Transcriptome profiling analysis of rare minnow (Gobiocypris rarus) gills after waterborne cadmium exposure

CBD-00400; No of Pages 9 Comparative Biochemistry and Physiology, Part D xxx (2016) xxx–xxx

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Transcriptome profiling analysis of rare minnow (Gobiocypris rarus) gills after waterborne cadmium exposure Zhi-Jian Wang 1, Xiao-Hong Liu 1, Li Jin, De-Yong Pu, Jing Huang, Yao-Guang Zhang ⁎ Key Laboratory of Freshwater Fish Reproduction and Development Ministry of Education, Key Laboratory of Aquatic Science of Chongqing, Southwest University School of Life Sciences, 400715 Chongqing, China

a r t i c l e

i n f o

Article history: Received 13 November 2015 Received in revised form 9 May 2016 Accepted 22 May 2016 Available online xxxx Keywords: Transcriptome Rare minnow (Gobiocypris rarus) Cadmium Digital gene expression Inflammation

a b s t r a c t Rare minnow (Gobiocypris rarus) is a widely used experimental fish in risk assessments of aquatic pollutants in China. Cadmium (Cd) is one of the most toxic heavy metals in the world; however, few studies have used fish gills, a multi-functional organ. In this study, we characterized the differential expression of adult female rare minnow gills after sub-chronic waterborne Cd (75 μg/L CdCl2) exposure for 35 d. A total of 452 genes (209 upregulated and 243 down-regulated) were identified by gene expression profiling using RNA-Seq before and after treatment. Of these differentially expressed genes, 75, 21, and 54 differentially expressed genes are related to ion transport, oxidation-reduction processes, and the immune response, respectively. The results of GO and KEGG enrichment analyses, together with the altered transcript levels of major histocompatibility complex (MHC) class I and class II molecules and the significant increases in the levels of serum tumor necrosis factor α (TNF-α), interleukin 1β (IL1β) and nuclear factor-κB (NF-κB), indicated a disruption of the immune system, particularly the induction of inflammation and autoimmunity. The significant down-regulation of coagulation factor XIII A1 polypeptide (F13A1), tripartite motif-containing protein 21 (TRIM21), and Golgi-associated plant pathogenesis-related protein (GAPr) during both acute (≤96 h) and sub-chronic (35 d) waterborne Cd exposure, as well as their dosage dependence, suggested that these three genes could be used as sensitive biomarkers for aquatic Cd risk assessment. © 2016 Elsevier Inc. All rights reserved.

1. Introduction Rare minnow (Gobiocypris rarus) is a small freshwater fish that is endemic to China. Its characteristics, such as its small size, convenient maintenance, and short life cycle, are similar to those of the widely used animal model, the zebrafish. In addition, rare minnow is very sensitive to aquatic pollutants (Zha et al., 2007). It is a proposed experimental fish listed in the Guidelines for the Testing of Chemicals and Determination Methods for Examination of Water and Wastewater provided by the Ministry of Environmental Protection of the People's Republic of China. In China, it is currently a widely used experimental fish in risk assessments of aquatic pollutants, particularly various types of endocrine disruptors, such as industrial materials (Fang et al., 2010; Zhang et al., 2008), fungicides (Yang et al., 2011), pesticides (Li et al., 2009b), herbicides (Li et al., 2009a), heavy metals (Wang et al., 2014; Zhu et al., 2011, 2014), and nano-materials (Zhu et al., 2015). Zebrafish are a well-known animal model in toxicology studies, but they are less sensitive than rare minnows to some contaminants, such ⁎ Corresponding author. E-mail addresses: [email protected], [email protected] (Y.-G. Zhang). 1 These authors contribute equally to this study.

as cadmium (Cd), mercury (Hg), copper (Cu) and zinc (Zn) (Wang et al., 2013). In addition, gonad transcriptome data for rare minnow have recently become available (Gao et al., 2015). RNA-Seq is a revolutionary approach used in deep-sequencing technologies for transcriptomics, and provides precise measurements of transcripts and their isoforms (Wang et al., 2009). The highthroughput sequencing of RNA is now a widely used technique. It is a cost-effective method for characterizing gene-associated SNPs, and a total of 56,972 putative SNPs have been identified in Takifugu rubripes (Cui et al., 2014). Moreover, this method is more effective and sensitive for the discovery of certain toxic effects: for example, after exposure to 5–50 μg/L nano-silver for 28 d, no morphological alterations were detected in the zebrafish gill, but the levels of some genes related to development, growth, DNA damage and repair were changed (Griffitt et al., 2013). Fish gills are sensitive respiratory ion-regulatory membranes and constitute key organs that come into contact with waterborne pollutants (Kamaruzzaman et al., 2010). This organ is morphologically and functionally complex tissue. It serves as the site of numerous physiological processes that are vital to the maintenance of systemic homeostasis in variable environmental conditions, and perform most of the functions that are controlled by pulmonary and renal processes

http://dx.doi.org/10.1016/j.cbd.2016.05.003 1744-117X/© 2016 Elsevier Inc. All rights reserved.

