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Transcriptional response of springtail (Folsomia candida) exposed to decabromodiphenyl ether-contaminated soil
Journal Pre-proofs Transcriptional response of springtail (Folsomia candida) exposed to decabromodiphenyl ether-contaminated soil Qian-Qian Zhang, Min Qiao PII: DOI: Reference:
S0048-9697(19)34851-X https://doi.org/10.1016/j.scitotenv.2019.134859 STOTEN 134859
To appear in:
Science of the Total Environment
Received Date: Revised Date: Accepted Date:
26 March 2019 29 September 2019 5 October 2019
Please cite this article as: Q-Q. Zhang, M. Qiao, Transcriptional response of springtail (Folsomia candida) exposed to decabromodiphenyl ether-contaminated soil, Science of the Total Environment (2019), doi: https://doi.org/ 10.1016/j.scitotenv.2019.134859
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Transcriptional
response
of
springtail
(Folsomia
candida)
exposed
to
decabromodiphenyl ether-contaminated soil
Qian-Qian Zhanga, b, Min Qiaoa, b*
a
State Key Lab of Urban and Regional Ecology, Research Center for
Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China b
University of Chinese Academy of Sciences, Beijing 100049, China
*
Corresponding author: Min Qiao, State Key Lab of Urban and Regional Ecology,
Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China. Tel: +86-10-62849158; Fax: +86-10-62923563; e-mail: [email protected]
Abstract
Decabromodiphenyl ether (BDE209) is a widely used brominated flame retardant that has become a common soil contaminant of concern due to its persistence and toxicity. However, little is known about molecular-level effects of BDE209 on soil invertebrates. Here, we detected changes in gene transcription of the soil springtail, Folsomia candida, exposed to BDE209 (0.81 mg/kg) in soil for 2, 7
and 14 days. We identified 16 and 771 significantly differentially expressed genes after 2 and 7 days of exposure respectively, and no significantly regulated genes were shared among the two time points. No genes were affected after 14 days of exposure. According to the annotation of the significantly differently expressed genes at 2 and 7 day exposure, we found that BDE209 affected the transcription of genes involved in moulting, neural signal transmission and detoxification. Our results suggeted that BDE209 could disrupt moulting of F. candida via the ecdysteroid pathway, and cause neurotoxicity through disrupting some neurotransmitter signalling pathways. This study provided insights into the toxic mechanism of BDE209 on F. candida.
Keywords
BDE209; Folsomia candida; Transcriptomics; Moulting; Neurotoxicity
1. Introduction
Polybrominated diphenyl ethers (PBDEs) are widely used as flame retardants in various products including textiles, furniture, electronic devices and building materials (Wang et al., 2005). Commercial PBDE mixures are usually produced at three levels of bromination, namely penta-BDE, octa-BDE and deca-BDE (La Guardia et al., 2006). In China, penta-BDE and octa-BDE are restricted while deca-BDE is still allowed (Wang et al., 2017). PBDEs have become widespread contaminants of concern due to their toxicity, persistence in the environment and bioaccumulation (Lee and Kim, 2015). Decabromodiphenyl ether (BDE209) is the
main component of commercial deca-BDE mixtures and has been the dominant congener detected in many soil samples (McGrath et al., 2017). For example, the average total concentration of 21 PBDEs (including BDE209) in soil samples from an electronic waste burning site in South China was 2283 μg/kg, while the average concentration of BDE209 alone was 2162 μg/kg (Nie et al., 2015). BDE209 can have a variety of adverse effects on organisms. For example, BDE209 (10 μg/L) can affect the expression of thyroid hormone (TH) and spermatogenesis associated genes in adult rare minnow (Gobiocypris rarus) following exposure for 21 days (Li et al., 2014). Proteins involved in normal brain development of mice had altered levels after exposure to 20.1 mg BDE209/kg body weight for 7 days, suggesting that BDE209 causes developmental neurotoxicity (Viberg et al., 2008). An in vitro study using neural stem cells (NSC) of rats showed that BDE209 can inhibit NSC proliferation and induce apoptosis (Zhang et al., 2016). Although the effects of BDE209 on vertebrates and in vitro cells have been widely reported, few studies are available on soil invertebrates. Springtails are widespread and abundant soil invertebrates that have been used extensively to assess the effects of chemicals on soil ecosystems (Fountain and Hopkin, 2005). The springtail Folsomia candida has been used as a model species in ecotoxicology studies (ISO, 1999). The effects of chemicals on F. candida are usually assessed by conventional bioassays, such as reproduction and avoidance tests (ISO, 2011, 2014). In recent years, genomic tools have been employed for studying
molecular effects, including toxic mechanisms. Zhang et al. (2012) reported that BDE209 can affect the reproduction and avoidance behaviours of F. candida, and that EC50 values were 0.81 and 1.27 mg/kg, respectively. However, the molecular effects of BDE209 on F. candida have not been reported. In this study, we aimed to assess the effects of BDE209 on gene transcription of F. candida and explore the mechanisms of toxicity.
