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Mul-tiomics analysis of cadmium stress on the ovarian function of the wolf spider Pardosa pseudoannulata
Journal Pre-proof Mul-tiomics analysis of cadmium stress on the ovarian function of the wolf spider Pardosa pseudoannulata Juan Wang, Xianjin Peng, Huilin Yang, Bo Lv, Zhi Wang, Qisheng Song PII:
S0045-6535(20)30096-5
DOI:
https://doi.org/10.1016/j.chemosphere.2020.125904
Reference:
CHEM 125904
To appear in:
ECSN
Received Date: 2 November 2019 Revised Date:
10 January 2020
Accepted Date: 11 January 2020
Please cite this article as: Wang, J., Peng, X., Yang, H., Lv, B., Wang, Z., Song, Q., Mul-tiomics analysis of cadmium stress on the ovarian function of the wolf spider Pardosa pseudoannulata, Chemosphere (2020), doi: https://doi.org/10.1016/j.chemosphere.2020.125904. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.
Credit author statement Zhi Wang: Conceptualization, Methodology Juan Wang: Data Curation, Writing Original Draft Xianjin Peng: Formal analysis Huilin Yang: Supervision Bo Lv: Visualization, Investigation Qisheng Song: Writing - Review & Editing
1
Mul-tiomics analysis of cadmium stress on the ovarian function of the wolf spider Pardosa pseudoannulata
2 3
Juan Wanga, Xianjin Penga, Huilin Yangb, Bo Lva, Zhi Wanga, Qisheng Songc
4
a. College of Life Sciences, Hunan Normal University, Changsha, Hunan 410081,
5
China
6
b. College of Bioscience and Biotechnology, Hunan Agriculture University, Changsha,
7
Hunan 410128, China
8
c. Division of Plant Sciences, University of Missouri, Columbia, MO 65211, USA
9 10
* Corresponding author
11
E-mail: [email protected]
12
Phone number: +86 15073164786
13
1
14
Abstract: Cadmium (Cd) pollution is widespread in paddy filed soil in China. In this
15
study, the toxicity of Cd with regard to the female reproductive system of paddy
16
spider Pardosa pseudoannulata was investigated by means of multi-omics analyses
17
(transcriptome, proteome, and miRNAs). Decreased activities of detoxifying enzymes
18
including peroxidase (POD), Glutathione S-transferases (GST), and superoxide
19
dismutase were detected in the ovary of P. pseudoannulata. Of these, GST and POD
20
were consistently down-regulated at the transcriptional and translational levels.
21
Vitellogenin content and fecundity of the spider were also reduced by Cd burden. Five
22
vitellogenin encodes genes were down-regulated while only vitellogenin-6 protein
23
was up-regulated. But protein lipovitellin-1, the main composition of vitellin, was
24
down-regulated. In addition, the correlation between the mitogen-activated protein
25
kinase (MAPK) signaling pathway and Cd stress was identified. A down-regulated
26
gene that encoding connector of kinase to AP-1 in the MAPK signaling pathway was
27
regulated
28
318>der-miR-318>dgr-miR-318>dme-miR-318-3p>dmo-miR-318>dpe-miR-318>dps
29
-miR-318>dse-miR-318>dsi-miR-318>dvi-miR-318>dwi-miR-318>dya-miR-318). In
30
conclusion, Cd stress possesses distinct female reproductive toxicity on P.
31
pseudoannulata through impairing antioxidant system and synthesis of vitellin.
32
Key words: P. pseudoannulata; Cadmium; Ovary; Multi-omics
by
the
up-regulated
miRNA
33 34 35 2
(miRNA
id:
miRNA
dan-miR-
36
1. Introduction
37
Rapid industrialization in China during the last three decades has resulted in
38
widespread heavy metal contamination in agricultural soils, like Cd, Pb, Zn, As, Cu,
39
Cr, Hg, and Ni. Survey shows that the mean concentration of these heavy metals in
40
the farmland soil of 31 cities were all higher than the National Background of Soil
41
Values in China (Chen et al. 1991, Xie et al. 2019). Cd is an extremely toxic metal
42
that exhibits substantial enrichment in agricultural soils, especially in southern China.
43
Liu et al. (2016) investigated the Cd distribution and contamination in Chinese paddy
44
soils on national scale, suggesting that Cd concentrations in paddy soils of China
45
ranged from 0.01 to 5.50 mg/kg, and the highest Cd concentrations were in several
46
southern provinces, including Hunan (0.73 mg/kg), Guangxi (0.70 mg/kg), and
47
Sichuan (0.46 mg/kg) (Liu et al. 2016). As a non-essential element for organism, Cd
48
can be absorbed by the root of plant from soil and transported to higher trophic levels
49
in a food web (Chen et al. 2016; Hemme et al. 2016).
50
Spiders are common arthropods in paddy fields, and are active in control of insect
51
pests such as leafhoppers and planthoppers (Yang et al. 2018). When spiders were
52
exposed to Cd stress, growth and reproduction might be strongly reduced due to an
53
increased detoxification effect. Li et al. (2016) assessed the ecotoxicological
54
responses of Pardosa pseudoannulata to Cd pollution, and showed that Cd stress
55
resulted in reduced body mass, delayed development, fewer eggs and increased
56
mortality (Li et al. 2016). Babczyńska et al. (2012) compared reproductive strategies
57
of Xerolycosa nemoralis and Agelena labyrinthica between polluted and unpolluted 3
58
sites, and revealed that heavy-containing spiders produced fewer but relatively
59
energy-rich eggs (Babczyńska et al. 2012). Chen et al. (2011) suggest that detoxifying
60
efforts by P. astrigera against metal toxicity incur the costs of fitness parameters,
61
including reduced reproduction and delayed development (Chen et al. 2011). Hence,
62
the reproductive system of spiders may be sensitive to Cd toxicity. Studies suggested
63
that Cd exposure decreased the number of ovulated oocytes and impaired oocyte
64
meiotic maturation rate and fertilization capacity in mice (Zhu et al. 2018). For
65
spiders, the damage of Cd stress on ovarian function was barely reported.
66
P. pseudoannulata is one of the most common species of wolf spiders in paddy
67
fields in southern China (Preap et al. 2001). In this study, ovarian tissues of spiders were
68
dissected for multi-omics analysis by means of High-throughput technologies.
69
Integrated analyses between data from multi-omics (transcriptome, proteome, and
70
miRNAs) and individual level (antioxidant enzyme activities and fecundity) were
71
conducted to reveal potential damage of Cd to the ovarian function of P.
72
pseudoannulata.
73 74
2. Methods
75
2.1 Insect and spider preparation
76
Fruit flies (Drosophila melanogaster) were provided by Hunan Normal
77
University and reared on a corn meal medium. The CdCl2 (1 g/L) solution was added
78
into the corn meal medium to obtain Cd-containing fruit flies. The final CdCl2
79
concentration in medium was 1 mg/L, which is equivalent to middle level of soil 4
80
pollution. Female spiders were collected from farmland without heavy metal pollution
81
in the Hunan Academy of Agricultural Science (Wang et al. 2018), feeding with
82
either normal (control group) or Cd containing fruit flies daily. Spiders were collected
83
every 5 days for Cd determination using Inductively Coupled Plasma (ICP) analysis.
84
Ovaries tissues were dissected from spiders that fed for 30 days, and used for
85
determination of enzymes activities and high-throughput sequencing (RNA-seq, small
86
RNA sequencing and proteome sequencing).
87
2.2 Determination of Cd content and enzyme activities
88
The Cd content in the spider were analyzed with the Plasma Atomic Emission
89
Spectrometer (ICPE-9000, SHIMADZU CORPORATIOM, JAPAN) as described by
90
Wang et al. (2018). Enzyme-linked immunosorbent assay (ELISA) kits (ZCI BIO,
91
China) including the Insect GST ELISA kit, Insect SOD ELISA kit, Insect POD
92
ELISA kit and Insect CAT ELISA kit were applied for determining activities of
93
glutathione S-transferase (GST), superoxide dismutase (SOD), peroxidase (POD), and
94
catalase (CAT) respectively. In addition, the Insect VTG ELISA kit (ZCI BIO, China)
95
and Insect MT ELISA kit (NJJCBIO, China) were used for determination of
96
vitellogenin content and metallothionein (MT) content in spider.
97
2.3 Mul-tiomics analysis
98
2.3.1 Transcriptome sequencing
99
A total of 180 spiders (Controls: 30/group, three replicates; Cd-treated: 30/group,
100
three replicates) were dissected for ovaries. Total RNA was extracted using the
101
TRIzol Reagent (Invitrogen, USA), and the purity was determined using the 5
102
NanoPhotometer® spectrophotometer (IMPLEN, CA, USA). cDNA libraries were
103
constructed using a NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB,
104
USA), and index codes were added to attribute sequences to each sample. These
105
libraries were sequenced on the Illumina sequencing platform, to generate the raw
106
reads. Reads were assembled by Trinity software and redundant sequences were
107
eliminated by TGIC software to obtain a set of available unigenes for subsequent
108
bioinformatics analyses (Pertea et al. 2003, Grabherr et al. 2011).