Please cite this article as: Wang, Z.-J., et al., Transcriptome profiling analysis of rare minnow (Gobiocypris rarus) gills after waterborne cadmium exposure, Comp. Biochem. Physiol., D (2016), http://dx.doi.org/10.1016/j.cbd.2016.05.003

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in mammals (Evans et al., 2005). Gills are also important organs in the response to pathogens and thermal stress of rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar) (Morrison et al., 2006; Rebl et al., 2013; Wynne et al., 2008). Transfer of green spotted puffer fish (Tetraodon nigoviridis) from 10 ppt salinity containing 2.9 mM Ca2 + to high (10 mM Ca2 +) and low (0.01 mM Ca2 +) calcium water resulted in the expression of genes involved in Ca2 + signaling/homeostasis, cytoskeleton, energy production/homeostasis and tissue remodeling to become altered (Pinto et al., 2010). Cd, one of the most toxic heavy metals in the world, is a ubiquitous pollutant in water systems. Waterborne Cd has been reported to enter the body via chloride cells in the filament epithelium (Olsson, 1998) or via Ca2+ channels in the apical membrane (Verbost et al., 1989). Moreover, in addition to the liver and kidney, the fish gill is an organ that accumulates high concentrations of Cd (Jezierska and Witeska, 2006). However, the data available on fish gills are limited. Although some histological, histochemical, and ultrastructural alterations of fish gills (Brunelli et al., 2011; Gargiulo et al., 1996) have been reported, the liver and kidney are the targets that are most widely investigated in studies of the toxicity of most pollutants, particularly heavy metals such as Cd. To further investigate the response of fish to Cd and the function of gills in this response, we analyzed the alterations in the transcriptome profiles of adult female rare minnow gills after sub-chronic waterborne Cd exposure using a de novo Illumina sequencing platform and identified the differentially expressed genes. These data will form the basis for further studies of Cd toxicity. 2. Materials and methods 2.1. Cd challenge and sample preparation Healthy adult female rare minnows were raised in glass tanks (5 L) for 7 d to ensure their acclimation to non-circulating water (CaCO3: 35.72 mg/L; alkalinity: 7.0–7.5; dissolved oxygen: 6.8–7.3 mg/L). 260 adult fish were then randomly divided into six groups [control (0), 12, 24, 48, 72, and 96 h] for the trials of acute exposure [2.0 mg/L, less than LC50 value (Wang et al., 2013)], and the other six groups [control (0), 5, 25, 50, 75 and 100 μg/L] were subjected to a 35-day subchronic treatment. The doses were selected based on the Water Quality Standard for Fisheries (5 μg/L) and Discharge Standard of Water Pollutants for Pharmaceutical Industry Chemical Synthesis Products Category (100 μg/L), and these doses were b5% of the 96-h LC50 value (Wang et al., 2013). Each group contained 18–20 fish (6–7 fish per tank). During the acute CdCl2 treatment period, all fish were fasted, whereas all of the fish in the sub-chronic exposure groups were fed a commercial diet twice a day. Based on a previous study (Li et al., 2014a), one fourth of the water used for the acute and sub-chronic treatments was replaced daily with new water containing the corresponding dose of the contaminant to maintain its concentration. The room temperature was controlled at 25 ± 0.5 °C under a 12L:12D artificial photoperiod. After anesthesia using MS222 (Sigma, St. Louis, MO, USA), blood from each fish was collected according to the methods of a previous study (Pedroso et al., 2012). The gills and other tissues of 12 individuals in each group were dissected on ice, immediately frozen by liquid nitrogen and stored at −80 °C until use. All procedures were performed according to the accepted standards for animal care of Southwest University, under a permit id: [2014]25.