2. Materials and methods
2.1 Test organism, soil and exposure experiment
The springtail Folsomia candida was cultured following methods described previously (Zhang and Qiao, 2018). Briefly, the springtail was cultured in Petri dishes on a moistened substrate consisting of plaster of Paris and activated carbon (9:1 weight ratio) under controlled conditions (20±1oC, 70±5% relative humidity and constant darkness). The springtails used for reproduction experiment and microarray experiment were 10-12 day old and 23 day old respectively. The soil used in the experiments was artificial soil, composed of 10% ground dry sphagnum peat sieved to 2mm, 20% kaolinite clay and 70% quartz sand. BDE209 (98% purity, Dr. Ehrenstorfer, German) was added to the soil using acetone and toluene (9:1 volume ratio) as carrier solvent, to reach a nominal concentration of 0.81 mg/kg dry soil. This nominal concentration in artificial soil was used because it reduced F. candida reproduction by 50% (EC50) after 28-day exposure (Zhang et al.,
2012). The solution of BDE209 was mixed with 10% of the test soil and the mixture was put in closed glass containers overnight. The glass containers were then opened and put in a fume hood for 24 hours to volatilize the acetone. The mixture (10% of the test soil and BDE209) was thoroughly mixed with the remaining soil (90%). The same volume of solvent (without BDE209) was added to the solvent control soil. Soil without solvent treatment was used as control soil. All soils were moistened using distilled water to 50% of the maximum water holding capacity. To verify that BDE209 significantly repressed the reproduction of F. candida at the reported EC50 concentration, a 28-day reproduction test was performed. Ten 10-12 day old juveniles were introduced into a 100 mL glass beaker containing 30 g wet soil (BDE209-polluted soil, solvent control soil or control soil), using four replicates for each treatment. These beakers were sealed with Para film and opened once per week to add food. After 28 days, the number of surviving adults and juveniles was counted. To detect gene transcription in F. candida exposed to BDE209-polluted soil, 30 adult springtails (23-day old) were exposed to BDE209-polluted soil or solvent control soil in 100 mL glass beakers for 2, 7 or 14 days. Three replicates were used for each treatment. Springtails were extracted from soil by means of floatation and collected in microcentrifuge tubes. They were snap frozen in liquid nitrogen and kept at -80 oC until RNA extraction.