109
The FPKM (Fragments Per Kilobase of transcript per Million mapped reads) of
110
each gene was calculated based on the length of the gene and reads count mapped to
111
this gene to quantify the expression level of gene (Trapnell et al. 2010). Differentially
112
expressed genes (DEGs) was screened using the DESeq2 R package (1.16.1) with
113
threshold value p-value <0.05, and absolute value of Log2foldchange value >1. All
114
genes were aligned by Blast searching against protein databases, Nr (non-redundant
115
protein database, NCBI), and Swiss-prot retrieving proteins with the highest sequence
116
similarity to the given genes along with their protein functional annotations. KEGG
117
(Kyoto Encyclopedia of Genes and Genomes) enrichment analysis was conducted to
118
understand the biological function of DEGs (Ogata et al. 2000). Transcriptome
119
sequencing and small RNA sequencing were conducted by OEbiotech Company
120
(Shanghai, China).
121
2.3.2 small RNA sequencing
122
Ovaries were dissected from P. pseudoannulata for small RNA sequencing.
123
Sequencing libraries were generated using the NEBNext® Multiplex Small RNA 6
124
Library Prep Set for Illumina® (NEB, USA.), and sequenced on an Illumina Hiseq
125
2000 platform. Single-end reads of 50 bp were generated. Raw reads containing poly
126
A or T or G or C and with low quality were removed to obtain cleans reads for the
127
bioinformatics analysis.
128
Clean reads were mapped on the transcriptome sequences of ovary from P.
129
pseudoannulata by Bowtie (Langmead et al., 2009) to analysis their expression and
130
distribution on the reference. miRbase database (http://www.mirbase.org/) was
131
applied to conduct known miRNA alignment, and software miREvo and mirdeep2
132
were integrated to predict novel miRNA (Friedländer et al. 2012, Wen 2012).
133
Software miRanda and DESeq R packages were applied to predict target genes of
134
miRNAs and screen differentially expressed miRNAs (DEMs, p<0.05) (Enright et al,
135
2003). KEGG enrichment analysis was used on the target gene candidates of
136
differentially expressed miRNAs by KOBAS software (Mao et al., 2005). The small
137
RNA sequencing was conducted by OEbiotech Company (Shanghai, China).
138
2.3.3 Proteomic study
139
Total proteins were extracted form ovaries of P. pseudoannulata for proteomic
140
study. Protein concentration was determined by Bradford protein assay (Bradford,
141
1976). Proteins were labeled using the iTRAQ® Reagent-8PLEX Multiplex Kit
142
(Sigma) for HPLC fractionation. LC-MS/MS analyses were performed using an
143
EASY-nLCTM 1200 UHPLC system (ThermoFisher) coupled with an Orbitrap Q
144
Exactive HF-X Mass Spectrometer (ThermoFisher) operating in the data-dependent
145
acquisition (DDA) mode. 7
146 147
For protein identification, database searches were performed by the search
148
engines, Proteome Discoverer 2.2 (PD 2.2, Thermo) on UniProt database
149
(http://www.uniprot.org). Protein with at least 1 unique peptide was identified at FDR
150
less than 1.0% on peptide and protein level, respectively. Proteins that contain similar
151
peptides, but could not be distinguished based on MS/MS analysis were grouped
152
separately as protein groups. Reporter Quantification (iTRAQ 8-plex) was used for
153
iTRAQ quantification. The protein quantitation results were statistically analyzed
154
using Mann-Whitney Test, the significant ratios, defined as p < 0.05 and ratio > 1.2,
155
were used to screen the differentially expressed proteins (DEPs). Here, proteomic
156
analysis was conducted by Novogene Biotech (Beijing, China).
157
2.4 Expression validation of DEGs and DEMs
158
In total of 8 miRNAs and 12 genes with different expression patterns were
159
selected for validation by quantitative real-time RT-PCR (qRT-PCR). Total RNA was
160
obtained from excess samples used in High-throughput Sequencing. qRT-PCR
161
analysis for DEGs was conducted as described in our previous study (Wang et al.
162
2019); Expression validation of mRNAs was carried out using miScript II Reverse
163
Transcription Kit (Qiagen, Germany) and QuantiFast® SYBR® Green PCR Kit
164
(Qiagen, Germany) according the manufacturer’s instructions. The expression levels
165
of mRNAs and miRNAs were quantified using the 2-
166
Schmittgen 2001).
167 8
Ct
method (Livak and
168 169
3. Results
170
3.1 Cd accumulation in spiders
171
ICP analysis showed that the Cd content in Cd-containing spiders increased with
172
feeding time, ranging from 4.059 ± 0.62 µg/g at day 5 to 7.27 ± 0.34 µg/g at day 30,
173
and declined to a lower level of 5.12 ± 0.96 µg/g at day 35 (Fig.1). In the following
174
studies, the samples from the spiders fed with the fruit flies for 30 days were used for
175
High-throughput sequencing.
176 177
Fig. 1 Bioaccumulation of cadmium in P. pseudoannulata. The histogram bars
178
indicate amounts of Cd (mean ± SE) detected using ICP analysis in P. pseudoannulata
179
adults fed on Cd -containing fruit flies for the indicated time periods. Bars annotated
180
with the same lowercase letters are not significantly different (one-way ANOVA, p<
181
0.05). 9
182
3.2 Expression analysis and qRT-PCR analysis
183
We obtained 92,778 genes with an average length of 1,104bp, 1,265 proteins, and
184
724 miRNAs with 714 known and 10 novel miRNAs from ovaries of P.
185
pseudoannulata by means of High-throughput sequencing (Fig. 2A). The raw reads of
186
transcriptome and miRNAs had been submitted into NCBI SRA database (Accession
187
numbers: PRJNA577066 and PRJNA577205). Expression analysis showed that there
188
were 4,535 DEGs with 1,795 up-regulated and 2,740 down-regulated, 231 DEPs with
189
82 up-regulated and 149 down-regulated, and 67 DEMs with 55 up-regulated and 11
190
down-regulated between the treated and control samples.
191
To confirm the results of the differential expression analyses, eight Cd-induced
192
miRNAs and 12 Cd-induced mRNAs were selected for qRT-PCR analysis. Primer
193
sequences of DEGs and DEMs, and target genes of DEMs were listed in Table S1. As
194
showed in Fig. 2B, the expression profiles of all DEGs and DEMs were consistent
195
with the RNA-seq data.
10
196 197
Fig. 2 Expression analysis and qRT-PCR verification. A Expression level analysis
198
for transcriptome, proteome and miRNA. B qRT-PCR verification of DEGs and
199
DEMs. A good correlation of qRT-PCR results and high-throughput sequencing data
200
were shown through comparing these results. The actual ID of DEGs and DEMs
201
corresponding to the miRNA-1 to miRNA-8, and mRNA-1 to mRNA-12 on the
202
X-axis were showed in Table S1.
203 204 205
3.3 Influencing of Cd on spider detoxifying enzyme activities Ovaries of P. pseudoannulata were dissected for detoxifying enzyme activity
206
determination. Results from the ELISA assays showed that activities of GST (controls:
207
21.880 ± 1.381 U, Cd-treated: 16.303 ± 0.517 U), POD (controls: 33.657 ± 6.173 U,
208
Cd-treated: 10.323 ± 1.333 U), and SOD (controls: 7.533 ± 0.175 U, Cd-treated:
209
3.907 ± 0.400 U) were significantly decreased in response to Cd toxicity, down by 11
210
25.5% for GST, down 69.3% for POD and down by 48.1% for SOD, while the
211
activity of CAT was unaffected (Fig. 3A). In addition, content of metallothionein was
212
increased in spider under Cd burden (controls: 245.963 ± 1.237 ng/g, Cd-treated:
213
353.083 ± 16.774).
214
At the transcriptional level, a total of 31, 11, 15, and 2 genes encoding GST, SOD,
215
POD and CAT respectively were obtained from transcriptome of spider ovarian
216
samples. Gene expression analysis showed that 8 GST genes with 6 down-regulated
217
and 2 up-regulated, 1 SOD gene, 1 POD gene, and 1 CAT gene were differentially
218
expressed in the ovaries of spiders triggered by Cd burden (Fig. 3B).
219
Correlation analysis between transcriptome and proteome suggested that six
220
proteins and their encoding genes were in an accordant expression trend (both
221
down-regulated), but two in the reverse trend (proteins up-regulated but encoding
222
genes down-regulated, Table 1). Of these, two proteins involved in activities of GST
223
and POD (A0A087U9G0, A0A087TPB8) were negatively expressed both in
224
transcriptional and translational levels ((p<0.05, Table 1).
225
12
226 227
Fig. 3 Effects of Cd on spider detoxifying enzyme activities. A Determination of
228
detoxifying enzyme activities using ELISA assay; B Integrated analysis between
229
DEGs and DEMs.
230
13
231
Table 1.
Genes encoding detoxifying enzymes in ovaries of the spider
Protein ID
Up or Down
mRNA ID
Up or Down
Functions
A0A087UNQ7
up
comp147789c0seq1
down
A0A087TCX6 A0A087U9G0
up down
comp96347c0seq1 CL19176Contig1
down down
A0A087TPB8
down
comp244344c0seq1
down
A0A087TW10
down
comp207851c0seq2
down
A0A087T6N3
down
CL1Contig1437
down
A0A087UE31 A0A087UJM0
down down
comp21618c0seq1 comp92783c0seq1
down down
Delta-1-pyrroline-5-carboxylate dehydrogenase ELAV-like protein 2 Glutathione S-transferase fragment Thioredoxin-dependent peroxide reductase Voltage-dependent anion-selective channel protein 2 Basement membrane-specific heparan sulfate proteoglycan core protein Collagen alpha-2(IV) chain FAS-associated factor 1
232 233
3.4 Influence of Cd-containing prey on spider fecundity
234
Egg sacs and ovaries were collected from the adult spiders to record the number
235
of eggs and determine the vitellogenin concentration, respectively. Significant
236
decrease in the number of eggs from 117 to 64 (down by 45.2%) and the
237
concentration of vitellogenin from 3,212 to 2,433 µg/g (down by 24.2%) were
238
observed on Cd-containing spiders when compared with controls (Table 2).
