Transcriptome analysis on chlorpyrifos detoxification in Uronema marinum (Ciliophora, Oligohymenophorea)
Chlorpyrifos (CPF) pollution has drawn widespread concerns in aquatic environments due to its risks to ecologic system, however, the response mechanisms of ciliates to CPF pollution were poorly studied. In our current work, the degradation of CPF by ciliates and the morphological changes of ciliates after CPF exposure were investigated. In addition, the transcriptomic profiles of the ciliate Uronema marinum, with and without exposure with CPF, were detected using digital gene expression technologies. De novo transcriptome assembly 166,829,634 reads produced from three groups (untreated, CPF treatment at 12 h and 24 h) by whole transcriptome sequencing (RNA-Seq). Gene ontology (GO) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathways were analyzed in all unigenes and different expression genes to identify their biological functions and processes. Furthermore, the results indicated that genes related to the stress response, cytoskeleton and cell structure proteins, and antioxidant systems might play an important role in the resistance mechanism of ciliates. The enzyme activities of SOD and GST after CPF stress were also analyzed, and the result showed the good antioxidant capacity of SOD and GST in ciliates inferred from the increase of the activities of the two enzymes. The ciliate Uronema marinum showed a resistance response to chlorpyrifos stress at the transcriptomic level in the present work, which indicates that ciliates can be considered as a potential bioremediation agent.
KeywordsUronema marinum Chlorpyrifos stress Response mechanism Transcriptome profiling
Organophosphorus (OP) pesticides are the most widely used pesticides in the world due to their efficacy in killing various crop pests (Chen et al. 2016). High levels of OP seep into rivers and oceans, affecting the aquatic environment (Abdel-Halim et al. 2006). Such toxic substances commonly accumulate through aquatic food chains and produce a toxic effect on zooplankton thus ultimately affect human health (Eddleston et al. 2008; Lee et al. 2016).
Chlorpyrifos [O, O-diethyl (O-3, 5, 6-trichloro-2-pyridyl) phosphorothioate, CPF] is a commonly used pesticide in both agriculture and residential environments (Al-Fanharawi et al. 2018; Marigoudar et al. 2018), and is classified as a moderately dangerous, class II insecticide (WHO 1997). Many studies have found the toxicity of CPF to aquatic vertebrates, including Salmo coruhensis (Kutluyer et al. 2017), Lates calcarifer (Marigoudar et al. 2018), and Salmo trutta caspius (Adel et al. 2017). Upon exposure to potentially growth-inhibiting external concentrations of chlorpyrifos (CPF), biochemical, and enzyme activities analysis suggests aquatic organisms respond with different resistance and detoxification mechanisms (Xing et al. 2013; Kim et al. 2016). In vertebrates such as carp, expression levels of 70-kDa heat shock protein (HSP70) and 70-kDa heat shock cognate protein (HSC70) with CPF treatment was significantly up-regulated, providing insights into the adaptive mechanisms to chlorpyrifos treatment (Xing et al. 2013). For invertebrates such as rotifers, the defensive system composition of cytochrome P450 complements, HSP70, and antioxidant enzymatic systems was significantly induced in response to CPF (Kim et al. 2016), indicating potential detoxification mechanisms. While there is an increasing awareness that multiple vertebrates and invertebrates can have a response mechanism such as adaptability and endurance to OP poisoning (Dawkar et al. 2016; Xing et al. 2012), few experimental studies focus on how OP pesticides interact with protozoa.
Ciliated protists, as major consumers of zooplankton and unicellular eukaryotic organisms, are abundant in terrestrial, freshwater as well as marine environments (Kim et al. 2017). They respond more sensitively to various environmental stressors than metazoa (Dayeh et al. 2005; Gutiérrez et al. 2003). As they share characteristics of eukaryotic cells, their biological responses to environmental factors were always be used to predict the responses of higher organisms (Trielli et al. 2007). The ciliate, Uronema marinum, can be cultured rapidly, always has a high cell density in a variety of media and culture conditions, and is adapted to microbial laboratory work. Many studies showed that U. marinum could be easily found in contaminated water (Coppellotti 1994; Holubar et al. 2000), indicating its certain resistance to environmental toxins. So far, however, none of the U. marinum genes participated in CPF stress regulation has been identified, as a result of hindering our understanding of CPF resistance mechanisms.
