Transcriptional response to sulfide in the Echiuran Worm Urechis unicinctus by digital gene expression analysis
© Liu et al. 2015
Received: 25 February 2015
Accepted: 15 October 2015
Published: 21 October 2015
Urechis unicinctus, an echiuran worm inhabiting the U-shaped burrows in the coastal mud flats, is an important commercial and ecological invertebrate in Northeast Asian countries, which has potential applications in the study of animal evolution, coastal sediment improvement and marine drug development. Furthermore, the worm can tolerate and utilize well-known toxicant-sulfide. However, knowledge is limited on the molecular mechanism of U. unicinctus responding to sulfide due to deficiency of its genetic information.
In this study, we performed Illumina sequencing to obtain the first Urechis unicinctus transcriptome data. Sequenced reads were assembled and then annotated using blast searches against Nr, Nt, Swiss-Prot, KEGG and COG. The clean tags from four digital gene expression (DGE) libraries were mapped to the U. unicinctus transcriptome. DGE analysis and functional annotation were then performed to reveal its response to sulfide. The expressions of 12 candidate genes were validated using quantitative real-time PCR. The results of qRT-PCR were regressed against the DGE analysis, with a correlation coefficient and p-value reported for each of them.
Here we first present a draft of U. unicinctus transcriptome using the Illumina HiSeqTM 2000 platform and 52,093 unique sequences were assembled with the average length of 738 bp and N50 of 1131 bp. About 51.6 % of the transcriptome were functionally annotated based on the databases of Nr, Nt, Swiss-Prot, KEGG and COG. Then based on the transcriptome, the digital gene expression analysis was conducted to examine the transcriptional response to sulfide during 6, 24 and 48 h exposure, and finally 1705, 1181 and 1494 tag-mapped genes were identified as differentially expressed genes in the 6-h, 24-h and 48-h libraries, then were further subjected to pathway analyses.
In the DGE database of U. unicinctus, the alterations in certain known sulfide-related pathways indicate similar changes in response to sulfide. For more than 80 % of the identified pathway members, this is the first report on their association with sulfide stress, among which glycolysis pathway and PIDD involving pathways were unique and discussed in details, and were thought to play important roles in the sulfide tolerance of U. unicinctus. All the results are helpful to explain the mechanism of sulfide tolerance and detoxification.
KeywordsDigital gene expression analysis Sulfide Transcriptional profile Urechis unicinctus
Exogenous sulfide, a common environmental toxin, harms organisms by reducing the affinity of hemoglobin for oxygen , inhibiting the cytochrome c and succinate oxidase complexes [2, 3], depolarizing mitochondria , inducing apoptosis , and causing oxidative damage to RNA and DNA . Moreover, sulfide is produced by mammals in vivo [7, 8] and endogenous sulfide is involved in multiple physiological functions, including inflammation , up-regulation of the antioxidant system , and activation of KATP channels . Thus, exploring the physiological effects of sulfide and organismal sulfide tolerance mechanisms is important.
Responses to sulfide exposure are distinct in different organisms. The nematode Caenorhabditis elegans survives in the presence 50 ppm sulfide, which increases its thermotolerance and lifespan . Mice exposed to 80 ppm sulfide enter a hibernation-like state to overcome the conditions inducing a low metabolic rate and decreased core body temperature without any apparent ill effects . Organisms living in sulfide-rich habitats adopt a variety of defensive responses to avoid prolonged toxic exposure to sulfide; for example, endosymbiotic bacteria sulfide metabolism and mitochondrial sulfide oxidation are two primary strategies for sulfide tolerance in invertebrates . The hydrothermal vent tube worm Riftia pachyptila and the clam Solemya velum use hemoglobin to deliver sulfide to their endosymbiotic bacteria [15–17]. Metatranscriptome pyrosequencing of the endosymbiotic bacteria of the clam S. velum revealed 28 genes involved in diverse pathways associated with sulfide metabolism, including the dissimilatory sulfite reductase pathway, the adenosine-5′-phosphosulfate pathway and the sulfur oxidation pathway . For some species that lack endosymbionts, such as echiuran worm Urechis unicinctus, mitochondrial sulfide oxidation is important for their survival in sulfide-rich habitats, and several key enzymes, including sulfide:quinone oxidoreductase (SQR), sulfur dioxygenase (SDO), and sulfur transferase (ST), play important roles in sulfide detoxification [19–22]; however, due to the lack of sufficient high-throughput molecular data, the full complement of genes and signalling pathways involved in sulfide tolerance and utilization and the mechanisms that regulate these pathways are unknown.