2.2 Microarray and RT-qPCR experiment
The microarray experiment was performed by the Shanghai Biotechnology Corporation using a custom Agilent 4 × 44 K microarray (Design ID 045772). The microarray contained 6175 different probes spotted randomly for seven times, representing 6175 different ESTs (Expressed sequence tags) from Collembase (Timmermans et al., 2007). Total RNA of each replicate (30 animals) was extracted using RNAiso Plus (Cat. No. 9109, TAKARA, Japan). RNA integrity was inspected by Agilent Bioanalyzer 2100 (Agilent technologies, USA). Qualified total RNA was purified by RNeasy Micro Kit (Cat. No. 74004, QIAGEN, Germany) and RNase-Free DNase Set (Cat. No. 74004, QIAGEN, Germany). Total RNA was amplified and labeled using Low Input Quick Amp Labeling Kit (One color, Cat. No. 5190-2305, Agilent technologies, USA) and was then purified using RNeasy Mini Kit (Cat. No. 74106, QIAGEN, Germany). Cy3-labled cRNA was hybridized with the microarray in a Hybridization Oven (Cat. No. G2545A, Agilent technologies, USA) using the Gene Expression Hybridization Kit (Cat. No. 5188-5242, Agilent technologies, USA). After hybridization, the microarrays were washed by Gene Expression Wash Buffer Kit (Cat. No. 5188-5327, Agilent technologies, USA) and then scanned by Agilent Microarray Scanner (Cat. No. G2565CA, Agilent technologies, USA). To validate the microarray data, reverse transcription real-time quantitative polymerase chain reaction (RT-qPCR) on six target genes (Fcc00966, Fcc04911, Fcc03521, Fcc01821, Fcc01655, Fcc01574) and two reference genes (YWHAZ and
SDHA (de Boer et al., 2009)) were performed for the total RNA samples from springtails which were exposed for 7 days using the same exposure method as in the microarray experiment. Reverse transcription reaction was performed with about 1 μg total RNA using FastQuant RT Kit (Cat. No. KR106, Tiangen Biotech, China) according to the manufacturer’s protocol. The obtained cDNA was diluted 10 times and 2 μL was used in the 20 μL qPCR volume. The qPCR reaction was performed with SuperReal PreMix Plus (SYBR Green) Kit following the manufacturer’s protocol (Cat. No. FP205, Tiangen Biotech, China) using the LightCycler 480 system (Roche, Switzerland). The qPCR conditions were as follows: initial denaturation at 95 oC
for 15 min; 40 cycles of 95 oC denaturation for 10s, 60 oC annealing for 20s and
72oC elongation for 20s. Detection of melting curves was performed after amplification. Primer sequences and efficiencies were summarized in Table S8.
2.3 Data analysis
To verify the effect of BDE209 (0.81 mg/kg) on reproduction of F. candida after 28-day exposure, juvenile numbers in the BDE209-polluted soil, solvent control soil or control soil were compared using one-way ANOVA followed by Tukey test. The scanned images of microarrays were analyzed by Feature Extraction software 10.7 (Agilent technologies, USA), and the detected and undetected genes were determined by this software. The data are accessible in NCBI's Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE137740). The raw data obtained were normalized by quantile algorithm using “limma” package in R
software. The significantly differentially expressed genes were determined by the Significance
Analysis
of
Microarrays
(SAM)
method
(http://statweb.stanford.edu/~tibs/SAM/) (Tusher et al., 2001). Genes with q-value (false discovery rate-corrected p-value) < 0.05 were considered as significantly differentially expressed genes. Annotations of all genes were performed using BLASTN (e-value < 10-5) against the F. candida online genomic database (https://collembolomics.nl/collembolomics/folsomia/index.php). GO term enrichment analysis was performed for significantly differentially expressed genes using Fisher’s exact test (p < 0.05) in Blast2GO (version 4.1.9) (Conesa et al., 2005). From the RT-qPCR data, relative expression values of the six target genes were calculated according to Pfaffl (2001) and the values were log2 transformed. The log2 fold change values of the six target genes measured by RT-qPCR and microarray were analyzed with Pearson correlation in OriginPro 2015.
3. Results
3.1 Effects of BDE209 on reproduction and gene expression
To verify that BDE209 had a significant effect on the reproduction of F. candida at the reported EC50 concentration (0.81 mg/kg), we performed a 28-day reproduction test using BDE209-polluted soil, solvent control soil and control soil. No significant difference was found in adult mortality and juvenile numbers between the solvent control and control group. Compared with the solvent control, the juvenile
numbers in BDE209-polluted soil reduced significantly by 37% (Fig. S1), and no significant adult mortality was observed, indicating that the reduction of juvenile numbers was not caused by the presence of fewer adults. Statistical analysis of gene expression changes in F. candida exposed to BDE209-polluted soil identified 16 and 771 significantly differentially expressed genes after 2 and 7 days of exposure, respectively (Table S1-3). No significantly differentially expressed genes were found at 14 days. At the 2 and 7 day time points, there were no common regulated genes (Fig. 1). To validate the microarray data, we performed RT-qPCR on six target genes and two reference genes. The expression changes of the six target genes measured by microarray and RT-qPCR were similar (Fig. 2), and values of the two tests were significantly correlated (Pearson 0.92, p < 0.05, Fig. S2).