With the development of next-generation sequencing technologies, transcriptomic approach has been able to become a valuable tool for life sciences research, which improves the efficiency and pace of gene discovery (Liu et al. 2016). In this study, cells of ciliate (U. marinum) were selected to be exposed to CPF. We confirmed the possibility that CPF can be degraded by ciliates, and we used a transcriptome-based analysis of differentially expressed genes (DEGs) to obtain the mechanism underlying the physiological changes induced by CPF treatment in ciliates. Besides, functional enrichment analysis was conducted to explain the critical processes/pathways in response mechanism to CPF exposure. Therefore, the novel data was supplied in the present study in order to better understand the molecular response mechanisms of U. marinum upon exposure with and without CPF pollution, which will contribute to provide insight into the potential detoxification and degradation of CPF by ciliates.
Sample collection and cultivation
The ciliate Uronema marinum was obtained from the Ocean University of China and clonal cultures were established by the Aquatic Laboratory, Harbin Normal University, China. A single individual was isolated, reared, and maintained in a 500-ml flask with seawater (> 14 h sunlight/day, water temperature 20–25 °C; salinity 20‰; pH 7). Rice grains were added to enrich the natural bacteria which were the food source for the ciliated protozoa. Species identification and the morphology and molecular information of Uronema marinum had been reported in Pan et al. (2015). Prevention of bacterial contamination was made before RNA extraction using lysozyme following Xiong et al. (2015). When ciliates reached the exponential phase of growth, they were supplemented with 0.05 mg / L of chlorpyrifos. The cell density was measured by direct counting, using an Axio Imager 2 microscope (Carl Zeiss, Germany) at 500× magnification with hemocytometer during the assay. Cells were concentrated by centrifugation before RNA extraction, then fixed in 5× volumes of RNAlater (Qiagen, USA), and frozen at −80 °C until RNA extraction.
Measuring CPF using UPLC
CPF (O, O-diethyl O-3, 5, 6-trichloropyridin-2-yl phosphorothioate, 99%) used for exposure and analyses were purchased from Aladdin (Aladdin Co. Ltd., China). About 1 × 104 individuals of U. marinum were collected after 0 h, 12, and 24 h CPF exposure (0.05 mg / L) and centrifuged at 12000 rpm. The supernatant was filtered by filter membrane, the filtrate was determined at 25 °C using UPLC. UPLC were performed in a Waters ACQUITY UPLC™ system (Waters Corp., Milford, USA) using a 100 mm × 2.1 mm ACQUITY BEH C18 1.7 μm column and a PDA detector set at 293 nm. A sample volume of 2 μL was injected with an Acquity UPLCTM autosampler. Three independent experiments (three replicates in each) were performed. The mobile phase was HPLC grade acetonitrile: water in 90:10 (v/v), and the flow rate was maintained at 0.5 mL min−1. Under these conditions, the retention time for CPF was about 2.5 min. The detection limit of UPLC of CPF was 0.006 mg/L.
Profiling of transcriptome in response to CPF by RNA-Seq
RNA extraction and mRNA purification
Ciliates were exposed with 0.05 mg/L CPF before RNA extraction. Samples treated with 12, 24 h CPF and control group (from a single individual) were thawed on ice and centrifuged (5000g, 5 min) at 4 °C to remove the RNAlater. The total mRNA was extracted using the RNeasy Protect Cell Mini Kit according to the manufacturer’s instructions (Qiagen, Valencia, CA). In addition, the mRNA quality and quantity were estimated using Thermo Scientific NanoDrop2000 (NanoDrop Technologies, Wilmington, USA). RNA integrity was verified using gel electrophoresis in denaturing 1.0% Agarose. A total of nine biological replicates were performed in this study.