Cd_treated_G_1 and Cd_treated_G_2 were the biological replicates of the Cd-treated group, and control_non-treated_G_1 and control_nontreated_G_2 were the replicates of the control group. Total RNA was extracted using the TRIzol reagent according to the manufacturer's recommended protocol (Invitrogen, Burlington, ON, Canada). The RNA integrity was confirmed based on the 28S and 18S rRNA bands on 1% agarose gels, and the RNA purity was checked using a NanoPhotometer® spectrophotometer (IMPLEN, CA, USA). The concentration of extracted total RNA was measured using a Qubit® RNA Assay Kit in Qubit® 2.0 fluorometer (Life Technologies, CA, USA), and the RNA integrity was assessed using a RNA 6000 Nano Assay Kit with a Bioanalyzer 2100 (Agilent Technologies, CA, USA). The average RIN value of the abovementioned four samples was 8.5 ± 0.4 (range from 8.1 to 8.9). 2.3. Library preparation, clustering, and sequencing 3 μg of RNA per sample (Cd_treated_G_1, Cd_treated_G_2, control_non-treated_G_1, and control_non-treated_G_2) was used as input material for RNA sample preparations. Transcriptome libraries were generated using a NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, USA) following the manufacturer's recommendations, and index codes were added to attribute sequences in each sample. Briefly, after its purification and fragmentation, the mRNA was used for cDNA synthesis. First strand cDNAs were synthesized using a random hexamer primer and M-MuLV Reverse Transcriptase (RNaseH−), and then subjected to second strand cDNA synthesis using DNA Polymerase I and RNase H. To select cDNA fragments that were preferentially 150– 200 bp in length, the library fragments were purified with the AMPure XP system (Beckman Coulter, Beverly, MA, USA). 3 μL of USER Enzyme (NEB, USA) was added to the size-selected, adaptor-ligated cDNA, and the mixture was incubated at 37 °C for 15 min, and then at 95 °C for 5 min. PCR was then performed with Phusion high-fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. The PCR products were purified (AMPure XP system), and the library quality was assessed with an Agilent Bioanalyzer 2100 system using an Agilent DNA 12000 kit (Agilent Technologies, CA, USA). Clustering of the index-coded samples was performed using a cBot Cluster Generation System with a TruSeq PE Cluster Kit v3-cBot-HS (Illumina), according to the manufacturer's instructions. After cluster generation, the library preparations were sequenced on an Illumina HiSeq 2500 platform, and paired-end reads were generated. 2.4. Transcriptome data analysis, assembly, and annotation Raw data (raw reads) in the FASTQ format were processed using inhouse Perl scripts to remove reads that contained adapters or poly-N and low-quality reads. The Q20, Q30, GC content, and sequence duplication level of the clean data were calculated. All downstream analyses were based on the high-quality clean sequences. Transcriptome assembly was accomplished using Trinity (Grabherr et al., 2011) with the min_kmer_cov value set to 2 by default, and all of the other parameters were set to the default values. The gene functions were annotated based on the following databases: Nr (NCBI non-redundant protein sequences), Nt (NCBI non-redundant nucleotide sequences), Pfam (protein family), KOG/COG (clusters of orthologous groups of proteins), Swiss-Prot (a manually annotated and reviewed protein sequence database), KO (KEGG Ortholog database), and GO (gene ontology).

2.2. RNA extraction, qualification, and quantification

2.5. Quantification and differential expression analysis of transcripts

After sample collection, the gills of three individuals in both the control and 75 μg/L groups were mixed to obtain pooled samples, and four pools, termed Cd_treated_G_1, Cd_treated_G_2, control_nontreated_G_1, and control_non-treated_G_2, were used for library construction. Two biological replicates were used for each group:

The gene expression levels in each sample were estimated by RSEM (Li and Dewey, 2011). First, clean data were mapped back onto the assembled transcriptome. The read count for each gene was then obtained from the mapping results. FPKM (expected number of fragments per kilobase of transcript sequence per millions base pairs

Please cite this article as: Wang, Z.-J., et al., Transcriptome profiling analysis of rare minnow (Gobiocypris rarus) gills after waterborne cadmium exposure, Comp. Biochem. Physiol., D (2016), http://dx.doi.org/10.1016/j.cbd.2016.05.003

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sequenced), which was calculated based on the mapped transcript fragments, the transcript length, and the sequencing depth, was used to quantify the transcript expression level. A differential expression analysis of these two groups was performed using the DESeq R package (1.10.1) (Anders and Huber, 2010). DESeq provides statistical routines for determining differential expressions in digital gene expression data using a model based on a negative binomial distribution. The resulting P values were adjusted using the approach developed by Benjamini and Hochberg for controlling the false discovery rate. Genes with an adjusted P-value (Padj) b0.05, as determined by DESeq were determined to be differentially expressed. The gene ontology (GO) enrichment analysis of the differentially expressed genes (DEGs) was implemented using the GO Seq R packages based on Wallenius' non-central hyper-geometric distribution (Young et al., 2010), which can adjust for gene length bias in DEGs. KOBAS (Mao et al., 2005) software was used to test the significance of the enrichment of differentially expressed genes in KEGG pathways.

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variation were b15%. No significant cross-reactivity or interference was observed in any assay. 2.8. Semi-quantitative RT-PCR To verify the expression of reproduction-related genes in the gill, semi-quantitative RT-PCR was performed. In brief, the total RNA of gills and other tissues from both Cd-treated, Cd-untreated and nonacclimatized rare minnows were extracted (N = 6 for each group), and 1 μg of total RNA of each sample was reverse transcribed to firststrand cDNA as mentioned above and then subjected to PCR amplification. A 20-μL PCR mixture containing 0.4 μM of both the upstream and downstream primers (Table S1), 1 μL of cDNA, 2 μL of 10× buffer, 1 U of rTaq (Takara, Dalian, China), 100 μM dNTPs (Takara, Dalian, China) was performed in a Veriti® 96-well Thermal Cycler (Applied Biosystems, USA), and each PCR product was subjected to electrophoresis on a 1.2% agarose gel. 2.9. Statistical analysis