239
Vitellogenin is the precursor molecule of vitellin, which is deposited in the
240
growing spider oocytes of the maturating female, and is also a main step of the
241
reproductive process in spider (Pourié and Trabalon 2003, Guo et al. 2018). A total of
242
10 vitellogenin encoding genes were obtained through Swiss-prot annotation, of
243
which 5 genes were down-regulated by Cd burden (p<0.05, absolute value of Log2
244
Fold change >1, Table 3). For proteome, the precursor protein vitellogenin-6 was 14
245
up-regulated, but the lipovitellin-1 (C0HJA5), a main composition of vitellin, was
246
up-regulated (p<0.05, Table 4).
247 248
Table 2. Effects of Cd on fecundity of the spider at individual level Vg* Concentration Fecundity (µg/g) Controls 3211.68 ± 51.957 117.18 ± 12.544 Cd-treated spiders
2433.11 ± 94.814*
64.22 ±26.1093*
249 250
Vg*: vitellogenin
251
Table 3 Effects of Cd on fecundity of the spider at transcriptional level Gene id Gene FPKM FPKM annotation value-Cd-treated calue-controls CL1751Contig1 Vitellogenin 51.5948 2.8984 comp174356_c0_seq1 Vitellogenin-5 16.0196 1.2146 comp178497_c0_seq1 Vitellogenin-2 10.4701 0.0000 3.8933 0.4200 comp212587_c0_seq1 Vitellogenin comp21587_c0_seq1 Vitellogenin 154.2527 0.3362 CL3318Contig1 Vitellogenin-6 30.2484 10.9338 0.0000 comp174358_c0_seq1 Vitellogenin-5 2.8248 comp174360_c0_seq1 Vitellogenin-5 30.1711 3.1042 0.5803 0.0000 comp219203_c0_seq1 Vitellogenin comp4775_c0_seq1 Vitellogenin 112.4606 19.4026
Up or Down Down Down Down Down Down / / / / /
pval 3.1E-12 0.048205 1.23E-08 0.000387 0.019362 0.064454 0.134049 0.06283 0.22662 0.319279
252 253
Table 4 Effects of Cd on fecundity of the spider at translational level Accession Description Average Up or Down CDLC/CKLC A0A087T463 Vitellogenin-6 1.655715113 Up C0HJA5 Lipovitellin-1 0.431626468 Down
t test p-value 0.0447143 0.0117556
254 255
3.5 Effect of Cd on the MAPK signaling pathway
256
The mitogen-activated protein kinase (MAPK) signaling cascades are highly
257
conserved from yeast to mammals, and play a crucial role in oocyte meiosis and
258
maturation in ovary (Motola et al 2010, Liu et al 2019). Gene expression analysis
259
indicated that 24 genes were differentially expressed in the MAPK signaling pathway 15
260
(ko04013), with 21 down-regulated (marked with green box) and 3 up-regulated
261
(market with red box, Fig. 4). These DEGs were acting on 18 nodes of ko04013,
262
including Tor (tyrosine-protein kinase receptor torso), Egfr (epidermal growth factor
263
receptor), Drk (growth factor receptor-binding protein 2), Dos (protein daughter of
264
sevenless), Ras85D (GTPase KRas), Ksr (kinase suppressor of Ras 2), Pointed
265
(C-ets-1), Hkb (huckebein), Pros (prospero homeobox 1), Btk29A (tyrosine-protein
266
kinase Tec), Rac (Ras-related C3 botulinum toxin substrate 1), dCyld (ubiquitin
267
carboxyl-terminal hydrolase CYLD), dTak1(mitogen-activated protein kinase kinase
268
kinase 7), Raw (raw), Cka (striatin 1/3/4), Dpp (bone morphogenetic), and Mef2
269
(MADS-box transcription enhancer factor 2A).
270
miRNA is a small non-coding RNA molecule that functions in RNA silencing
271
and post-transcriptional regulation of gene expression, by base-pairing with
272
complementary sequences within mRNA molecules. KEGG enrichment analysis was
273
applied for target genes of DEMs between Cd-treated spiders and controls. There
274
were 17 enriched pathways mapped by 115 target genes of DEMs, of which the
275
MAPK signaling pathway (ko04013) was most enriched, followed by EGFR tyrosine
276
kinase inhibitor resistance (ko01521), Novobiocin biosynthesis (ko00401), and ErbB
277
signaling pathways (ko04012, FDR<0.01, Table S2). Here, in the MAPK signaling
278
pathway, three nodes such as Egfr, Cka, and Omb (T-box protein 2) (marked with blue
279
shading) were mapped by target genes of 5 known miRNAs, including
280
dan-miR-318>der-miR-318>dgr-miR-318>dme-miR-318-3p>dmo-miR-318>dpe-mi
281
R-318>dps-miR-318>dse-miR-318>dsi-miR-318>dvi-miR-318>dwi-miR-318>dya-m 16
282
iR-318,
xbo-miR-92a,
283
ame-miR-278>cqu-miR-278>dan-miR-278>der-miR-278>dgr-miR-278>dme-miR-27
284
8-3p>dmo-miR-278>dpe-miR-278>dps-miR-278>dse-miR-278>dsi-miR-278>dvi-mi
285
R-278-3p>dwi-miR-278>dya-miR-278,
286
between
287
dan-miR-318>der-miR-318>dgr-miR-318>dme-miR-318-3p>dmo-miR-318>
288
dpe-miR-318>dps-miR-318>dse-miR-318>dsi-miR-318>dvi-miR-318>dwi-miR-318
289
>dya-miR-318 had negative regulation on its target gene CL1Contig670, while
290
expression level of other target genes were not changed (Fig. 4). According to the map
291
04013, this pathway was involved in oogenesis or egg polarity in animals (Fig. 4).
DEGs
and
prd-miR-235-3p,
DEMs
and
aae-miR-278-3p>aga-miR-278>
odi-miR-92a.
suggested
that
Integration only
one
analysis miRNA
292
Integrated analysis between DEGs and DEMs also indicated that genes encoding
293
serine/threonine-protein kinase mTOR, glycine dehydrogenase, and tyrosine
294
aminotransferase were also negatively regulated by five DEMs (Table 5).
17
295 296
Fig. 4. Effect of Cd on the MAPK signaling pathway. Proteins or genes marked
297
with green or red boxes were mapped by DEGs, and marked with blue shading were
298
mapped by target genes of DEMs. One box may be mapped by several genes.
299 300
18
301
Table 5. Integration analysis for DEMs and DEGs p
Up or
DEMs
Up or
p
Down
value
Target genes value
Down
KO_Definition
Serine/threonineCL36Contig2
Down
0.016
protein kinase mTOR
dpe-miR-317>dps-miR-317
0.022
Up
comp171382_c0_seq1
0.028 Down Glycine
comp186723_c0_seq3
0.017 Down
dehydrogenase
comp248081_c0_seq1
Down
0.038
dan-miR-318>der-miR318>dgr-miR-318>dmemiR-318-3p>dmo-miR318>dpe-miR-318>dps0.089
Up
CL1Contig670
Down
0.033
0.305
Up
comp102460_c0_seq2
Down
0.005
Striatin 1/3/4
miR-318>dse-miR-318>dsimiR-318>dvi-miR318>dwi-miR-318>dyamiR-318 Tyrosine dpe-miR-31b>dps-miR-31b
aminotransferase dme-miR-986-5p>dps-miR0.309
Up
comp210471_c0_seq2
Down
0.015
Up
comp7518_c0_seq1
Down
0.005
986-5p>dsi-miR-986-5p dme-miR-958-3p>dps-miR4E958-3p>dsi-miR-958-
09 3p>dvi-miR-958-3p comp171382_c0_seq1
Down
0.028
comp186723_c0_seq3
Down
0.017
dan-miR-317>der-miR317>dgr-miR-317>dme-
Glycine 0.011
Up
dehydrogenase
miR-317-3p>dse-miR317>dsi-miR-317>dvi-miR-
comp248081_c0_seq1
Down
0.038
302 303 304 305
4. Discussion Cd is toxic heavy metal widely distributed in the environment (Kim and Koo 19
306
2008). After Cd enters the various trophic levels via food web, there is little excretion
307
due to the lack of an effective Cd elimination pathway in organisms, and the
308
remaining Cd is accumulated in target organs (Seebaugh et al. 2005, Cai et al. 2009).
309
In this study, the Cd content in the spider increased with feeding time, then decreased
310
slightly, when fed with Cd-containing fruit flies that reared on corn meal medium
311
supplemented with Cd at the medium Cd pollution level. This is similar to the report
312
by Li et al (2016), when P. pseudoannulata was exposed to low and high CdCl2
313
solutions (Li et al. 2016). Antioxidant enzymes include SOD, CAT, GST and POD
314
were found extensively in organisms. These enzymes are generally considered
315
sensitive biomarkers of an organism’s antioxidant response and protect cells from
316
reactive oxygen species (ROS) toxicity (Lei et al. 2011, Li and Sunde 2016).