The echiuran worm, U. unicinctus, inhabiting “U-shaped” burrows in intertidal and subtidal mudflats, is widely distributed in Russia, Japan, the Korean Peninsula and China, which belongs to phylum Echiura, a special group of invertebrate protostomes. Preliminary studies have been presented that the worm U. unicinctus is able to metabolize exogenous sulfide and utilize it as energy [23–25]. Moreover, characterizations of the key genes for mitochondrial sulfide oxidation [21, 22, 26–28] have been revealed. Here, a high-throughput methods—digital gene expression (DGE) technique was conducted to analyze the transcriptional response to sulfide in U. unicinctus. Before the DGE analysis, the transcriptome data of U. unicinctus is necessary, so we performed a large-scale transcriptome sequencing of U. unicinctus by Illumina HiSeqTM 2000 platform (BGI, Shenzhen, China) to obtain the first version of the U. unicinctus transcriptome.
The aims of the present study are to (i) characterize the U. unicinctus transcriptome; (ii) identify the genes and signalling pathways responding to sulfide in U. unicinctus. The results will provide a comprehensive understanding for further exploring the molecular mechanism of sulfide metabolism in U. unicinctus.
Output statistics of sequencing
Total raw reads
Total clean reads
Total clean nucleotides (nt)
Summary of the de novo assembly for U. unicinctus transcriptome
Total number of raw reads
Total number of clean reads
Total clean nucleotides (nt)
Average clean read length (bp)
Total number of contigs
Mean length of contigs (bp)
Contig size N50 (bp)
Total number of distinct clusters
Total number of distinct singletons
Total number of distinct unigenes
Unigene size N50 (bp)
Sequences with E-value <10−5
KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis showed that 18,884 unigenes (72.73 % of the total annotated sequences) were mapped to 254 KEGG pathways. Of these, metabolic pathways contained 2820 unigenes and were no doubt larger than other pathways, such as pathways in focal adhesion (717), cancer (654) and regulation of the actin cytoskeleton (645). Approximately 18 % of the unigenes were related to such pathways as immunity, stress, and growth.
DGE library sequencing and tag mapping
DGE sequencing statistics
Control 0 h
Exposure to Sulfide
Distinct tag number
Distinct tag number
All tags mapping to a gene
Total % of clean tags
Distinct tag number
Distinct tag % of clean tags
Unambiguous tags mapping to a gene
Total % of clean tag
Distinct tag number
Distinct tag % of clean tags
All tag-mapped genes
% of reference genes
Unambiguous tag-mapped genes
% of reference genes
Total % of clean tags
Distinct tag number
Distinct tag % of clean tags
To elucidate the molecular events underlying the DGE profiles, the clean tags were mapped to the previous transcriptome. In the control, 6-h, 24-h and 48-h libraries, 51.39, 45.48, 47.89 and 46.23 % of the distinct clean tags uniquely mapped to the reference sequence, generating 21,121, 20,526, 20,129 and 20,111 tag-mapped genes, respectively (Table 3).
DEG (differentially expressed gene) identification, validation and pathway enrich analysis
KEGG pathway analysis of the DEGs revealed that 175, 166 and 176 metabolic pathways were affected in U. unicinctus exposed to 50 μM sulfide for 6, 24, and 48 h, respectively and the pathways including oxidative phosphorylation, ribosome, proteasome, complement and coagulation cascades, and metabolism were all significantly enriched in the 6-h, 24-h and 48-h library (Additional file 2).