Library preparation for transcriptome sequencing
Sequencing libraries were generated using NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations and index codes were added to attribute sequences to each sample. Firstly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Secondly, the mRNA was cleaved into short fragments with divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5X) after purification. Then, the cleaved fragments were reverse transcribed to create the final cDNA library, and cDNA fragments of preferentially 150~200 bp in length was selected by AMPure XP system (Beckman Coulter, Beverly, USA). The quality of the sample library was assessed by the Agilent 2100 Bioanalyzer System. Furthermore, Illumina HiSeqTM2000 instrument was used to sequencing the library products.
De novo assembly and unigene functional annotation
Sequencing data were transformed by raw reads, which were filtered by discarding adaptors, unknown or low quality reads to obtain clean reads. At the same time, Q20, Q30, GC-content, and sequence duplication level of the clean data were calculated. Transcriptome assembly was accomplished using Trinity software (Grabherr et al. 2011) under default parameters and with min_kmer_cov set to 2 by default. For gene expression levels, clean data mapped back onto the assembled transcriptome and readcount for each gene was obtained from the mapping results following RSEM method (Li and Dewey 2011). All assembled transcripts were compared with six public databases, including Non-redundant (Nr), Non-redundant nucleotide database (Nt), Swiss-prot protein (Swiss-Prot), Cluster of Orthologous Group of proteins (COG), and Kyoto Encyclopedia of Gene and Genomes (KEGG) using BLASTX or BLASTn alignment (cut-off E-value< 10−5) to search for homologs.
Unigene expression difference analysis
The unigene transcription levels were calculated as fragments per kilobase of transcript per million fragments mapped (FPKM) method (Trapnell et al. 2010), in order to identify significant changes in transcript expression levels. The false discovery rate (FDR) ≤0.001 and an absolute value of log2 ratio ≥ 1 (1.5-fold change) were set as the threshold to judge the significance of gene expression differences.
qRT-PCR to validate the DEGs
To validate the RNA-seq data, qRT-PCR was conducted out on ABI 7500 System (Applied Biosystems; Foster City, CA, USA). The total of 200 mL of the ciliates cultures were collected and centrifuged at 12000g for 1 min at 4 °C. The Transcript One-Step gDNA Removal and cDNA Synthesis Supermix kit (TransGenBiotech, Beijing, China) was used to synthesize first-strand cDNA using a UltraSYBR Mixture (with ROX) Kit (CWBIO; Jiangsu, China). The cycling procedure was processed as the followings: 95 °C for 3 min, 40 cycles of 95 °C for 15 s and 55 °C for 15 s, 72 °C for 15 s; 72 °C elongation for 10 min. qRT-PCR analysis was performed for β-tubulin, HSC70, HSP90, and CYP321A1. The primers sequences for qRT-PCR analysis were provided in Table S1, and 18S RNA was used as the reference gene. Average amplification efficiency varied from 97.42 to 98.73% (Table S1). Three biological replicates and three technical replicates were performed in this study. The relative expression of the candidate genes was determined using the 2−ΔΔCT method (Livak and Schmittgen 2001).
Antioxidant enzyme assay
After treatment with 0.05 mg L−1 CPF at 0 h, 12 h and 24 h, 50 mL of the ciliates cultures were collected and centrifuged at 12000g for 3 min at 4 °C. Superoxide dismutase (SOD) activity was determined according to (Beauchamp and Fridovich 1971), using nitro blue tetrazolium chloride as substrate, at 560 nm. Enzyme activity was expressed as nmol NBT conjugate formed × 10−5 U cells. Glutathione-S-transferase (GST) activity was measured using protocol of (Keen et al. 1976), using 1-chloro-2, 4-dinitrobenzene (CDNB) as substrate, at 340 nm. Enzyme activity was expressed as nmol CDNB conjugate formed × 10−5 U cells.