317
Significant correlations between the concentration of heavy metals and activities of
318
GST, GPOX, and GSTPx were observed in spiders A. labyrinthica and P. lugubris
319
(Wilczek et al. 2004); Activities of SOD, CAT and GST were increased in P.
320
pseudoannulata, when they exposed to Cd for 7 days (Li et al. 2016). Reproduction
321
organs are one of the most important target organs for Cd accumulation and
322
intoxication, causing a variety of physiological changes in ovary (Li et al. 2010, Yang
323
et al. 2012). Here in the present study, Cd stress decreased the activities of SOD, GST
324
and POD in the ovary of P. pseudoannulata. Meanwhile, expression level of
325
detoxifying enzyme encoding genes were up or down regulated, and proteins related
326
to activities of detoxifying enzymes were down-regulated. Among them, two proteins
327
involved in activities of GST and POD (A0A087U9G0, A0A087TPB8) were 20
328
negatively expressed both at transcriptional and translational levels. This implied that
329
antioxidant system in the ovary of the spider may be impaired, due to decreased
330
activity and negative expression of related genes and proteins.
331
Cd is known as the inhibitor of protein synthesis in vitro (Norton and Kench
332
1977). In arthropod, Cd has been reported to inhibit the vitellogenesis leading to
333
reduction of yolk synthesis and egg production (Płachetka-Bożek et al 2018. Cervera
334
2005, Zhao 2016). Vitellogenin is the precursor molecule of vitellin, its synthesis
335
could be inhibited by Cd burden in the ovary of P. pseudoannulata. In the present
336
study, multi-omics data indicated that 5 vitellogenin encoding genes were down
337
regulated. Although protein vitellogenin-6 (A0A087T463) was up-regulated, but
338
protein lipovitellin-1 (C0HJA5) is down regulated, which was the main composition
339
of vitellin. Vitellin is needed for the survival of embryos before the offspring can feed
340
independently (Engelmann 1979, Cabrera et al. 2009). In the present study, the
341
decreased fecundity of P. pseudoannulata was triggered by Cd induced inhibition of
342
vitellin synthesis.
343
The MAPK signaling cascade is highly conserved from yeast to mammals, and
344
plays a crucial role in oocyte meiosis and maturation in ovary (Motola et al 2010,
345
Liu et al 2019). In oocytes, MAPK can play a role as a component of cytostatic factor
346
to suppress DNA replication between meiosis I and II; And the vitellogenin
347
transcription is also mediated by the MAPK-dependent cascades (Chen et al., 2018).
348
But Cd exposures would alter the MAPK pathway by disturbing the state of
349
phosphorylation of Cdc2 and histone H3, and then cause delayed oocyte maturation of 21
350
the African clawed frog Xenopus laevis (Slaby et al. 2017). Cd treatment can alter the
351
splicing of the kitl pre-mRNA in ovarian granulosa cells, and miRNAs play regulatory
352
roles in the alternative splicing of kitl. These miRNAs are enriched in several
353
pathways like the MAPK signaling pathway, the Ras signaling pathway,and the Foxo
354
signaling pathway (Wang et al. 2017). In the present study, integrate analysis was
355
conducted for DEGs and DEMs. A correlation between the expression of the MAPK
356
signaling pathway and Cd stress was identified in P. pseudoannulata. A
357
down-regulated gene (CL1Contig670) was regulated by the up-regulated miRNA
358
(dan-miR-318>der-miR-318>dgr-miR-318>dme-miR-318-3p>dmo-miR-318>dpe-mi
359
R-318>dps-miR-318>dse-miR-318>dsi-miR-318>dvi-miR-318>dwi-miR-318>dya-m
360
iR-318), which encodes protein Cka in the MAPK signaling pathway. This provides a
361
new aspect for exploring the mechanism related to Cd-induced ovarian toxicity in
362
spider.
363
5. In conclusion
364
Cd stress possesses the distinct female reproductive toxicity on P. pseudoannulata.
365
Antioxidant system in the ovary of the spider may be impaired by decreased activities
366
of GST, POD and SOD. Inhibition of vitellin synthesis and negative regulation of the
367
MAPK signaling pathway by Cd may contribute to reduced egg production of the
368
spider.
369 370 371 22
372
Declaration of Competing Interest
373
The authors declare that they have no known competing financial interests or
374
personal relationships that could have appeared to influence the work reported in this
375
paper.
376
Acknowledgment
377
This work was supported by the Natural Science Foundation of China
378
(No.31472017,31272339), by the Open Research Fund of Key Laboratory of Tropical
379
Disease Control and Research, Ministry of Education in China (2018 kfkt03), and by
380
Project supported by the Hunan Provincial Education Department (18A024).
381 382
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28
Table 1. Genes encoding detoxifying enzymes in ovaries of the spider Protein ID
Up or Down
mRNA ID
Up or Down
Functions
A0A087UNQ7 up
comp147789c0seq1
down
Delta-1-pyrroline-5-carboxylate dehydrogenase
A0A087TCX6
up
comp96347c0seq1
down
ELAV-like protein 2
A0A087U9G0
down
CL19176Contig1
down
Glutathione S-transferase fragment
A0A087TPB8
down
comp244344c0seq1
down
Thioredoxin-dependent peroxide reductase
A0A087TW10
down
comp207851c0seq2
down
Voltage-dependent anion-selective channel protein 2
A0A087T6N3
down
CL1Contig1437
down
Basement membrane-specific heparan sulfate proteoglycan core protein
A0A087UE31
down
comp21618c0seq1
down
Collagen alpha-2(IV) chain
A0A087UJM0
down
comp92783c0seq1
down
FAS-associated factor 1
Table 2. Effects of Cd on fecundity of the spider at individual level Vg* Concentration (µg/g)
Fecundity
Controls
3211.68 ± 51.957
117.18 ± 12.544
Cd-treated spiders
2433.11 ± 94.814*
64.22 ±26.1093*
Vg*: vitellogenin
Table 3 Effects of Cd on fecundity of the spider at transcriptional level Gene id
Gene annotation
FPKM valueCd-treated
FPKM caluecontrols
Up or Down
pval
CL1751Contig1
Vitellogenin
51.5948
2.8984
Down
3.1E-12
comp174356_c0_seq1 Vitellogenin5
16.0196
1.2146
Down
0.048205
comp178497_c0_seq1 Vitellogenin2
10.4701
0.0000
Down
1.23E-08
comp212587_c0_seq1 Vitellogenin
3.8933
0.4200
Down
0.000387
comp21587_c0_seq1
Vitellogenin
154.2527
0.3362
Down
0.019362
CL3318Contig1
Vitellogenin6
30.2484
10.9338
/
0.064454
comp174358_c0_seq1 Vitellogenin5
2.8248
0.0000
/
0.134049
comp174360_c0_seq1 Vitellogenin5
30.1711
3.1042
/
0.06283
comp219203_c0_seq1 Vitellogenin
0.5803
0.0000
/
0.22662
comp4775_c0_seq1
112.4606
19.4026
/
0.319279
Vitellogenin
Table 4 Effects of Cd on fecundity of the spider at translational level Accession
Description
Average CDLC/CKLC
Up or Down
t test p-value
A0A087T463
Vitellogenin6
1.655715113
Up
0.0447143
C0HJA5
Lipovitellin1
0.431626468
Down
0.0117556
Table 5. Integration analysis for DEMs and DEGs DEMs
dpe-miR-317>dps-miR-317
p value
0.022
Up or Down
Up
Up or Down
p value
KO_Definition
CL36Contig2
Down
0.016
Serine/threonineprotein kinase mTOR
comp171382_c0_seq1
Down
0.028
comp186723_c0_seq3
Down
0.017
comp248081_c0_seq1
Down
0.038
Target genes
Glycine dehydrogenase
dan-miR-318>der-miR318>dgr-miR-318>dmemiR-318-3p>dmo-miR318>dpe-miR-318>dpsmiR-318>dse-miR-318>dsimiR-318>dvi-miR318>dwi-miR-318>dyamiR-318
0.089
Up
CL1Contig670
Down
0.033
Striatin 1/3/4
dpe-miR-31b>dps-miR-31b
0.305
Up
comp102460_c0_seq2
Down
0.005
Tyrosine aminotransferase
dme-miR-986-5p>dps-miR986-5p>dsi-miR-986-5p
0.309
Up
comp210471_c0_seq2
Down
0.005
dme-miR-958-3p>dps-miR958-3p>dsi-miR-9583p>dvi-miR-958-3p
0.015
Up
comp7518_c0_seq1
Down
4E09
comp171382_c0_seq1
Down
0.028
comp186723_c0_seq3
Down
0.017
comp248081_c0_seq1
Down
0.038
dan-miR-317>der-miR317>dgr-miR-317>dmemiR-317-3p>dse-miR317>dsi-miR-317>dvi-miR317-3p>dwi-miR-317>dyamiR-317
0.011
Up
Glycine dehydrogenase
Highlight Cd stress possesses the distinct female reproductive toxicity on P. pseudoannulata Cd exposures damage the antioxidant system of ovary in P.pseudoannulata Vt synthesis was affected by Cd both at transcriptional and translational levels
Expression of MAPK signaling pathway was negative regulated by miRNA under Cd stress
Conflict of Interest Statement The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
S0045-6535(20)30096-5
DOI:
https://doi.org/10.1016/j.chemosphere.2020.125904
Reference:
CHEM 125904
To appear in:
ECSN
Received Date: 2 November 2019 Revised Date:
10 January 2020
Accepted Date: 11 January 2020
Please cite this article as: Wang, J., Peng, X., Yang, H., Lv, B., Wang, Z., Song, Q., Mul-tiomics analysis of cadmium stress on the ovarian function of the wolf spider Pardosa pseudoannulata, Chemosphere (2020), doi: https://doi.org/10.1016/j.chemosphere.2020.125904. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.