3.2 Functional analysis of differentially expressed genes
BDE209 significantly affected many biological processes and molecular functions (GO terms) in F. candida at 2 and 7 days (Tables S4-7). At 2 days, ten biological process terms and eight molecular function terms were up-regulated (Table S4), and one biological process term and one molecular function term were down-regulated (Table S5). A transcript encoding cuticlin-1 (Fcc00252), a component of the cuticles, was up-regulated (Table S2). At 7 days, 53 biological process terms and 15 molecular function terms were up-regulated (Table S6), and 13 biological process terms and 11 molecular function
terms were down-regulated (Table S7). The biological process term “translation” was up-regulated, indicating that translation was induced (Table S6). Four biological process terms related to apoptosis, including “programmed cell death”, “regulation of apoptotic process”, “regulation of programmed cell death” and “apoptotic process” (Table S6), suggesting apoptosis was induced by BDE209. Six transcripts related to moulting were up-regulated, including nuclear hormone receptor E75 (Fcc04094), ecdysteroid-regulated 16 kDa protein (Fcc04359), chitinase (Fcc00789), cuticle protein (Fcc05439 and Fcc05711) and cuticlin-1 (Fcc01731) (Table S3). Five transcripts related to nervous system were affected, including four up-regulated transcripts: neurotransmitter-gated ion-channel ligand-binding domain (Fcc00888), gamma-aminobutyric acid receptor (GABA receptor, Fcc02667), dopamine N-acetyltransferase (DAT, Fcc03190) and neuroendocrine convertase 1 (Fcc01701), and one down-regulated transcript: dopamine N-acetyltransferase (Fcc04605) (Table S3). Four transcripts (Fcc00299, Fcc01329, Fcc05556 and Fcc04311) encoding phase I detoxification enzyme cytochrome P450s (CYPs) were up-regulated (Table S3). The transcripts encoding phase II detoxification enzymes UDP-glucuronosyltransferase (UGT, Fcc05333) and glutathione S-transferase (GST, Fcc01166) were up-regulated (Table S3).
4. Discussion
BDE209 is a common soil contaminant of concern, but little is known about its
effects and toxic mechanisms at the molecular level on soil invertebrates. Genomic technologies such as microarray have become effective tools for studying molecular toxic effects and mechanisms of contaminants (Qiao et al., 2015; Chen et al., 2015). Here we detected changes in the transcriptional profiles of F. candida in response to BDE209 exposure using microarray test, which can provide useful information about the molecular effects of BDE209 on F. candida. 4.1 Effects on moulting-related genes The up-regulation of chitinase gene (Fcc00789) revealed that chitin degradation was affected. Chitin is a principal component of arthropod cuticle and peritrophic membrane and chitinase degrade chitin during moulting (Merzendorfer, 2003). The peritrophic membrane is involved in digestion and located in the gut (Fountain and Hopkin, 2005). For growth and development, F. candida periodically replace cuticle and gut epithelium through the moulting process (Thimm et al., 1998; Timmermans et al., 2009). Genes encoding cuticle protein (Fcc05439 and Fcc05711) and cuticlin-1 (Fcc01731 and Fcc00252) that are also major components of cuticle were observed to be up-regulated (Sebastiano et al., 1991; Charles, 2010). From the transcriptional level changes of chitinase, cuticle protein and cuticlin-1 genes, it could be inferred that the moulting of F. candida was affected by BDE209 exposure. However, contrasting with our results, BDE209 (0.3-500μg/L) did not affect moulting frequency of the aquatic invertebrate Daphnia magna following ten-day exposure (Davies and Zou, 2012). This may have been due to the experimental conditions applied (e.g. time