Chlorpyrifos stimulated the proliferation of Uronema marinum cells
Sequencing and de novo assembly
Summary of generated transcriptome data
Mean length of unigenes (bp)
GC content (%)
Overview of functional annotation of assembled unigenes
Number of annotated unigenes
Percentage of annotated unigenes (%)a
Functional annotation of unique genes
GO analysis and KEGG pathways
As shown in Fig. S2, a total of 37,162 unigenes were functionally assigned to 55 groups, including biological process (BP), molecular function (MF), and cellular component (CC). Among the biological processes category, “cellular processes” (20,805; 55.98%), “metabolic processes” (22,371; 61.48%) were the most predominant groups. For the cellular components category, “cell” (11,381; 30.63%) as well as “cell part” (11,380; 30.62%) was the most overrepresented groups. With molecular function category, “binding” (18,406; 49.53%), “catalytic activity” (16,812; 45.24%) groups were clustered a large proportion of unigenes.
Using the KEGG database, a total of 16,054 unigenes were grouped into 230 pathways. The most represented pathway was “signal transduction” (2572 unigenes, 16.02% of the total), followed by “translation” (2090 unigenes, 13.02% of the total) and “endocrine system” (1618 unigenes, 10.08% of the total, Fig. S3).
A total of 20,920 unigenes were assigned to KOG classification to classify the genes and to predict their functions. Results showed that these unigenes were functionally classified into 26 protein families (Fig. S4). The cluster predicting for posttranslational modification, protein turnover, and chaperones (3275 unigenes, 15.65% of the total) was the largest group, followed by translation, ribosomal structure, and biogenesis (2914 unigenes, 13.93% of the total).
Identification of the DEGs of U. marinum
SOD and GST enzyme activity after CPF exposure
To date, many transcriptomes of various aquatic organisms after pollution stress have been sequenced, including microalgae, clam, scallop, and copepod (Ghiselli et al. 2012; Meng et al. 2013; Simon et al. 2013; Wang et al. 2017). Ciliates, such as Tetrahymena thermophila, T. pyriformis, and Euplotes crassus have been widely used as bio-indicators, particularly in ecotoxicology and in water quality monitoring studies (Kim et al. 2017; Mortimer et al. 2010; Zou et al. 2013). However, studies on organophosphorus pesticides impacts in ciliates have been very limited. It is shown in Fig. 1A that the number of cells in the CPF treatment group was almost the same as that of the control group at 12 h, indicating that there might be a regulation or detoxification mechanism in the cells. At the same time, it is found that the morphological characters changed at 24 h, which might demonstrate that CPF exhibit cytotoxic in ciliates at 24 h (Fig. 1B). Hussain et al. (2008) reported that the well-known ciliate Paramecium caudatum was deformation of cell body after the pesticide stress. In addition, it is well known that the half-life of chlorpyrifos in distilled water was 12.3 and 8.12 days, at 16 and 40 °C, respectively (Hui et al. 2010), which showed that the half-life was longer than our experimental period (36 h). In the present study, UPLC analysis showed that the chlorpyrifos concentration was lower than that of the control group (0 h) at 12 h, indicating that chlorpyrifos could be degraded by ciliates (Fig. 1C). Our results confirmed that ciliates could degrade pesticides, which was also mentioned by Feng et al. (2014). The transcriptome of Uronema marinum with and without exposure to CPF at 12 h and 24 h were compared, providing ecological knowledge for the effects of CPF pollution on ciliates. Since the degradation of chlorpyrifos might occur during the 12 h treatment period by ciliates, the up-regulated genes involved in and resistance regulation processes at 12 h were discussed in the present work.
Expression profile of genes involved in CPF detoxification
The cytochrome P450 (cytochrome P450s or P450s, Phase 1 detoxification enzyme) plays important roles in metabolizing or degrading contaminants (Jensen and Moller 2010). Many cytochrome P450 isoforms are well known as important enzymes catalyzing oxidation-reduction reactions which can modify the molecules of xenobiotics, thus conferring a detoxification capability (Kebeish et al. 2014; Tan et al. 2015). Studies in higher plants have shown that P450s could catalyze a detoxification process, through dealkylation and hydroxylation, in alfalfa exposed to atrazine (Tan et al. 2015). A study of common carp gill found that cytochrome P450 content and enzyme activity could be induced by atrazine and chlorpyrifos exposure (Fu et al. 2013). In this study, two cytochrome P450s genes were shown to be up-regulated under CPF exposure at 12 h (Fig. 5 and Table S2), suggesting that these genes were most likely involved in resistance and detoxification of U. marinum to CPF 12 h after treatment.