2.6. Quantitative real-time PCR (RT-qPCR) The total RNA of the gills from all of the groups (N = 6 for each group) was also prepared separately for non-library construction use. The total RNA extraction, purification, cDNA synthesis and RT-qPCR protocols were based on our previous report (Liu et al., 2016). In brief, total RNA was analyzed using 1% agarose gels and a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific Inc., USA) for the assessment of RNA degradation, contamination, purity, and concentration. A 20-uL volume of first-strand cDNA was synthesized from 1 μg of total RNA of each sample using the PrimeScript® RT reagent kit with gDNA Eraser (Perfect Real Time, Takara, Dalian, China) according to the user's manual. RT-qPCR was performed to validate the identified differentially expressed genes. The primers (Table S1) used for RT-qPCR were designed using the Primer 5 software (PREMIER Biosoft, Palo Alto, CA, USA), and the PCR products were ligated to the PMD 19-T vector (Takara, Dalian, China) according to the manufacturer's instructions and then transformed into Escherichia coli DH5a (Tiangen, Beijing, China). At least three positive clones were sequenced by BGI Tech (Shenzhen, China). A 20-μL RT-qPCR reaction containing 0.4 μM of both the upstream and downstream primers, 1 μL of cDNA, and 10 μL of 2× SYBR® green Premix Ex Taq™ II (Tli RNaseH Plus) was prepared. The cycling conditions consisted of an initial denaturation cycle of 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 30 s. After RT-PCR, a default melting curve analysis was performed to verify the absence of non-specific amplification. The housekeeping gene β-actin was used as a reference for normalization. Each sample was run in triplicate. Standard curves were generated using a series of 10−3–10−8 dilutions of the plasmids for each gene to calculate the efficiency (E, listed in Table S1) using StepOne™ software, version 2.2.2 (Applied Biosystems, USA). The relative expression levels of the target genes were analyzed using the classic 2−ΔΔCt method (Livak and Schmittgen, 2001). 2.7. Enzyme-linked immunosorbent assay (ELISA) The sera from six individuals from each group were isolated from blood after centrifugation (2500 rpm) at 4 °C for 20 min. The isolated serum was used for the detection of tumor necrosis factor α (TNF-α), interleukin 1β (IL1β), nuclear factor-κB (NF-κB), and interleukin 6 (IL6) using corresponding ELISA kits (Yuan Ye Biotechnology, Shanghai, China) according to the user's manual or was stored at −80 °C until use. The catalogue numbers of the abovementioned four ELISA kits were CKE93557F, CK-E90016F, CK-E93558F and CK-E93552F, respectively. All of these assays were designed based on the sequences of the corresponding proteins in common carp (Cyprinus carpio). The assay sensitivity of the four markers was 1.0 pg/mL, and the intra-assay coefficients of

All of the data are expressed as the mean ± S.E.M. The data were analyzed through either one-way ANOVA followed by Turkey's multiple comparison post-hoc test or unpaired Student's t-test using Prism software (GraphPad Software Inc., San Diego, CA, USA). Differences were considered significant at P b 0.05. 3. Results 3.1. RNA sequencing In the present study, 3.62 to 3.65 G bases were obtained, and 29,437,222 to 29,735,573 raw reads were generated from four libraries of adult female rare minnow gills (Table S2). The sequencing raw data have been submitted to the Sequence Read Archive (SRA) under Accession Number SRR2805762. After quality control, 28,964,832 to 29,173,627 clean reads were obtained. The error probability of all the samples was ≤ 0.04%. The clean reads were then subjected to further analysis, and approximately 74.54% to 76.94% of the reads in each sample were mapped to a reference, which was an assembly pool of reads from the gills and three other tissues: brains, ovaries and livers (unpublished data). 3.2. Identification of highly expressed transcripts in rare minnow gills A scatter plot showed that the transcript expression levels of all four samples were similar based on the log10 (FPKM) value (Fig. S1). Approximately 2000 genes were highly expressed in both treated and untreated gills, with an FPKM value N50. The top 30 annotated transcripts, with average FPKM reads N2000 in the four libraries, included 14 genes encoding ribosome subunits, five genes encoding fertilization-related proteins [e.g., zona pellucid glycoproteins (ZPs), fertilization envelope outer layer protein (FEOLP)], three genes encoding immune response-related proteins (e.g., complementary factor D, a-z-macroglobulin-like precursor), three genes encoding cytochrome c oxidase subunits (COX), three unannotated genes, and two genes encoding proteins involved in other functions (ubiquitin, elongation factor-1-alpha) in both Cd-treated and untreated gills (Fig. S2). To verify the high expression of fertilization-related genes in the gills, we performed semi-quantitative PCR and found that ZP3, ZP2, ZP1, and FEOLP were highly expressed in both the gills and the ovaries of the control and the Cd-treated groups, whereas ZP1L (zona pellucida 1-like) was moderately expressed in these two tissues (Fig. S3A). Because our experiments were conducted in 5-L glass tanks, to avoid space limitation effects, we further detected the expression of these genes in nine tissues of adult female rare minnows that were fed in

Please cite this article as: Wang, Z.-J., et al., Transcriptome profiling analysis of rare minnow (Gobiocypris rarus) gills after waterborne cadmium exposure, Comp. Biochem. Physiol., D (2016), http://dx.doi.org/10.1016/j.cbd.2016.05.003

