthworm guts and inhabited soil (t-test, P < 0.05)

310

(Figure S4). The relative abundance of Rhizobiales in the soil (7.8%) was 14


Page 15 of 33


311

significantly higher than that in the gut (1.8%) (t-test, P < 0.01). The relative

312

abundance of the shared bacterial families shows a statistically significant difference

313

between the gut and soil sources (t test, P < 0.05) (Figure 3). Bacillaceae (17.3%),

314

Mycobacteriaceae (15.5%), Streptomycetaceae (15.4%), and Microbacteriaceae

315

(13.9%) were the four most abundant families in the gut, whereas their abundance

316

was only 3.1%, 7.8%, 3.2%, and 3.0% in soil, respectively. Sphingobacteriaceae

317

(12.7%) and Xanthomonadaceae (7.9%) were the most abundance families detected in

318

soil (Figure 3). In addition, the dominant genera in soil and earthworm gut

319

microbiome were different (Figure S7). The relative abundance of Bacillus in the gut

320

(12.2%) was significantly higher than that in the soil (4.4%) (t-test, P < 0.01).

321

The Chao1 index shows that the diversity of the soil bacterial community is

322

significantly higher than that in gut (t-test, P < 0.01) (Figure S5). The result was

323

further confirmed by the PD whole tree and Shannon index measures (Figure S5).

324

PCoA analysis (Figure 4) demonstrates a significant shift (Adonis test, P < 0.01)

325

between gut contents and soil along with PC1 (which explained 30.8% of the total

326

variance).

327

Effect of Arsenic on the M. sieboldi Gut Microbial Community. The maximum

328

OTUs (26 452) and minimum OTUs (12 071) were found in G280 and G70,

329

respectively. Only 2 202 OTUs were shared (Figure S3B) between gut contents from

330

different treatments, accounting for 4.4% of the total OTUs in gut samples. The

331

unique OTUs in the GC, G70, G140 and G280 account for 11.7%, 8.6%, 11.0% and

332

27.6% of the total OTUs in gut contents, respectively. At the phylum level, the 15



Page 16 of 33

333

abundance of Bacteroidetes increased significantly with increasing arsenic

334

concentration (t-test, P < 0.05) in the earthworm gut (Figure S6). Compared to the

335

control (GC), Acidobacteria and Gemmatimonadetes in G280 were both notably

336

increased (t-test, P < 0.05). At the family level, the abundance of Flavobacteriaceae,

337

Cytophagaceae, Chitinophagaceae, Weeksellaceae, and Sphingobacteriaceae in G280

338

dramatically exceeded that in gut samples from other arsenic concentration treatments

339

(t-test, P

Effects of Arsenic on Gut Microbiota and Its Biotransformation Genes in

Recommend Documents