U. unicinctus, belonging to the phylum Echiura, can tolerate high concentrations of sulfide living in the “U-shaped” burrows in intertidal and subtidal mudflats. In this study, we first demonstrated a de novo assembly of the U. unicinctus transcriptome using Illumina Hiseq2000 platform. This is also the first transcriptome report in echiuran worms. In the obtained transcriptome of U. unicinctus, the assembly of the 53,874,422 clean reads produced 52,093 unique sequences with an average size of 738 bp which is comparable with other studies generating transcriptome data [29–31]. The number of protein-coding genes in the U. unicinctus genome is unknown; however, if we assume general conservation based on estimates for the representative of the phylum Annelida Capitella teleta (32,415 genes; JGI annotation pipeline), ~83 % of protein-coding genes (26,889) were assembled in this study. All the transcriptome data obtained in U. unicinctus indicated that it is appropriate to conduct DGE analysis to identify transcriptional response to sulfide in U. unicinctus.
DEGs and their associated pathways in U. unicinctus exposed to sulfide
U. unicinctus Unigene accession number
Differential expression multiplication log2 Ratio (TPM time/ TPM control)
NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 2-like
NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 9-like
succinate dehydrogenase [ubiquinone] iron-sulfur subunit
ubiquinol-cytochrome c reductase
cytochrome c oxidase subunit I
mitochondrial H+ ATPase a subunit
In this study, several unique pathways and more than 80 % of the identified pathway members are the first report, which are associated with sulfide stress in U. unicinctus exposed to sulfide. Among these pathways, ATP generated pathways (glycolytic and oxidative phosphorylation pathway) as well as the PIDD (p53-induced protein with a death domain) induced pathways involving in the DNA damage response attracted our interests.
Usually the anaerobic glycolytic pathway typically replaces oxidative phosphorylation for ATP production and the maintenance of the intracellular ATP balance when mouse or human cells are cultured in media containing 10 μM sulfide, and the expression of nearly all of the genes in the anaerobic glycolytic pathway are up-regulated [39, 40]. In U. unicinctus, it is confirmed that activities of key enzymes in the glycolytic pathway, phosphorylase, phosphofructokinase, and pyruvate kinase become markedly high after fertilization in U. unicinctus eggs [41, 42], and enzymes associated with oxidative phosphorylation for ATP production have also been elucidated in echiuran respiration [43–47]. In this study, we identified a large number of genes related to glycolytic and oxidative phosphorylation pathways, such as pyruvate kinase, phosphorylase, phosphofructokinase, ADP-dependent glucokinase and phosphoglycerate kinase (Table 4). Interestingly, the expressions of most unigenes involved in glycolysis such as ADP-dependent glucokinase and phosphoglycerate kinase were significantly down-regulated when the worms were exposed to sulfide (Table 4); moreover, the mRNA levels of both complex I and complex II were also down-regulated (Table 4). These results suggested unlike mammals, U. unicinctus may adopt a non-glycolytic pathway to produce ATP when sulfide exposure. Several sulfide-tolerant organisms—such as the ribbed mussel Geukensia demissa, echiuran worm U. unicinctus and the lugworm Arenicola marina—utilize sulfide to produce ATP [24, 48–50]. Hildebrandt  suggested that a super-complex composed of SQR and complexes III and IV initiates ATP production using sulfide as a material resource when sulfide enters the respiratory chain of rat mitochondria. In this study, the genes encoding SQR and complexes III and IV (Table 4) were all up-regulated in U. unicinctus exposed to sulfide. Therefore, we propose that the super-complex (SQR, complexes III and IV) catalytic activity by using sulfide as a material resource replaces the glycolysis to maintain the ATP balance when these worms are exposed to sulfide. Further studies are needed to confirm these suggestions.