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tanks with water circulation (35 cm × 35 cm × 40 cm). The results showed that ZP3, ZP2, and FEOLP presented a wide expression spectrum in various tissues: in fact they were detected in the heart, hepatopancreas, spleen, kidney, intestine, gill, muscle, brain, and ovary (Fig. S3B). 3.3. Identification of differentially expressed genes (DEGs) after Cd exposure To better understand Cd-induced toxicity, the DEGs in Cd-treated (75 μg/L Cd treated for 35 d) and untreated rare minnow gills were identified. A total of 452 DEGs (209 up-regulated and 243 downregulated) were detected after Cd exposure using Padj b 0.05 as the cutoff values (Fig. 1). Of these DEGs, 75 are related to cellular ion binding or transport, and these ions include zinc (27), calcium (24), copper, iron, magnesium, potassium, sodium, nickel, and H+. Furthermore, 21 and 54 of the identified DEGs are related to the oxidation-reduction process and immune response, respectively. Of the 54 immune-related genes, only six including genes encoding the MHC I complex protein and virion proteins were up-regulated. The others which are associated with immunoglobulin chain mRNA, MHC II molecules, B cell and T cell receptors, T-lymphocyte activation antigen, T cell activation Rho-activating protein were down-regulated (Table S3). 3.4. Functional distribution of differentially expressed genes Based on the GO categories, a total of 1696 clusters were annotated with GO terms. The identified DEGs were categorized into three major functional groups: cellular components, molecular functions, and biological processes. Ten categories were significantly clustered using a corrected P-value b 0.05. The most abundant categories included antigen processing and presentation, membrane, carbohydrate binding, MHC protein complex, protein nitrosylation, peptidyl-cysteine Snitrosylation, and biological adhesion (Fig. 2A).

To further investigate the functions of the DEGs, all of the DEGs were mapped to the KEGG database. A total of 452 genes were categorized into 170 pathways, and the following nine of these pathways were significantly enriched (corrected P-value b0.05): autoimmune thyroid disease, graft-versus-host disease, viral myocarditis, allograft rejection, type I diabetes mellitus, antigen processing and presentation, intestinal immune network for IgA production, cell and molecule adhesion, and asthma (Fig. 2B). 3.5. RT-qPCR validation of differentially expressed genes RT-qPCR was used to validate selected differentially expressed genes identified from the RNA-Seq data. Major histocompatibility complex I (MHC I, c122936_g4), coagulation factor XIII, A1 polypeptide (F13A1, c153851_g1), tripartite motif-containing protein 21 (TRIM21, c146402_g4), Golgi-associated plant pathogenesis-related protein (GAPr, c147577_g2), MHC II (c147674_g1), and integrin subunits beta (ITGb, c148294_g1) were selected from the set of DEGs, and these included up- and down-regulated genes in the two groups. The RT-qPCR results confirmed the expression pattern of the selected DEGs in Cdtreated and untreated gills of adult female rare minnows (Fig. 3). To explore whether these genes could be used as biomarkers for risk assessments of aquatic Cd pollution, we detected the levels of F13A1, TRIM21, and GAPr in the gills of other groups subjected to sub-chronic waterborne Cd exposure (0, 5, 25, 50, and 100 μg/L for 35 d) and groups subjected to acute exposure (2.0 mg/L) for 0, 12, 24, 48, 72 and 96 h. The results showed that acute or sub-chronic exposure with traces or high doses of waterborne Cd significantly down-regulated all three genes in the gills (Fig. 4). 3.6. Serum concentration of inflammation markers after waterborne Cd exposure During acute waterborne Cd exposure, levels of serum TNF-α, IL1β, NF-κB, and IL6 were decreased after 12 h, whereas the levels of these cytokines were moderately increased from 24 to 96 h (Fig. 5A). After exposure to 75 μg/L Cd for 35 d, serum TNF-α, IL1β, and NF-κB levels were significantly increased (P b 0.05). Exposure to 5 and 25 μg/L Cd led to a moderate increase in these factors, whereas 50 or N50 μg/L Cd significantly increased their concentrations in the circulation. The IL6 concentration was moderately increased in all of the groups (Fig. 5B). 4. Discussion

Fig. 1. Volcano plot of unigenes in Cd-treated gills compared with control non-treated gills of female rare minnows (Gobiocypris rarus). In total, 452 unigenes were identified as differentially expressed (adjusted P-value b 0.05). The genes in blue under the dotted line represent genes that were not affected by exposure to 75 μg/L waterborne cadmium for 35 d. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

The fish gill is the first organ to come into contact with aquatic pollutants, and the gill transcriptome responses to pathogens as well as supplementation with Zn and nanoparticulate silver have previously been studied by microarray or RNA sequencing (Griffitt et al., 2013; Morrison et al., 2006; Zheng et al., 2010). In this study, we analyzed the transcriptional data of rare minnow gills exposed to Cd. The two main functions of fish gills are gas exchange and osmoregulation and the combination of mitochondria-rich cells and the blood system is necessary for the completion of these two processes (Evans et al., 2005). The high expression of mitochondria-derived proteins and hemoglobin α chain in rare minnow gills provides a molecular basis for the function of fish gills. Complementary factor D is the initial obligatory and ratelimiting catalytic component in the alternative complement pathway, and plays a well-known role in the humoral suppression of infectious agents (Biesma et al., 2001). Its high expression suggests that fish gills constitute an important part of the immune system. Mucus synthesis is the only strategy for the protection of fish gills against the outer environment; therefore, the detected high expression of ribosomal subunits might be related to mucus synthesis because the ribosome is the site for translation of mucin and other secretory proteins (Pluta et al., 2012). In contrast, the extremely high expression of ZPs and FEOLP in gills is quite unexpected. These genes are mainly expressed in the ovaries or