intervals between observations, exposure concentration and duration of experiment). Alternatively, despite the lack of changes in moulting frequency, there may have been changes in other parameters related to moulting which were not tested, such as cuticle abnormality. Studies on the effects of other PBDEs on moulting-related genes or proteins are far more common than those on BDE209 alone. For example, in the aquatic invertebrate Gammarus pulex, the activity of chitobiase and chitinolytic enzymes differed after exposure for 96h to BDE47 and BDE99 (0.1 and 1 μg/L) (Gismondi and Thomé, 2014). Genes encoding ecdysteroid-regulated 16 kDa protein (Fcc04359) and nuclear hormone receptor E75 (Fcc04094) that were involved in the ecdysteroid signalling were up-regulated, indicating BDE209 could disrupt moulting of F. candida via endocrine disruption, specifically in the ecdysteroid pathway, a hormone pathway in arthropods which regulates moulting (Chan, 1998; Nakagawa and Henrich, 2009). Other PBDEs are known to affect genes involved in the ecdysteroid pathway. For example, in an in vitro test using epidermis of the aquatic invertebrate Callinectes sapidus, BDE47 (100 nM) induced the transcription of N-acetyl-β-glucosaminidase (NAG), an enzyme involved in ecdysteroid signalling (Booth and Zou, 2016). 4.2 Effects on nervous system We observed that five genes related to nervous system were affected, including genes
encoding
neurotransmitter-gated
ion-channel
ligand-binding
domain
(Fcc00888), GABA receptor (Fcc02667), DAT ( Fcc03190 and Fcc04605) and neuroendocrine convertase 1 (Fcc01701). GABA receptor is a kind of ligand-gated ion channel (LGIC) that participate in fast synaptic transmission (Beg and Jorgensen, 2003; Collingridge et al., 2009). Down- or up-regulation of GABA receptor transcription in F. candida exposed to BDE209 could cause protein level changes of the receptor in neuron membrane, and thus may affect neural signal transmission associated with GABA receptor. In other organisms, PBDEs affect molecules involved in signalling pathways mediated by GABA receptor. For example, the commercial PBDE mixture DE-71 was found to reduce the level of GABA(A) 2α receptor subunit and GAD67 (GABA synthesizing enzyme), and increase the level of vGAT (vesicular GABA transporter) in the frontal cortex of mice exposed to DE-71 (30 mg/kg) for 30 days (Bradner et al., 2013). Although the study used different organism and other PBDEs instead of BDE209, it also indicated that PBDEs could result in neurotoxicity probably through disrupting neurotransmitter signalling pathway, which was in line with our results. DAT catalyses the acetylation of dopamine (a neurotransmitter) and it is involved in neurotransmitter metabolism and cuticle sclerotization (Klein, 2007). The down-regulation of DAT in F. candida exposed to BDE209 indicated that BDE209 could affect dopamine metabolism, thereby disrupting dopaminergic signalling. Wang et al. (2015) studied the impact of exposure to the PBDE mixture DE-71 (0-100 μg/L, 5d) on zebrafish, and found that DE-71 reduced the level of whole-body dopamine
and dihydroxyphenylacetic acid (DOPAC, metabolite of dopamine), the transcription of genes involved in dopaminergic neuron development and the protein levels of tyrosine hydroxylase and dopamine transporter in dopaminergic neurons. These results suggested that PBDEs can influence dopaminergic signalling by disrupting dopamine metabolism, which was similar to our results. The transcription of DAT gene in F. candida was also found to be affected by other xenobiotics. For example, Pentachlorophenol (PCP, 87 mg/kg dry soill) can repress the translation of DAT (Fcc04605) in F. candida after 1 and 2 day exposure (Qiao et al., 2015), suggesting that PCP also can disrupt dopaminergic signalling like BDE209. Neuroendocrine convertases (also called prohormone convertases and proprotein convertases, PCs) aid in activation of protein hormones and neuropeptides from their inactive precursors (Rodrigues et al., 2017). The functions of PCs in springtails are unknown, but PCs play important roles in other invertebrates. RNA interference with a PC-encoding gene in the invertebrate Schmidtea mediterranea paralyzed animals (Reddien et al., 2005). Prohormone convertase 1 (PC1) in the invertebrate Haliotis diversicolor supertexta is involved in reproductive behaviour (Zhou and Cai, 2010). The up-regulation of PC1 in F. candida exposed to BDE209 could affect F. candida reproduction and behaviour through hormone and neuropeptide regulation processes. 4.3 Effects on the detoxification system The detoxification enzyme genes CYP (Fcc00299, Fcc01329, Fcc05556 and Fcc04311), UGT (Fcc05333) and GST (Fcc01166) were up-regulated, indicating that