It is known sulfide generates significant oxidative damage to genomic DNA . Typically, PIDD, a key member of the p53 DNA damage response pathway, activates two distinct pathways in response to DNA damage. One is to activate NF-κB via the ubiquitination of NEMO to stimulate DNA repair and promote organismal survival [53–55]; alternatively, PIDD induces genotoxic stress-mediated apoptosis and activates the mitochondrial apoptosis pathway to protect genome integrity . In this study, all the members involving in this pathway were all in up-regulated expression when U. unicinctus were exposed to sulfide (Table 4). Furthermore, it is suggested that PIDD acts in response to sulfide-induced DNA damage based on probably the increase of the PIDD mRNA level. Further researches need to be conducted to verify the suggestions from the DGE analysis.
In this study, a transcriptome from the U. unicinctus adult tissues was sequenced. This database is currently the largest in echiuran worms, and 52,093 unigenes are assembled. Many unigenes have been identified and are involved in various fields. Then based on the transcriptome, transcriptional profile of U. unicinctus exposed to 50 μM was revealed via DGE analysis. Approximately 1705, 1181 and 1494 DEGs were identified at 6, 24 and 48 h after sulfide exposure, respectively. These DEGs are involved in many biological processes. In the DGE database of U. unicinctus, the alterations in certain known sulfide-related pathways indicate similar changes in response to sulfide, whereas U. unicinctus exhibited differences from other species with respect to its modulation of glycolysis in response to sulfide. In addition, this is the first report on PIDD in response to sulfide-induced DNA damage in U. unicinctus. These data facilitates the identification of sulfide-related genes and elucidates the molecular mechanisms by which organisms tolerate sulfide stress. This study provides a framework for additional functional studies to examine sulfide toxicology in this system.
Each of the procedures that were used to handle and treat the Echiuran worms during this study was approved by the Ocean University of China Institutional Animal Care and Use Committee (OUC-IACUC) prior to the initiation of the study.
Animals and sulfide treatment
Urechis unicinctus (mean fresh mass of 33.4 ± 10.4 g) were purchased from an aquatic product market, which collected worms from a coastal intertidal flat in Yantai, China. The worms were temporarily maintained in aerated seawater (17 ± 1 °C, pH 8.0, and salinity 32 PSU) for a week without feeding. Then 30 healthy worms were randomly assigned to three groups (10 worms per group), and for each group the worms were maintained in a sealed aquarium containing 30 L of seawater. The sulfide concentration was maintained at 50 μM by adding a sulfide stock solution (10 mM Na2S, pH 8.0) every 2 h as necessary based on the sulfide concentration, which was determined using the methylene blue method . Six worms were sampled (i.e., two individuals from each aquarium) before sulfide addition (control) and at 6, 24 and 48 h after the initiation of sulfide exposure, frozen in liquid nitrogen, and stored at −80 °C for subsequent RNA extraction.
RNA extraction and quality control
Total RNAs from each stored tissues were extracted with TRIzol® Reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. The yield, purity and integrity of the RNA samples were analyzed using a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA).
cDNA library construction and illumina sequencing
The mRNA was purified using a PolyATract® mRNA Isolation System (Ambion, Austin, TX, USA). Fragmentation buffer was added to interrupt mRNA to short fragments. With these short fragments as templates, random hexamer-primer was used to synthesize the first-strand cDNA. The second-strand cDNA was synthesized using buffer, dNTPs, RNase H and DNA polymerase I. Short fragments were purified with QiaQuick PCR extraction kit (Qiagen, Hilden, Germany) and resolved with EB buffer for end reparation and tailing A. Next, the short fragments were connected with sequencing adapters. After agarose gel electrophoresis, suitable fragments were selected for the PCR amplification as templates. Finally, the library could be sequenced using Illumina HiSeq™ 2000, which could generate paired-end (PE) reads with a length of 90 bp.
De novo assembly and transcriptome annotation
Raw reads from Illumina sequencing were first pre-processed for quality control, such as filtering of high-quality reads based on the score value given in fastq files, removal of reads containing primer/adaptor sequences and trimming the read length using common programs. After comparison, the SOAP-denovo program was most effective for the assembly of 25-bp segments that are randomly clipped from the reads obtained in long parts with the help of the de Bruijn graph algorithm and was then used for de novo assembly of our PE reads and generation of non-redundant unique sequences.