Credit author statement Zhi Wang: Conceptualization, Methodology Juan Wang: Data Curation, Writing Original Draft Xianjin Peng: Formal analysis Huilin Yang: Supervision Bo Lv: Visualization, Investigation Qisheng Song: Writing - Review & Editing
1
Mul-tiomics analysis of cadmium stress on the ovarian function of the wolf spider Pardosa pseudoannulata
2 3
Juan Wanga, Xianjin Penga, Huilin Yangb, Bo Lva, Zhi Wanga, Qisheng Songc
4
a. College of Life Sciences, Hunan Normal University, Changsha, Hunan 410081,
5
China
6
b. College of Bioscience and Biotechnology, Hunan Agriculture University, Changsha,
7
Hunan 410128, China
8
c. Division of Plant Sciences, University of Missouri, Columbia, MO 65211, USA
9 10
* Corresponding author
11
E-mail: [email protected]
12
Phone number: +86 15073164786
13
1
14
Abstract: Cadmium (Cd) pollution is widespread in paddy filed soil in China. In this
15
study, the toxicity of Cd with regard to the female reproductive system of paddy
16
spider Pardosa pseudoannulata was investigated by means of multi-omics analyses
17
(transcriptome, proteome, and miRNAs). Decreased activities of detoxifying enzymes
18
including peroxidase (POD), Glutathione S-transferases (GST), and superoxide
19
dismutase were detected in the ovary of P. pseudoannulata. Of these, GST and POD
20
were consistently down-regulated at the transcriptional and translational levels.
21
Vitellogenin content and fecundity of the spider were also reduced by Cd burden. Five
22
vitellogenin encodes genes were down-regulated while only vitellogenin-6 protein
23
was up-regulated. But protein lipovitellin-1, the main composition of vitellin, was
24
down-regulated. In addition, the correlation between the mitogen-activated protein
25
kinase (MAPK) signaling pathway and Cd stress was identified. A down-regulated
26
gene that encoding connector of kinase to AP-1 in the MAPK signaling pathway was
27
regulated
28
318>der-miR-318>dgr-miR-318>dme-miR-318-3p>dmo-miR-318>dpe-miR-318>dps
29
-miR-318>dse-miR-318>dsi-miR-318>dvi-miR-318>dwi-miR-318>dya-miR-318). In
30
conclusion, Cd stress possesses distinct female reproductive toxicity on P.
31
pseudoannulata through impairing antioxidant system and synthesis of vitellin.
32
Key words: P. pseudoannulata; Cadmium; Ovary; Multi-omics
by
the
up-regulated
miRNA
33 34 35 2
(miRNA
id:
miRNA
dan-miR-
36
1. Introduction
37
Rapid industrialization in China during the last three decades has resulted in
38
widespread heavy metal contamination in agricultural soils, like Cd, Pb, Zn, As, Cu,
39
Cr, Hg, and Ni. Survey shows that the mean concentration of these heavy metals in
40
the farmland soil of 31 cities were all higher than the National Background of Soil
41
Values in China (Chen et al. 1991, Xie et al. 2019). Cd is an extremely toxic metal
42
that exhibits substantial enrichment in agricultural soils, especially in southern China.
43
Liu et al. (2016) investigated the Cd distribution and contamination in Chinese paddy
44
soils on national scale, suggesting that Cd concentrations in paddy soils of China
45
ranged from 0.01 to 5.50 mg/kg, and the highest Cd concentrations were in several
46
southern provinces, including Hunan (0.73 mg/kg), Guangxi (0.70 mg/kg), and
47
Sichuan (0.46 mg/kg) (Liu et al. 2016). As a non-essential element for organism, Cd
48
can be absorbed by the root of plant from soil and transported to higher trophic levels
49
in a food web (Chen et al. 2016; Hemme et al. 2016).
50
Spiders are common arthropods in paddy fields, and are active in control of insect
51
pests such as leafhoppers and planthoppers (Yang et al. 2018). When spiders were
52
exposed to Cd stress, growth and reproduction might be strongly reduced due to an
53
increased detoxification effect. Li et al. (2016) assessed the ecotoxicological
54
responses of Pardosa pseudoannulata to Cd pollution, and showed that Cd stress
55
resulted in reduced body mass, delayed development, fewer eggs and increased
56
mortality (Li et al. 2016). Babczyńska et al. (2012) compared reproductive strategies
57
of Xerolycosa nemoralis and Agelena labyrinthica between polluted and unpolluted 3
58
sites, and revealed that heavy-containing spiders produced fewer but relatively
59
energy-rich eggs (Babczyńska et al. 2012). Chen et al. (2011) suggest that detoxifying
60
efforts by P. astrigera against metal toxicity incur the costs of fitness parameters,
61
including reduced reproduction and delayed development (Chen et al. 2011). Hence,
62
the reproductive system of spiders may be sensitive to Cd toxicity. Studies suggested
63
that Cd exposure decreased the number of ovulated oocytes and impaired oocyte
64
meiotic maturation rate and fertilization capacity in mice (Zhu et al. 2018). For
65
spiders, the damage of Cd stress on ovarian function was barely reported.
66
P. pseudoannulata is one of the most common species of wolf spiders in paddy
67
fields in southern China (Preap et al. 2001). In this study, ovarian tissues of spiders were
68
dissected for multi-omics analysis by means of High-throughput technologies.
69
Integrated analyses between data from multi-omics (transcriptome, proteome, and
70
miRNAs) and individual level (antioxidant enzyme activities and fecundity) were
71
conducted to reveal potential damage of Cd to the ovarian function of P.
72
pseudoannulata.
73 74
2. Methods
75
2.1 Insect and spider preparation
76
Fruit flies (Drosophila melanogaster) were provided by Hunan Normal
77
University and reared on a corn meal medium. The CdCl2 (1 g/L) solution was added
78
into the corn meal medium to obtain Cd-containing fruit flies. The final CdCl2
79
concentration in medium was 1 mg/L, which is equivalent to middle level of soil 4
80
pollution. Female spiders were collected from farmland without heavy metal pollution
81
in the Hunan Academy of Agricultural Science (Wang et al. 2018), feeding with
82
either normal (control group) or Cd containing fruit flies daily. Spiders were collected
83
every 5 days for Cd determination using Inductively Coupled Plasma (ICP) analysis.
84
Ovaries tissues were dissected from spiders that fed for 30 days, and used for
85
determination of enzymes activities and high-throughput sequencing (RNA-seq, small
86
RNA sequencing and proteome sequencing).
87
2.2 Determination of Cd content and enzyme activities
88
The Cd content in the spider were analyzed with the Plasma Atomic Emission
89
Spectrometer (ICPE-9000, SHIMADZU CORPORATIOM, JAPAN) as described by
90
Wang et al. (2018). Enzyme-linked immunosorbent assay (ELISA) kits (ZCI BIO,
91
China) including the Insect GST ELISA kit, Insect SOD ELISA kit, Insect POD
92
ELISA kit and Insect CAT ELISA kit were applied for determining activities of
93
glutathione S-transferase (GST), superoxide dismutase (SOD), peroxidase (POD), and
94
catalase (CAT) respectively. In addition, the Insect VTG ELISA kit (ZCI BIO, China)
95
and Insect MT ELISA kit (NJJCBIO, China) were used for determination of
96
vitellogenin content and metallothionein (MT) content in spider.
97
2.3 Mul-tiomics analysis
98
2.3.1 Transcriptome sequencing
99
A total of 180 spiders (Controls: 30/group, three replicates; Cd-treated: 30/group,
100
three replicates) were dissected for ovaries. Total RNA was extracted using the
101
TRIzol Reagent (Invitrogen, USA), and the purity was determined using the 5
102
NanoPhotometer® spectrophotometer (IMPLEN, CA, USA). cDNA libraries were
103
constructed using a NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB,
104
USA), and index codes were added to attribute sequences to each sample. These
105
libraries were sequenced on the Illumina sequencing platform, to generate the raw
106
reads. Reads were assembled by Trinity software and redundant sequences were
107
eliminated by TGIC software to obtain a set of available unigenes for subsequent
108
bioinformatics analyses (Pertea et al. 2003, Grabherr et al. 2011).