they were involved in the metabolism of BDE209. Effects of PBDEs on detoxification enzymes were also found in other organisms. For example, Usenko et al. (2013) found that the transcriptions of some CYPs and UGTs in zebrafish were affected by PBDEs. Zhang et al. (2014) reported that the GST activity of earthworm was repressed by exposure to BDE209. Other xenobiotics also can affect the transcription of the detoxification enzyme genes in F. candida. For example, PCP (87 mg/kg dry soil) induced the CYP gene (Fcc00299) after 1 and 7 day exposure (Qiao et al., 2015). Diclofenac (50 mg/kg dry soil) also induced the CYP gene (Fcc00299) after 2 day exposure (Chen et al., 2015). 4.4 Comparison among exposure durations The transcriptional responses of F. candida to BDE209 differed greatly at 2, 7 and 14 days. The number of significantly differentially expressed genes at 2 day was far fewer than that at 7 day. Qiao et al. (2015) studied the transcriptional profile of F. candida exposed to PCP (87 mg/kg dry soil) for 1, 2 and 7 days, and found that the number of affected genes decreased with exposure time, which was different from this study. One possible reason was that the BDE209 concentration used in this study was far lower (0.81 mg/kg dry soil). Besides, the BDE209 concentration we used was close to the environmental concentration in some polluted area (Nie et al., 2015), suggesting that springtail community in these polluted area would be affected. No genes were affected by BDE209 at 14 days. BDE209 was persistent in soil (Liu et al., 2011), so the degradation of BDE209 cannot explain the result. F. candida may have
experienced acclimation after 14-day exposure to BDE209.
5. Conclusions
In this study, we demonstrated the effects of BDE209 on the gene transcription of the soil invertebrate F. candida. The transcriptional profiles provided insights into the toxic mechanism of BDE209 on F. candida. We found that BDE209 affects the transcription of genes related to moulting, neural signal transmission and detoxification. It seems that BDE209 could disrupt moulting of F. candida via the ecdysteroid
pathway,
and
cause
neurotoxicity
through
disrupting
some
neurotransmitter signalling pathways. Our study could give useful information when assessing the environmental risk of BDE209 and other similar chemicals in soil.
Acknowledgements
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Fig. 1. Venn diagrams of differentially expressed genes between springtails (Folsomia candida) cultured in BDE209-polluted soil and control soil for 2 and 7 days (SAM, q-value (%) < 5). Numbers in red represent up-regulated genes, and numbers in green represent down-regulated genes.
Fig. 2. Comparison of Folsomia candida gene expression measured using microarray and RT-qPCR. Values shown are average log2 fold changes and standard error (n = 3).
Transcription responses of F. candida differed greatly at 2, 7 and 14 days.
BDE209 affected moulting, nervous and detoxification system.
BDE209 could disrupt moulting of F. candida via the ecdysteroid pathway. BDE209 caused neurotoxicity by disrupting some neurotransmitter signaling pathways.
26
S0048-9697(19)34851-X https://doi.org/10.1016/j.scitotenv.2019.134859 STOTEN 134859
To appear in:
Science of the Total Environment
Received Date: Revised Date: Accepted Date:
26 March 2019 29 September 2019 5 October 2019
Please cite this article as: Q-Q. Zhang, M. Qiao, Transcriptional response of springtail (Folsomia candida) exposed to decabromodiphenyl ether-contaminated soil, Science of the Total Environment (2019), doi: https://doi.org/ 10.1016/j.scitotenv.2019.134859
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© 2019 Elsevier B.V. All rights reserved.