Please cite this article as: Wang, Z.-J., et al., Transcriptome profiling analysis of rare minnow (Gobiocypris rarus) gills after waterborne cadmium exposure, Comp. Biochem. Physiol., D (2016), http://dx.doi.org/10.1016/j.cbd.2016.05.003

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Fig. 2. Enrichment analysis of differentially expressed genes. A: Functional gene ontology categories of differentially expressed genes. CC: cellular component; MF: molecular function; BP: biological process. B: Scatter plot of differentially expressed genes enriched in KEGG pathways. The enrichment factor represents the ratio of the number of DEGs to the number of all the unigenes in the pathway; the q value represents the corrected P-value.

livers of fish such as the gilthead seabream (Sparus aurata) (Modig et al., 2006), and have a dominant function in the fertilization process. The genome includes several ZP genes, and the ZPB of rare minnows has diverse isoforms, all of which are expressed only in the ovaries or livers (Wu et al., 2012). ZP3 have a relatively wide expression pattern. ZP3b can be detected in hearts, brains, muscles, spleens, gills, and intestines of female half-smooth tongue sole (Cynoglossus semilaevis) (Sun et al., 2010), and in rare minnows, this gene can be detected at very low levels in the gills (Yuan et al., 2013). In addition, the expression of sturgeon ZP is age-dependent (Chuang-Ju et al., 2011). Although our results from the transcriptome analysis differ from those detailed in previous reports (Wu et al., 2012; Yuan et al., 2013), they confirm the expression of these genes in non-ovary or non-liver tissues. The discrepancies between our results and those obtained in other studies might be attributed to different ages or feeding systems and it is also possible that fertilizationrelated genes may play an unknown role in rare minnows under certain conditions. Based on results of investigations on mammals, ZPs are related to the stimulation of the acrosome reaction of mammalian sperm, by an elevated Ca2+ influx through T-type Ca2+ channels [reviewed by Darszon et al.

(2001)]. In addition, ZP2 and ZP3 can directly interact with voltagedependent anion channel 2 (VDAC2) (Petit et al., 2013). Because ion transport-based osmoregulation is the major function of the fish gill (Evans et al., 2005), we hypothesized that the expression of ZPs might be related to the ion transport of rare minnow gills, and this hypothesis constitutes the basis of our next investigation. Through two-dimensional gel electrophoresis, a total of 77 differentially expressed proteins were previously identified in the zebrafish liver cell line ZFL after Cd exposure. These proteins were found to be related to stress responses, transporters, transcription regulation, redox homeostasis, or different signal pathways, and half of the identified proteins are capable of binding metal ions (Zhu and Chan, 2012). In the present study, we identified more DEGs in the gills, and found that 75 of the identified DEGs have ion-binding properties. This result suggests that exposure to 75 μg/L waterborne Cd severely disturbs the cellular ion balance. Previous studies found that, after entering cells, Cd can function as a blocker of Ca2 + channels in chicken sensory neurons (Swandulla and Armstrong, 1989) or interact with calmodulin (CaM), an intracellular Ca2+ binding protein, thereby interfering with the functioning of CaM (Powlin et al., 1997) and ultimately disrupting Ca2 +

Fig. 3. Validation of the transcriptome data (N = 6). A: Relative expression level of differentially expressed genes (F13A1, TRIM21, GAPr, MHC II and ITGb) in the gills of female rare minnows after 75 μg/L waterborne Cd exposure for 35 d, as validated by RT-qPCR. B: Relative expression of MHC I. * P b 0.05 and ** P b 0.01; the asterisk indicates the significant difference between the control and Cd- treated groups. Error bars represent standard errors.

Please cite this article as: Wang, Z.-J., et al., Transcriptome profiling analysis of rare minnow (Gobiocypris rarus) gills after waterborne cadmium exposure, Comp. Biochem. Physiol., D (2016), http://dx.doi.org/10.1016/j.cbd.2016.05.003

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Fig. 4. Expression of F13A1, TRIM21 and GAPr in gills of female rare minnows (Gobiocypris rarus) after acute or sub-chronic waterborne cadmium exposure (N = 6). A: Expression of F123A1 in gills after acute exposure to 2.0 mg/L waterborne cadmium. B: Expression of TRIM21 in gills after acute exposure to 2.0 mg/L waterborne cadmium. C: Expression of GAPr in gills after acute exposure to 2.0 mg/L waterborne cadmium. D: Expression of F123A1 in gills after exposure to 0–100 μg/L waterborne cadmium for 35 d. E: Expression of TRIM21 in gills after exposure to 0–100 μg/L waterborne cadmium for 35 d. F: Expression of GAPr in gills after exposure to 0–100 μg/L waterborne cadmium for 35 d. * P b 0.05, ** P b 0.01, and *** P b 0.001; the asterisks indicate a significant difference between the control and Cd-treated groups. Error bars represent standard errors.