Unigenes were firstly aligned for homologues by blastX (E-value < 10−5) to protein databases Nr, Swiss-Prot, KEGG and COG and then aligned by blastn to nucleotide databases Nt (E-value < 10−5). GO terms were assigned utilizing Blast2GO  for blast output obtained from the Nr database. After getting GO annotation for every unigene, WEGO software  was used to do GO functional classification for all unigenes and to understand the distribution of gene functions of the species from the macro level. With the help of KEGG database, pathway annotation for unigenes was obtained.
DGE library construction, sequencing and annotation
Four DGE libraries were constructed from the total RNA sample of the worms exposed to 50 μM sulfide for 0, 6, 24 or 48 h. For all libraries, mRNA was purified from 6 μg of total RNA sample using oligo(dT) magnetic beads and was reverse-transcribed into double-stranded cDNA (SuperScript II, Invitrogen). Two restriction enzymes were used to generate sequencing tags. The bead-bound cDNA was digested using NlaIII (CATG recognition site) and ligated to Illumina adaptor 1. The junction of Illumina adapter 1 and CATG generated the recognition site for MmeI. Thereafter, MmeI was used to cut the cDNA fragments 17 bp downstream of the CATG site. After removing the 3′ fragment via magnetic bead precipitation, Illumina adapter 2 was introduced at the site of MmeI cleavage, resulting in a library of tagged cDNA containing different adaptors on each end. Fifteen cycles of PCR amplification were performed to enrich the 21-bp tags in the samples. Massively parallel sequencing-by-synthesis was performed using an Illumina HiSeq™ 2000. Clean tags were obtained by filtering the adaptor sequences and low-quality tags using the Illumina data processing pipeline. The distinct clean tags were classified according to their copy number in the libraries, and the saturation of the four libraries was analyzed. The distinct clean tags were mapped to the transcriptome of U. unicinctus sequenced before. Only the tags displaying zero or one mismatch were annotated based on the reference genes.
Identification and validation of DEGs
To compare the gene expression in U. unicinctus at different periods of sulfide exposure, the number of raw clean tags in each library was first normalized to the number of transcripts per million clean tags (TPM). The expression analysis was then carried out using TPM of sulfide treatment groups compared with that of the control group. The thresholds for determining significant differences in gene expression were a FDR (false discovery rate) ≤ 0.001 and an absolute value of log2 Ratio ≥1. All DEGs were subjected to KEGG pathway enrichment analysis compared with the transcriptome background using hypergeometric test. The calculated P-value goes through as a threshold. KEGG pathway fulfilling corrected P-value ≤ 0.05 from Bonferroni correction were defined as significantly enriched terms in DEGs.
The expression of tag-mapped genes in the four libraries was validated using quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR). Primers were designed using Primer 5.0 software for 12 selected genes, including 8 up-regulated genes, 3 down-regulated genes and 1 gene not displaying differential expression (Additional file 3: Table S1). Total RNA sample (1 μg) from the stock samples used to construct the sequencing libraries was reverse-transcribed into cDNA using the Primescript™ RT Reagent Kit with gDNA Eraser (Takara, Otsu, Japan). All amplification reactions were performed using a LightCycler™ 480 (Roche, Basel, Switzerland) in a 20-μL volume containing 1 × SYBR Premix Ex Tap (Takara), 0.2 mM of each primer and 1.5 μL of cDNA. The amplification was conducted according to the following standard protocol: 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s, 60 °C for 1 min, and a thermal denaturing step to generate the melting curves for the verification of amplification specificity. The U. unicinctus β-actin (GenBank accession number: GU592178.1) was used as an internal standard, and the relative gene expression levels were calculated using the 2-ΔΔCt method, where -ΔCt is the difference in the Ct between β-actin and the target gene.
Availability of supporting data
Raw data was submitted to NCBI’s short read archive (SRA, SRA122323). The gene expression data induced by the sulfide are available in the online Supporting Information.
This work was supported by the National Natural Science Foundation of China (31372506 and 31072191).
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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