Transcriptional
response
of
springtail
(Folsomia
candida)
exposed
to
decabromodiphenyl ether-contaminated soil
Qian-Qian Zhanga, b, Min Qiaoa, b*
a
State Key Lab of Urban and Regional Ecology, Research Center for
Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China b
University of Chinese Academy of Sciences, Beijing 100049, China
*
Corresponding author: Min Qiao, State Key Lab of Urban and Regional Ecology,
Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China. Tel: +86-10-62849158; Fax: +86-10-62923563; e-mail: [email protected]
Abstract
Decabromodiphenyl ether (BDE209) is a widely used brominated flame retardant that has become a common soil contaminant of concern due to its persistence and toxicity. However, little is known about molecular-level effects of BDE209 on soil invertebrates. Here, we detected changes in gene transcription of the soil springtail, Folsomia candida, exposed to BDE209 (0.81 mg/kg) in soil for 2, 7
and 14 days. We identified 16 and 771 significantly differentially expressed genes after 2 and 7 days of exposure respectively, and no significantly regulated genes were shared among the two time points. No genes were affected after 14 days of exposure. According to the annotation of the significantly differently expressed genes at 2 and 7 day exposure, we found that BDE209 affected the transcription of genes involved in moulting, neural signal transmission and detoxification. Our results suggeted that BDE209 could disrupt moulting of F. candida via the ecdysteroid pathway, and cause neurotoxicity through disrupting some neurotransmitter signalling pathways. This study provided insights into the toxic mechanism of BDE209 on F. candida.
Keywords
BDE209; Folsomia candida; Transcriptomics; Moulting; Neurotoxicity
1. Introduction
Polybrominated diphenyl ethers (PBDEs) are widely used as flame retardants in various products including textiles, furniture, electronic devices and building materials (Wang et al., 2005). Commercial PBDE mixures are usually produced at three levels of bromination, namely penta-BDE, octa-BDE and deca-BDE (La Guardia et al., 2006). In China, penta-BDE and octa-BDE are restricted while deca-BDE is still allowed (Wang et al., 2017). PBDEs have become widespread contaminants of concern due to their toxicity, persistence in the environment and bioaccumulation (Lee and Kim, 2015). Decabromodiphenyl ether (BDE209) is the
main component of commercial deca-BDE mixtures and has been the dominant congener detected in many soil samples (McGrath et al., 2017). For example, the average total concentration of 21 PBDEs (including BDE209) in soil samples from an electronic waste burning site in South China was 2283 μg/kg, while the average concentration of BDE209 alone was 2162 μg/kg (Nie et al., 2015). BDE209 can have a variety of adverse effects on organisms. For example, BDE209 (10 μg/L) can affect the expression of thyroid hormone (TH) and spermatogenesis associated genes in adult rare minnow (Gobiocypris rarus) following exposure for 21 days (Li et al., 2014). Proteins involved in normal brain development of mice had altered levels after exposure to 20.1 mg BDE209/kg body weight for 7 days, suggesting that BDE209 causes developmental neurotoxicity (Viberg et al., 2008). An in vitro study using neural stem cells (NSC) of rats showed that BDE209 can inhibit NSC proliferation and induce apoptosis (Zhang et al., 2016). Although the effects of BDE209 on vertebrates and in vitro cells have been widely reported, few studies are available on soil invertebrates. Springtails are widespread and abundant soil invertebrates that have been used extensively to assess the effects of chemicals on soil ecosystems (Fountain and Hopkin, 2005). The springtail Folsomia candida has been used as a model species in ecotoxicology studies (ISO, 1999). The effects of chemicals on F. candida are usually assessed by conventional bioassays, such as reproduction and avoidance tests (ISO, 2011, 2014). In recent years, genomic tools have been employed for studying
molecular effects, including toxic mechanisms. Zhang et al. (2012) reported that BDE209 can affect the reproduction and avoidance behaviours of F. candida, and that EC50 values were 0.81 and 1.27 mg/kg, respectively. However, the molecular effects of BDE209 on F. candida have not been reported. In this study, we aimed to assess the effects of BDE209 on gene transcription of F. candida and explore the mechanisms of toxicity.