Environmental contaminants can stimulate the overproduction of reactive oxygen species (ROS) in organisms. These include hydrogen peroxide (H2O2), superoxide anion (O2•—), and singlet oxygen (1O2), all of which cause oxidative injury (Schutzendubel and Polle 2002). In order to prevent the damage caused by ROS, aerobic organisms are able to maintain very low levels of ROS by means of several biochemical mechanisms. Accordingly, the enzymatic and non-enzymatic anti-oxidant systems are involved with scavenging ROS and maintaining redox homeostasis (Guemouri et al. 1991; Thounaojam et al. 2012). At present, there are few studies reporting the antioxidant system-related enzymes of protozoa after exposure to exogenous substances. Kim et al. (2011, 2014) pointed out that ciliates could maintain population growth in Cu- and Zn-contaminated environments, and this was associated with high activities of antioxidant enzymes (Kim et al. 2011, 2014). However, studies of antioxidant systems in ciliates exposed to organophosphorus pesticide stress have not been reported. In the present study, we found that some of the antioxidant system-related gene expression levels were significantly up-regulated after CPF treatment (GST, GSTM1, GR, SOD, and PHGPx) at 12 h (Fig. 2 and Table S2). Among them, glutathione-S-transferase is an important phase II enzyme, which has been demonstrated to play a central role in detoxification of many injurious compounds in a wide range of animal and plant species (Edwards et al. 2000; Piper et al. 1998; Sillapawattana and Schäffer 2017). In green algae, GST activities in Scenedesmus platydiscus and Selenastrum capricornutum increase significantly as pyrene concentrations increasing, showing their resistance and ability to metabolize contaminants (Lei et al. 2003). Superoxide dismutase (SOD), the first line of the defense system against oxygen free radicals, catalyzes the dismutation of ROS into hydrogen peroxide and oxygen (Zelko et al. 2002). Our previous study found that sod gene expression was significantly up-regulated with oxytetracycline (OTC) treatment in Pseudocohnilembus persalinus, which is also consist with the present study (Wang et al. 2017). In addition, GPx is a family of enzymes that catalyze the reduction of hydrogen peroxide (H2O2), using reduced glutathione (GSH) as an electron donor (Prabhakar et al. 2005; Reiter et al. 2000). The PHGPx is essential for protection from radiation and oxidative damage in mice (Yant et al. 2003). It is also reported to regulate ferroptotic cancer cell death (Yang et al. 2014b). A study in the common carp found that the up-regulation of PHGPx occurs after a period of stress, rather than during the initial stress time, which was similar to our results (Hermesz and Ferencz 2009). At the same time, the expression level of PHGPx showed an obviously species and stress specificity (Wang et al. 2012). Besides, the results of the present study showed that the SOD and GST enzyme activity increased after CPF exposure comparing to control group, indicating that the two enzymes play roles in defense mechanism.
ATP-binding cassette (ABC) transporters, members of one of the largest protein superfamily, are known to contribute to detoxification through transporting endogenous metabolites and exogenous xenobiotics out of cells, belonging to the phase 3 detoxification enzymes (Huang et al. 2009; Kim et al. 2017; Schwartz et al. 2010). In the present study, ABC transporter after 12 h of stress was highly express, indicated that they played an important role in resistance mechanism of ciliates.