homeostasis (Matsuo et al., 2005). Waterborne Cd enters the fish body by first entering the gill via Ca2 + channels in the apical membrane (Verbost et al., 1989) or via chloride cells in the gills (Olsson, 1998). Thus, the disruption of most genes related to Ca2+ binding or transport is plausible, and Ca2+ homeostasis must be restricted. However, genes related to a vast variety of metal ions, including Zn, Mg, K, Na, Fe, Cu, and Ni, and even to non-metal ion (H+) binding or transport, were found to also be influenced by 35-day exposure to 75 μg/L waterborne Cd. This result indicates that Cd-induced toxicity is very complicated

and that much remains unclear, even at the ion homeostasis level. These results are in accordance with those described by Zhu et al. (2014), who proposed that Cd has various protein-binding activities and may exert its toxicity by binding and interfering with some essential metal metabolism. Zn and Mg, which are essential elements for some cellular enzymes, together with Ca, have been reported to act as Cd antagonists through (1) the induction of further synthesis of MT, which increases Cd sequestration, (2) their antioxidant properties, (3) an increase in antioxidants, including MT and GSH, and (4) a

Fig. 5. Serum concentration of inflammation markers in female rare minnows (Gobiocypris rarus) (N = 6). A: Serum concentration of TNF-α, IL1β, NF-κB, and IL6 in adult female rare minnows after acute exposure to waterborne cadmium (2.0 mg/L) for 0, 12, 24. 48, 72, and 96 h. B: Serum concentrations of TNF-α, IL1β, NF-κB, and IL6 in adult female rare minnows after sub-chronic exposure to waterborne cadmium (0, 5, 25, 50, 75 and 100 μg/L) for 35 d. * P b 0.05, and ** P b 0.01; the asterisks indicate a significant difference between the control and Cd-treated groups. The dotted line indicates the corresponding concentration in the control group and error bars represent standard errors.

Please cite this article as: Wang, Z.-J., et al., Transcriptome profiling analysis of rare minnow (Gobiocypris rarus) gills after waterborne cadmium exposure, Comp. Biochem. Physiol., D (2016), http://dx.doi.org/10.1016/j.cbd.2016.05.003

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decrease in the activity of antioxidant enzymes to prevent Cd-induced cellular toxicity in the kidneys (Babaknejad et al., 2014; Jihen et al., 2010). Our data provide another possibility: the protective role of additional Zn and Mg might exert beneficial effects on the recovery of the expression of ion-binding or ion transport-related genes for attaining ion homeostasis. Because Cu, Zn, and Mg are essential elements of some antioxidant enzymes and Ca2 + homeostasis is also involved in redox signaling (Hidalgo and Donoso, 2008), the disruption of metal ion homeostasis must have effects on the redox system. Indeed, exposure to cadmium causes the induction of reactive oxygen species or the alteration of antioxidant responses both in vitro (Chen et al., 2011; Oh and Lim, 2006) and in vivo (Li et al., 2014b; Sevcikova et al., 2011). In this study, a total of 21 DEGs were found to be involved in the oxidation-reduction process. NADH/NAD + and NADPH/NADP + are important members of the pyridine nucleotide redox system and participate in antioxidant defense by controlling cellular oxidative stress and the GSH/GSSG redox balance (Circu and Aw, 2010). NAD (P) transhydrogenase (NNT), all-trans-retinol dehydrogenase (NAD +) (SDR16C5) (Liden and Eriksson, 2006), NADH-ubiquinone oxidoreductase chain 4 (ND4) (Vinogradov, 2008), prostaglandin reductase 1 (PTGR1, LTB4DH) (Dick et al., 2001), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Pariona-Llanos et al., 2015) are regulators of the NADH/ NAD+ and NADPH/NADP + balance. The significant up-regulation of these genes detected in this study suggests the redox system of rare minnow gills is disrupted after sub-chronic waterborne Cd exposure. Oxidative phosphorylation and anti-oxidant defense-related genes have also been reported to be altered in the hepatopancreas of freshwater crab (Sinopotamon henanense) (Sun et al., 2016). The immune response can be triggered and shaped by redox reactions, which can be further involved in the termination and initialization of cellular restorative processes (Gostner et al., 2013). Although Cd has been reported to be a pro-inflammation factor in humans and rodents (Olszowski et al., 2012), relevant studies in fish are rare. Only some clues regarding the proinflammatory property of Cd in fish are available: (1) after exposure to 1.68 mg/L CdCl2 for 20 d, the infiltration of large numbers of inflammatory cells was observed in the intestines of Nile tilapia through a histochemistry study (Younis et al., 2015) and (2) waterborne Cd exposure can alter macrophage-mediated immune functions of rainbow trout in a time-dependent manner (Zelikoff et al., 1995). In the present study, approximately 54 DEGs were found to be related to the immune response, confirming that the immune system of rare minnows is disrupted by sub-chronic waterborne Cd exposure. With the exception of MHC I, most genes, including those encoding MHC class II molecules and Ig heavy chain, were downregulated. A further analysis based on the KEGG database showed that the pathway related to immune function was significantly enriched. Both MHC I and MHC II conduct antigen processing signals, though in an opposite manner. MHC I glycoproteins are present in almost every cell of the body and are involved in a cytosolic pathway, mainly in response to endogenous antigens originating from the cytoplasm, such as self proteins and foreign proteins produced by a virus, leading to the activation of cytotoxic (CD8) T cells (Hewitt, 2003). MHC II proteins present exogenous antigens that originated extracellularly from foreign bodies, such as bacteria, and can be recognized by a different type of T cell (Robinson and Delvig, 2002). MHC II-mediated antigen presentations to CD4 T cells by dendritic cells (DCs) and B cells have been reported to have different outcomes in vivo: mature DCs die, whereas B cells are activated to participate in immune responses. The death effect is important in the termination of immune responses to avoid autoimmunity (Jin et al., 2008). Thus, a decrease in MHC class II molecules might lead to increased autoimmunity and a decreased immune response to pathogens. Constitutively expressed MHC class I molecules can attenuate TLR-triggered innate inflammatory responses (Xu et al., 2012). The detected up-regulation of MHC I, viral capsid and viral nucleocapsid