109
The FPKM (Fragments Per Kilobase of transcript per Million mapped reads) of
110
each gene was calculated based on the length of the gene and reads count mapped to
111
this gene to quantify the expression level of gene (Trapnell et al. 2010). Differentially
112
expressed genes (DEGs) was screened using the DESeq2 R package (1.16.1) with
113
threshold value p-value <0.05, and absolute value of Log2foldchange value >1. All
114
genes were aligned by Blast searching against protein databases, Nr (non-redundant
115
protein database, NCBI), and Swiss-prot retrieving proteins with the highest sequence
116
similarity to the given genes along with their protein functional annotations. KEGG
117
(Kyoto Encyclopedia of Genes and Genomes) enrichment analysis was conducted to
118
understand the biological function of DEGs (Ogata et al. 2000). Transcriptome
119
sequencing and small RNA sequencing were conducted by OEbiotech Company
120
(Shanghai, China).
121
2.3.2 small RNA sequencing
122
Ovaries were dissected from P. pseudoannulata for small RNA sequencing.
123
Sequencing libraries were generated using the NEBNext® Multiplex Small RNA 6
124
Library Prep Set for Illumina® (NEB, USA.), and sequenced on an Illumina Hiseq
125
2000 platform. Single-end reads of 50 bp were generated. Raw reads containing poly
126
A or T or G or C and with low quality were removed to obtain cleans reads for the
127
bioinformatics analysis.
128
Clean reads were mapped on the transcriptome sequences of ovary from P.
129
pseudoannulata by Bowtie (Langmead et al., 2009) to analysis their expression and
130
distribution on the reference. miRbase database (http://www.mirbase.org/) was
131
applied to conduct known miRNA alignment, and software miREvo and mirdeep2
132
were integrated to predict novel miRNA (Friedländer et al. 2012, Wen 2012).
133
Software miRanda and DESeq R packages were applied to predict target genes of
134
miRNAs and screen differentially expressed miRNAs (DEMs, p<0.05) (Enright et al,
135
2003). KEGG enrichment analysis was used on the target gene candidates of
136
differentially expressed miRNAs by KOBAS software (Mao et al., 2005). The small
137
RNA sequencing was conducted by OEbiotech Company (Shanghai, China).
138
2.3.3 Proteomic study
139
Total proteins were extracted form ovaries of P. pseudoannulata for proteomic
140
study. Protein concentration was determined by Bradford protein assay (Bradford,
141
1976). Proteins were labeled using the iTRAQ® Reagent-8PLEX Multiplex Kit
142
(Sigma) for HPLC fractionation. LC-MS/MS analyses were performed using an
143
EASY-nLCTM 1200 UHPLC system (ThermoFisher) coupled with an Orbitrap Q
144
Exactive HF-X Mass Spectrometer (ThermoFisher) operating in the data-dependent
145
acquisition (DDA) mode. 7
146 147
For protein identification, database searches were performed by the search
148
engines, Proteome Discoverer 2.2 (PD 2.2, Thermo) on UniProt database
149
(http://www.uniprot.org). Protein with at least 1 unique peptide was identified at FDR
150
less than 1.0% on peptide and protein level, respectively. Proteins that contain similar
151
peptides, but could not be distinguished based on MS/MS analysis were grouped
152
separately as protein groups. Reporter Quantification (iTRAQ 8-plex) was used for
153
iTRAQ quantification. The protein quantitation results were statistically analyzed
154
using Mann-Whitney Test, the significant ratios, defined as p < 0.05 and ratio > 1.2,
155
were used to screen the differentially expressed proteins (DEPs). Here, proteomic
156
analysis was conducted by Novogene Biotech (Beijing, China).
157
2.4 Expression validation of DEGs and DEMs
158
In total of 8 miRNAs and 12 genes with different expression patterns were
159
selected for validation by quantitative real-time RT-PCR (qRT-PCR). Total RNA was
160
obtained from excess samples used in High-throughput Sequencing. qRT-PCR
161
analysis for DEGs was conducted as described in our previous study (Wang et al.
162
2019); Expression validation of mRNAs was carried out using miScript II Reverse
163
Transcription Kit (Qiagen, Germany) and QuantiFast® SYBR® Green PCR Kit
164
(Qiagen, Germany) according the manufacturer’s instructions. The expression levels
165
of mRNAs and miRNAs were quantified using the 2-
166
Schmittgen 2001).
167 8
Ct
method (Livak and
168 169
3. Results
170
3.1 Cd accumulation in spiders
171
ICP analysis showed that the Cd content in Cd-containing spiders increased with
172
feeding time, ranging from 4.059 ± 0.62 µg/g at day 5 to 7.27 ± 0.34 µg/g at day 30,
173
and declined to a lower level of 5.12 ± 0.96 µg/g at day 35 (Fig.1). In the following
174
studies, the samples from the spiders fed with the fruit flies for 30 days were used for
175
High-throughput sequencing.
176 177
Fig. 1 Bioaccumulation of cadmium in P. pseudoannulata. The histogram bars
178
indicate amounts of Cd (mean ± SE) detected using ICP analysis in P. pseudoannulata
179
adults fed on Cd -containing fruit flies for the indicated time periods. Bars annotated
180
with the same lowercase letters are not significantly different (one-way ANOVA, p<
181
0.05). 9
182
3.2 Expression analysis and qRT-PCR analysis
183
We obtained 92,778 genes with an average length of 1,104bp, 1,265 proteins, and
184
724 miRNAs with 714 known and 10 novel miRNAs from ovaries of P.
185
pseudoannulata by means of High-throughput sequencing (Fig. 2A). The raw reads of
186
transcriptome and miRNAs had been submitted into NCBI SRA database (Accession
187
numbers: PRJNA577066 and PRJNA577205). Expression analysis showed that there
188
were 4,535 DEGs with 1,795 up-regulated and 2,740 down-regulated, 231 DEPs with
189
82 up-regulated and 149 down-regulated, and 67 DEMs with 55 up-regulated and 11
190
down-regulated between the treated and control samples.
191
To confirm the results of the differential expression analyses, eight Cd-induced
192
miRNAs and 12 Cd-induced mRNAs were selected for qRT-PCR analysis. Primer
193
sequences of DEGs and DEMs, and target genes of DEMs were listed in Table S1. As
194
showed in Fig. 2B, the expression profiles of all DEGs and DEMs were consistent
195
with the RNA-seq data.
10
196 197
Fig. 2 Expression analysis and qRT-PCR verification. A Expression level analysis
198
for transcriptome, proteome and miRNA. B qRT-PCR verification of DEGs and
199
DEMs. A good correlation of qRT-PCR results and high-throughput sequencing data
200
were shown through comparing these results. The actual ID of DEGs and DEMs
201
corresponding to the miRNA-1 to miRNA-8, and mRNA-1 to mRNA-12 on the
202
X-axis were showed in Table S1.
203 204 205
3.3 Influencing of Cd on spider detoxifying enzyme activities Ovaries of P. pseudoannulata were dissected for detoxifying enzyme activity
206
determination. Results from the ELISA assays showed that activities of GST (controls:
207
21.880 ± 1.381 U, Cd-treated: 16.303 ± 0.517 U), POD (controls: 33.657 ± 6.173 U,
208
Cd-treated: 10.323 ± 1.333 U), and SOD (controls: 7.533 ± 0.175 U, Cd-treated:
209
3.907 ± 0.400 U) were significantly decreased in response to Cd toxicity, down by 11
210
25.5% for GST, down 69.3% for POD and down by 48.1% for SOD, while the
211
activity of CAT was unaffected (Fig. 3A). In addition, content of metallothionein was
212
increased in spider under Cd burden (controls: 245.963 ± 1.237 ng/g, Cd-treated:
213
353.083 ± 16.774).
214
At the transcriptional level, a total of 31, 11, 15, and 2 genes encoding GST, SOD,
215
POD and CAT respectively were obtained from transcriptome of spider ovarian
216
samples. Gene expression analysis showed that 8 GST genes with 6 down-regulated
217
and 2 up-regulated, 1 SOD gene, 1 POD gene, and 1 CAT gene were differentially
218
expressed in the ovaries of spiders triggered by Cd burden (Fig. 3B).
219
Correlation analysis between transcriptome and proteome suggested that six
220
proteins and their encoding genes were in an accordant expression trend (both
221
down-regulated), but two in the reverse trend (proteins up-regulated but encoding
222
genes down-regulated, Table 1). Of these, two proteins involved in activities of GST
223
and POD (A0A087U9G0, A0A087TPB8) were negatively expressed both in
224
transcriptional and translational levels ((p<0.05, Table 1).
225
12
226 227
Fig. 3 Effects of Cd on spider detoxifying enzyme activities. A Determination of
228
detoxifying enzyme activities using ELISA assay; B Integrated analysis between
229
DEGs and DEMs.
230
13
231
Table 1.
Genes encoding detoxifying enzymes in ovaries of the spider
Protein ID
Up or Down
mRNA ID
Up or Down
Functions
A0A087UNQ7
up
comp147789c0seq1
down
A0A087TCX6 A0A087U9G0
up down
comp96347c0seq1 CL19176Contig1
down down
A0A087TPB8
down
comp244344c0seq1
down
A0A087TW10
down
comp207851c0seq2
down
A0A087T6N3
down
CL1Contig1437
down
A0A087UE31 A0A087UJM0
down down
comp21618c0seq1 comp92783c0seq1
down down
Delta-1-pyrroline-5-carboxylate dehydrogenase ELAV-like protein 2 Glutathione S-transferase fragment Thioredoxin-dependent peroxide reductase Voltage-dependent anion-selective channel protein 2 Basement membrane-specific heparan sulfate proteoglycan core protein Collagen alpha-2(IV) chain FAS-associated factor 1
232 233
3.4 Influence of Cd-containing prey on spider fecundity
234
Egg sacs and ovaries were collected from the adult spiders to record the number
235
of eggs and determine the vitellogenin concentration, respectively. Significant
236
decrease in the number of eggs from 117 to 64 (down by 45.2%) and the
237
concentration of vitellogenin from 3,212 to 2,433 µg/g (down by 24.2%) were
238
observed on Cd-containing spiders when compared with controls (Table 2).