Cytoskeleton and cell structure proteins involved in resistance mechanism of Uronema marinum
In U. marinum, growth and development-associated genes (such as α-tubulin 1 and β-tubulin) were up-regulated after CPF exposure at 12 h. The α-tubulin proteins, which are building blocks of microtubules, are act as a key component of the cytoskeleton responsible for maintaining cellular architecture, regulation cell shape, and motility (Janke and Kneussel 2010; Maria et al. 2013; Miura et al. 2005). Tubulins combined with microtubule binding proteins and related proteins work together in many cellular functions, such as maintenance of protein movement, microtubule polymerization and depolymerization, cell growth, shaping, and signaling (Calligaris et al. 2010; Castoldi and Popov 2003; Lauritano et al. 2011). The α-tubulin proteins are thought to be involved in resistance to contaminants in aquatic environments, and also exhibit up-regulation in response to contaminant (such as heavy metals) exposure (Jiang et al. 2013; Wang et al. 2011). For example, after contaminant stress in bivalves, protein analysis confirmed that tubulin was involved in the cytoskeleton enhancement mechanism to prevent the deleterious effects of contaminants on organisms (Maria et al. 2013). Our results showed that the increase in cell growth of ciliates at 12 h might be related to the rapid synthesis of cytoskeleton through up-regulation of the tubulin beta chain. In addition, the down-regulation of the expression of the tubulin alpha-1A and tubulin alpha-1C (24 h) might contribute to the apoptosis and ultimately reduced the cell density (Fig. 5 and Table S2).
In addition, it is interesting to note that many studies have shown that tubulin genes were involved in the effects of chlorpyrifos on neural responses due to their chlorpyrifos axon binding in human and rodents (Grigoryan and Lockridge 2009; Prendergast et al. 2007). However, a neural mechanism of ciliates remains still unclear, whether neurotrophic response involved in the resistance response to toxic in ciliates demanded to be further discussion, and one limitation of this work might be the cells cultured from a single individuals, thus more studies are needed for understanding the neural mechanisms of ciliates.
Stress gene response to CPF in U. marinum
Many studies have shown that in the environmental stress, the cells protective systems would produce a variety of mechanisms to deal with adverse reactions, such as the heat shock response (Santoro 2000; Martindale & Holbrook 2002). HSPs play crucial roles on the stabilization of proteins and membranes produced by cells to defend themselves in response to multiple environmental stressors (Wang et al. 2004). Our results revealed that heat shock protein genes (HSP70, HSC70 and HSP90) expression were significantly down-regulated after CPF treatment at 12 h and 24 h (Table S2). It is well known that these three proteins are highly conserved and participated in many biological processes, such as membrane translocation, degradation of mis-folded proteins (Liu et al. 2015; Mayer and Bukau 2005). HSP70 expression acts as a component of the physiological mechanisms which is normally used by aquatic organism to cope with environmental challenges, such as microalgae and rotifers (Lewis et al. 2001; Yang et al. 2014a). Generally, HSP70 transcripts are greatly up-regulated during stress and maintained at low levels in normal cells, HSC70 is constitutively expressed in unstressed cells but is slightly induced under stress conditions (Ahamed et al. 2010; Deane and Woo 2006; Rabergh et al. 2000; Xing et al. 2013). Interestingly, the present results showed that HSP70 and HSC70 genes were notably down-regulated after the ciliates exposed to CPF, which was consist with the study of common carp after atrazine stress (Xing et al. 2013).
To date, this is the first report on transcriptome sequencing of ciliates under CPF stress using the Illumina sequencing platform. Our results revealed that some morphological characters of ciliates were changed after a short period of CPF stress, and there was a degradation of CPF in the well-known ciliate Uronema marinum. In addition, we found that in U. marinum, cytoskeleton and cell structure proteins, detoxification and antioxidant systems genes played an important role in the degradation and resistance of CPF. These findings will provide helpful information for studying the potential of CPF bioremediation using ciliates U. marinum.
We would like to thank Professor Lijie Yu and Xiaoxia Jin (Harbin Normal University) for providing help in this study. We thank to Li Zhenxiang for the help in the pre-experiment and initial transcriptome extraction in this work.
This study was funded by The National Natural Science Funds of China (31601866), Program of Natural Science of Heilongjiang Province of R. P. China (QC2017017) and University Nursing Program for Young Scholars with Creative Talents in Heilongjiang Province (UNPYSCT-2017178).
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