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and the down-regulation of antiviral effector MX1 (interferon-induced GTP-binding protein) suggest that the anti-virus ability of fish gill cells declined after sub-chronic waterborne Cd exposure. The decline of the anti-pathogen response was also confirmed by the significant downregulation of immunoglobulin, B and T cell receptors, F13A1, and TRIM21, which are key genes in the regulation of immune responses (Nikolajsen et al., 2014; Ozato et al., 2008). The down-regulation of immunity-related genes was also observed in Cd-treated freshwater crab hepatopancreas (Sun et al., 2016). In addition, the up-regulation of MHC class I is reportedly a diagnostic marker of inflammatory myopathies in humans (Salaroli et al., 2012). Therefore, we hypothesized that inflammation is induced after sub-chronic Cd exposure. TNF-α, IL1β, IL6, and NF-κB are commonly used biomarkers of inflammation (Elsabahy and Wooley, 2013; Xu et al., 2015), and TNF-α, IL1β, and IL6 are predominant pro-inflammatory cytokines in fish (Kum and Sekkin, 2011). The significant up-regulation of TNF-α, IL1β, and NF-κB by treatment with higher doses of Cd suggests an inflammatory response. Taken together, our results indicate that Cd can lead to inflammation indirectly through decreases in the anti-virus and anti-bacterial function of fish gills or directly by damage to cells that up-regulate autoimmune responses. The gills are one of the first tissues exposed to aquatic pollutants. Therefore, the markers identified from gills might be used as biomarkers for the rapid testing of contaminants. Biomarkers must be sensitive, specific, and respond in a dose-dependent manner (Hamza-Chaffai, 2014). The significant down-regulation of F13A1, TRIM21, and GAPr in both acutely and sub-chronically Cd-treated rare minnow gills, even under exposure to a safe concentration (b 10 μg/L), suggests that rare minnow gills are sensitive enough in response to Cd exposure. In addition, dose-dependent relationships could be observed after subchronic exposure to Cd concentrations ranging from 5 to 100 μg/L. Thus, these genes could be used as biomarkers, particularly for assaying the degree of aquatic Cd pollution. 5. Conclusions In summary, the presented data suggest that the processes of ion homeostasis, redox system, and the immune response are disrupted in the gills of adult female rare minnows following Cd exposure. The GO and KEGG enrichment analyses, and the observed increase in serum pro-inflammatory markers suggest the vital function of the immune response in Cd toxicity. F13A1, TRIM21, and GAPr could be used as biomarkers for risk assessments of aquatic Cd pollution. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.cbd.2016.05.003. Acknowledgements This work was funded by the Major Program of Science and Technology Commission Foundation of Chongqing (cstc2014yykfc80001), the Chongqing Graduate Student Research and Innovation Project (CYB14049), and the Fundamental Research Funds for the Central Universities (XDJK2015D033). The authors would like to thank Dr. Jian-Ping Xie for the writing suggestions provided and the members of the Zhang and Wang laboratories for the helpful discussion and the assistance with the sample collection. References Anders, S., Huber, W., 2010. Differential expression analysis for sequence count data. Genome Biol. 11, R106. Babaknejad, N., Moshtaghie, A.A., Shahanipour, K., Bahrami, S., 2014. The protective roles of zinc and magnesium in cadmium-induced renal toxicity in male wistar rats. Iran. J.Toxicol. 8, 1160–1167. Biesma, D.H., Hannema, A.J., van Velzen-Blad, H., Mulder, L., van Zwieten, R., Kluijt, I., Roos, D., 2001. A family with complement factor D deficiency. J. Clin. Invest. 108, 233–240.

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Please cite this article as: Wang, Z.-J., et al., Transcriptome profiling analysis of rare minnow (Gobiocypris rarus) gills after waterborne cadmium exposure, Comp. Biochem. Physiol., D (2016), http://dx.doi.org/10.1016/j.cbd.2016.05.003