239
Vitellogenin is the precursor molecule of vitellin, which is deposited in the
240
growing spider oocytes of the maturating female, and is also a main step of the
241
reproductive process in spider (Pourié and Trabalon 2003, Guo et al. 2018). A total of
242
10 vitellogenin encoding genes were obtained through Swiss-prot annotation, of
243
which 5 genes were down-regulated by Cd burden (p<0.05, absolute value of Log2
244
Fold change >1, Table 3). For proteome, the precursor protein vitellogenin-6 was 14
245
up-regulated, but the lipovitellin-1 (C0HJA5), a main composition of vitellin, was
246
up-regulated (p<0.05, Table 4).
247 248
Table 2. Effects of Cd on fecundity of the spider at individual level Vg* Concentration Fecundity (µg/g) Controls 3211.68 ± 51.957 117.18 ± 12.544 Cd-treated spiders
2433.11 ± 94.814*
64.22 ±26.1093*
249 250
Vg*: vitellogenin
251
Table 3 Effects of Cd on fecundity of the spider at transcriptional level Gene id Gene FPKM FPKM annotation value-Cd-treated calue-controls CL1751Contig1 Vitellogenin 51.5948 2.8984 comp174356_c0_seq1 Vitellogenin-5 16.0196 1.2146 comp178497_c0_seq1 Vitellogenin-2 10.4701 0.0000 3.8933 0.4200 comp212587_c0_seq1 Vitellogenin comp21587_c0_seq1 Vitellogenin 154.2527 0.3362 CL3318Contig1 Vitellogenin-6 30.2484 10.9338 0.0000 comp174358_c0_seq1 Vitellogenin-5 2.8248 comp174360_c0_seq1 Vitellogenin-5 30.1711 3.1042 0.5803 0.0000 comp219203_c0_seq1 Vitellogenin comp4775_c0_seq1 Vitellogenin 112.4606 19.4026
Up or Down Down Down Down Down Down / / / / /
pval 3.1E-12 0.048205 1.23E-08 0.000387 0.019362 0.064454 0.134049 0.06283 0.22662 0.319279
252 253
Table 4 Effects of Cd on fecundity of the spider at translational level Accession Description Average Up or Down CDLC/CKLC A0A087T463 Vitellogenin-6 1.655715113 Up C0HJA5 Lipovitellin-1 0.431626468 Down
t test p-value 0.0447143 0.0117556
254 255
3.5 Effect of Cd on the MAPK signaling pathway
256
The mitogen-activated protein kinase (MAPK) signaling cascades are highly
257
conserved from yeast to mammals, and play a crucial role in oocyte meiosis and
258
maturation in ovary (Motola et al 2010, Liu et al 2019). Gene expression analysis
259
indicated that 24 genes were differentially expressed in the MAPK signaling pathway 15
260
(ko04013), with 21 down-regulated (marked with green box) and 3 up-regulated
261
(market with red box, Fig. 4). These DEGs were acting on 18 nodes of ko04013,
262
including Tor (tyrosine-protein kinase receptor torso), Egfr (epidermal growth factor
263
receptor), Drk (growth factor receptor-binding protein 2), Dos (protein daughter of
264
sevenless), Ras85D (GTPase KRas), Ksr (kinase suppressor of Ras 2), Pointed
265
(C-ets-1), Hkb (huckebein), Pros (prospero homeobox 1), Btk29A (tyrosine-protein
266
kinase Tec), Rac (Ras-related C3 botulinum toxin substrate 1), dCyld (ubiquitin
267
carboxyl-terminal hydrolase CYLD), dTak1(mitogen-activated protein kinase kinase
268
kinase 7), Raw (raw), Cka (striatin 1/3/4), Dpp (bone morphogenetic), and Mef2
269
(MADS-box transcription enhancer factor 2A).
270
miRNA is a small non-coding RNA molecule that functions in RNA silencing
271
and post-transcriptional regulation of gene expression, by base-pairing with
272
complementary sequences within mRNA molecules. KEGG enrichment analysis was
273
applied for target genes of DEMs between Cd-treated spiders and controls. There
274
were 17 enriched pathways mapped by 115 target genes of DEMs, of which the
275
MAPK signaling pathway (ko04013) was most enriched, followed by EGFR tyrosine
276
kinase inhibitor resistance (ko01521), Novobiocin biosynthesis (ko00401), and ErbB
277
signaling pathways (ko04012, FDR<0.01, Table S2). Here, in the MAPK signaling
278
pathway, three nodes such as Egfr, Cka, and Omb (T-box protein 2) (marked with blue
279
shading) were mapped by target genes of 5 known miRNAs, including
280
dan-miR-318>der-miR-318>dgr-miR-318>dme-miR-318-3p>dmo-miR-318>dpe-mi
281
R-318>dps-miR-318>dse-miR-318>dsi-miR-318>dvi-miR-318>dwi-miR-318>dya-m 16
282
iR-318,
xbo-miR-92a,
283
ame-miR-278>cqu-miR-278>dan-miR-278>der-miR-278>dgr-miR-278>dme-miR-27
284
8-3p>dmo-miR-278>dpe-miR-278>dps-miR-278>dse-miR-278>dsi-miR-278>dvi-mi
285
R-278-3p>dwi-miR-278>dya-miR-278,
286
between
287
dan-miR-318>der-miR-318>dgr-miR-318>dme-miR-318-3p>dmo-miR-318>
288
dpe-miR-318>dps-miR-318>dse-miR-318>dsi-miR-318>dvi-miR-318>dwi-miR-318
289
>dya-miR-318 had negative regulation on its target gene CL1Contig670, while
290
expression level of other target genes were not changed (Fig. 4). According to the map
291
04013, this pathway was involved in oogenesis or egg polarity in animals (Fig. 4).
DEGs
and
prd-miR-235-3p,
DEMs
and
aae-miR-278-3p>aga-miR-278>
odi-miR-92a.
suggested
that
Integration only
one
analysis miRNA
292
Integrated analysis between DEGs and DEMs also indicated that genes encoding
293
serine/threonine-protein kinase mTOR, glycine dehydrogenase, and tyrosine
294
aminotransferase were also negatively regulated by five DEMs (Table 5).
17
295 296
Fig. 4. Effect of Cd on the MAPK signaling pathway. Proteins or genes marked
297
with green or red boxes were mapped by DEGs, and marked with blue shading were
298
mapped by target genes of DEMs. One box may be mapped by several genes.
299 300
18
301
Table 5. Integration analysis for DEMs and DEGs p
Up or
DEMs
Up or
p
Down
value
Target genes value
Down
KO_Definition
Serine/threonineCL36Contig2
Down
0.016
protein kinase mTOR
dpe-miR-317>dps-miR-317
0.022
Up
comp171382_c0_seq1
0.028 Down Glycine
comp186723_c0_seq3
0.017 Down
dehydrogenase
comp248081_c0_seq1
Down
0.038
dan-miR-318>der-miR318>dgr-miR-318>dmemiR-318-3p>dmo-miR318>dpe-miR-318>dps0.089
Up
CL1Contig670
Down
0.033
0.305
Up
comp102460_c0_seq2
Down
0.005
Striatin 1/3/4
miR-318>dse-miR-318>dsimiR-318>dvi-miR318>dwi-miR-318>dyamiR-318 Tyrosine dpe-miR-31b>dps-miR-31b
aminotransferase dme-miR-986-5p>dps-miR0.309
Up
comp210471_c0_seq2
Down
0.015
Up
comp7518_c0_seq1
Down
0.005
986-5p>dsi-miR-986-5p dme-miR-958-3p>dps-miR4E958-3p>dsi-miR-958-
09 3p>dvi-miR-958-3p comp171382_c0_seq1
Down
0.028
comp186723_c0_seq3
Down
0.017
dan-miR-317>der-miR317>dgr-miR-317>dme-
Glycine 0.011
Up
dehydrogenase
miR-317-3p>dse-miR317>dsi-miR-317>dvi-miR-
comp248081_c0_seq1
Down
0.038
302 303 304 305
4. Discussion Cd is toxic heavy metal widely distributed in the environment (Kim and Koo 19
306
2008). After Cd enters the various trophic levels via food web, there is little excretion
307
due to the lack of an effective Cd elimination pathway in organisms, and the
308
remaining Cd is accumulated in target organs (Seebaugh et al. 2005, Cai et al. 2009).
309
In this study, the Cd content in the spider increased with feeding time, then decreased
310
slightly, when fed with Cd-containing fruit flies that reared on corn meal medium
311
supplemented with Cd at the medium Cd pollution level. This is similar to the report
312
by Li et al (2016), when P. pseudoannulata was exposed to low and high CdCl2
313
solutions (Li et al. 2016). Antioxidant enzymes include SOD, CAT, GST and POD
314
were found extensively in organisms. These enzymes are generally considered
315
sensitive biomarkers of an organism’s antioxidant response and protect cells from
316
reactive oxygen species (ROS) toxicity (Lei et al. 2011, Li and Sunde 2016).
317
Significant correlations between the concentration of heavy metals and activities of
318
GST, GPOX, and GSTPx were observed in spiders A. labyrinthica and P. lugubris
319
(Wilczek et al. 2004); Activities of SOD, CAT and GST were increased in P.
320
pseudoannulata, when they exposed to Cd for 7 days (Li et al. 2016). Reproduction
321
organs are one of the most important target organs for Cd accumulation and
322
intoxication, causing a variety of physiological changes in ovary (Li et al. 2010, Yang
323
et al. 2012). Here in the present study, Cd stress decreased the activities of SOD, GST
324
and POD in the ovary of P. pseudoannulata. Meanwhile, expression level of
325
detoxifying enzyme encoding genes were up or down regulated, and proteins related
326
to activities of detoxifying enzymes were down-regulated. Among them, two proteins
327
involved in activities of GST and POD (A0A087U9G0, A0A087TPB8) were 20
328
negatively expressed both at transcriptional and translational levels. This implied that
329
antioxidant system in the ovary of the spider may be impaired, due to decreased
330
activity and negative expression of related genes and proteins.
331
Cd is known as the inhibitor of protein synthesis in vitro (Norton and Kench
332
1977). In arthropod, Cd has been reported to inhibit the vitellogenesis leading to
333
reduction of yolk synthesis and egg production (Płachetka-Bożek et al 2018. Cervera
334
2005, Zhao 2016). Vitellogenin is the precursor molecule of vitellin, its synthesis
335
could be inhibited by Cd burden in the ovary of P. pseudoannulata. In the present
336
study, multi-omics data indicated that 5 vitellogenin encoding genes were down
337
regulated. Although protein vitellogenin-6 (A0A087T463) was up-regulated, but
338
protein lipovitellin-1 (C0HJA5) is down regulated, which was the main composition
339
of vitellin. Vitellin is needed for the survival of embryos before the offspring can feed
340
independently (Engelmann 1979, Cabrera et al. 2009). In the present study, the
341
decreased fecundity of P. pseudoannulata was triggered by Cd induced inhibition of
342
vitellin synthesis.
343
The MAPK signaling cascade is highly conserved from yeast to mammals, and
344
plays a crucial role in oocyte meiosis and maturation in ovary (Motola et al 2010,
345
Liu et al 2019). In oocytes, MAPK can play a role as a component of cytostatic factor
346
to suppress DNA replication between meiosis I and II; And the vitellogenin
347
transcription is also mediated by the MAPK-dependent cascades (Chen et al., 2018).
348
But Cd exposures would alter the MAPK pathway by disturbing the state of
349
phosphorylation of Cdc2 and histone H3, and then cause delayed oocyte maturation of 21
350
the African clawed frog Xenopus laevis (Slaby et al. 2017). Cd treatment can alter the
351
splicing of the kitl pre-mRNA in ovarian granulosa cells, and miRNAs play regulatory
352
roles in the alternative splicing of kitl. These miRNAs are enriched in several
353
pathways like the MAPK signaling pathway, the Ras signaling pathway,and the Foxo
354
signaling pathway (Wang et al. 2017). In the present study, integrate analysis was
355
conducted for DEGs and DEMs. A correlation between the expression of the MAPK
356
signaling pathway and Cd stress was identified in P. pseudoannulata. A
357
down-regulated gene (CL1Contig670) was regulated by the up-regulated miRNA
358
(dan-miR-318>der-miR-318>dgr-miR-318>dme-miR-318-3p>dmo-miR-318>dpe-mi
359
R-318>dps-miR-318>dse-miR-318>dsi-miR-318>dvi-miR-318>dwi-miR-318>dya-m
360
iR-318), which encodes protein Cka in the MAPK signaling pathway. This provides a
361
new aspect for exploring the mechanism related to Cd-induced ovarian toxicity in
362
spider.
363
5. In conclusion
364
Cd stress possesses the distinct female reproductive toxicity on P. pseudoannulata.
365
Antioxidant system in the ovary of the spider may be impaired by decreased activities
366
of GST, POD and SOD. Inhibition of vitellin synthesis and negative regulation of the
367
MAPK signaling pathway by Cd may contribute to reduced egg production of the
368
spider.
369 370 371 22
372
Declaration of Competing Interest
373
The authors declare that they have no known competing financial interests or
374
personal relationships that could have appeared to influence the work reported in this
375
paper.
376
Acknowledgment
377
This work was supported by the Natural Science Foundation of China
378
(No.31472017,31272339), by the Open Research Fund of Key Laboratory of Tropical
379
Disease Control and Research, Ministry of Education in China (2018 kfkt03), and by
380
Project supported by the Hunan Provincial Education Department (18A024).
381 382
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Table 1. Genes encoding detoxifying enzymes in ovaries of the spider Protein ID
Up or Down
mRNA ID
Up or Down
Functions
A0A087UNQ7 up
comp147789c0seq1
down
Delta-1-pyrroline-5-carboxylate dehydrogenase
A0A087TCX6
up
comp96347c0seq1
down
ELAV-like protein 2
A0A087U9G0
down
CL19176Contig1
down
Glutathione S-transferase fragment
A0A087TPB8
down
comp244344c0seq1
down
Thioredoxin-dependent peroxide reductase
A0A087TW10
down
comp207851c0seq2
down
Voltage-dependent anion-selective channel protein 2
A0A087T6N3
down
CL1Contig1437
down
Basement membrane-specific heparan sulfate proteoglycan core protein
A0A087UE31
down
comp21618c0seq1
down
Collagen alpha-2(IV) chain
A0A087UJM0
down
comp92783c0seq1
down
FAS-associated factor 1
Table 2. Effects of Cd on fecundity of the spider at individual level Vg* Concentration (µg/g)
Fecundity
Controls
3211.68 ± 51.957
117.18 ± 12.544
Cd-treated spiders
2433.11 ± 94.814*
64.22 ±26.1093*
Vg*: vitellogenin
Table 3 Effects of Cd on fecundity of the spider at transcriptional level Gene id
Gene annotation
FPKM valueCd-treated
FPKM caluecontrols
Up or Down
pval
CL1751Contig1
Vitellogenin
51.5948
2.8984
Down
3.1E-12
comp174356_c0_seq1 Vitellogenin5
16.0196
1.2146
Down
0.048205
comp178497_c0_seq1 Vitellogenin2
10.4701
0.0000
Down
1.23E-08
comp212587_c0_seq1 Vitellogenin
3.8933
0.4200
Down
0.000387
comp21587_c0_seq1
Vitellogenin
154.2527
0.3362
Down
0.019362
CL3318Contig1
Vitellogenin6
30.2484
10.9338
/
0.064454
comp174358_c0_seq1 Vitellogenin5
2.8248
0.0000
/
0.134049
comp174360_c0_seq1 Vitellogenin5
30.1711
3.1042
/
0.06283
comp219203_c0_seq1 Vitellogenin
0.5803
0.0000
/
0.22662
comp4775_c0_seq1
112.4606
19.4026
/
0.319279
Vitellogenin
Table 4 Effects of Cd on fecundity of the spider at translational level Accession
Description
Average CDLC/CKLC
Up or Down
t test p-value
A0A087T463
Vitellogenin6
1.655715113
Up
0.0447143
C0HJA5
Lipovitellin1
0.431626468
Down
0.0117556
Table 5. Integration analysis for DEMs and DEGs DEMs
dpe-miR-317>dps-miR-317
p value
0.022
Up or Down
Up
Up or Down
p value
KO_Definition
CL36Contig2
Down
0.016
Serine/threonineprotein kinase mTOR
comp171382_c0_seq1
Down
0.028
comp186723_c0_seq3
Down
0.017
comp248081_c0_seq1
Down
0.038
Target genes
Glycine dehydrogenase
dan-miR-318>der-miR318>dgr-miR-318>dmemiR-318-3p>dmo-miR318>dpe-miR-318>dpsmiR-318>dse-miR-318>dsimiR-318>dvi-miR318>dwi-miR-318>dyamiR-318
0.089
Up
CL1Contig670
Down
0.033
Striatin 1/3/4
dpe-miR-31b>dps-miR-31b
0.305
Up
comp102460_c0_seq2
Down
0.005
Tyrosine aminotransferase
dme-miR-986-5p>dps-miR986-5p>dsi-miR-986-5p
0.309
Up
comp210471_c0_seq2
Down
0.005
dme-miR-958-3p>dps-miR958-3p>dsi-miR-9583p>dvi-miR-958-3p
0.015
Up
comp7518_c0_seq1
Down
4E09
comp171382_c0_seq1
Down
0.028
comp186723_c0_seq3
Down
0.017
comp248081_c0_seq1
Down
0.038
dan-miR-317>der-miR317>dgr-miR-317>dmemiR-317-3p>dse-miR317>dsi-miR-317>dvi-miR317-3p>dwi-miR-317>dyamiR-317
0.011
Up
Glycine dehydrogenase
Highlight Cd stress possesses the distinct female reproductive toxicity on P. pseudoannulata Cd exposures damage the antioxidant system of ovary in P.pseudoannulata Vt synthesis was affected by Cd both at transcriptional and translational levels
Expression of MAPK signaling pathway was negative regulated by miRNA under Cd stress
Conflict of Interest Statement The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.