2. Materials and methods
2.1 Test organism, soil and exposure experiment
The springtail Folsomia candida was cultured following methods described previously (Zhang and Qiao, 2018). Briefly, the springtail was cultured in Petri dishes on a moistened substrate consisting of plaster of Paris and activated carbon (9:1 weight ratio) under controlled conditions (20±1oC, 70±5% relative humidity and constant darkness). The springtails used for reproduction experiment and microarray experiment were 10-12 day old and 23 day old respectively. The soil used in the experiments was artificial soil, composed of 10% ground dry sphagnum peat sieved to 2mm, 20% kaolinite clay and 70% quartz sand. BDE209 (98% purity, Dr. Ehrenstorfer, German) was added to the soil using acetone and toluene (9:1 volume ratio) as carrier solvent, to reach a nominal concentration of 0.81 mg/kg dry soil. This nominal concentration in artificial soil was used because it reduced F. candida reproduction by 50% (EC50) after 28-day exposure (Zhang et al.,
2012). The solution of BDE209 was mixed with 10% of the test soil and the mixture was put in closed glass containers overnight. The glass containers were then opened and put in a fume hood for 24 hours to volatilize the acetone. The mixture (10% of the test soil and BDE209) was thoroughly mixed with the remaining soil (90%). The same volume of solvent (without BDE209) was added to the solvent control soil. Soil without solvent treatment was used as control soil. All soils were moistened using distilled water to 50% of the maximum water holding capacity. To verify that BDE209 significantly repressed the reproduction of F. candida at the reported EC50 concentration, a 28-day reproduction test was performed. Ten 10-12 day old juveniles were introduced into a 100 mL glass beaker containing 30 g wet soil (BDE209-polluted soil, solvent control soil or control soil), using four replicates for each treatment. These beakers were sealed with Para film and opened once per week to add food. After 28 days, the number of surviving adults and juveniles was counted. To detect gene transcription in F. candida exposed to BDE209-polluted soil, 30 adult springtails (23-day old) were exposed to BDE209-polluted soil or solvent control soil in 100 mL glass beakers for 2, 7 or 14 days. Three replicates were used for each treatment. Springtails were extracted from soil by means of floatation and collected in microcentrifuge tubes. They were snap frozen in liquid nitrogen and kept at -80 oC until RNA extraction.
2.2 Microarray and RT-qPCR experiment
The microarray experiment was performed by the Shanghai Biotechnology Corporation using a custom Agilent 4 × 44 K microarray (Design ID 045772). The microarray contained 6175 different probes spotted randomly for seven times, representing 6175 different ESTs (Expressed sequence tags) from Collembase (Timmermans et al., 2007). Total RNA of each replicate (30 animals) was extracted using RNAiso Plus (Cat. No. 9109, TAKARA, Japan). RNA integrity was inspected by Agilent Bioanalyzer 2100 (Agilent technologies, USA). Qualified total RNA was purified by RNeasy Micro Kit (Cat. No. 74004, QIAGEN, Germany) and RNase-Free DNase Set (Cat. No. 74004, QIAGEN, Germany). Total RNA was amplified and labeled using Low Input Quick Amp Labeling Kit (One color, Cat. No. 5190-2305, Agilent technologies, USA) and was then purified using RNeasy Mini Kit (Cat. No. 74106, QIAGEN, Germany). Cy3-labled cRNA was hybridized with the microarray in a Hybridization Oven (Cat. No. G2545A, Agilent technologies, USA) using the Gene Expression Hybridization Kit (Cat. No. 5188-5242, Agilent technologies, USA). After hybridization, the microarrays were washed by Gene Expression Wash Buffer Kit (Cat. No. 5188-5327, Agilent technologies, USA) and then scanned by Agilent Microarray Scanner (Cat. No. G2565CA, Agilent technologies, USA). To validate the microarray data, reverse transcription real-time quantitative polymerase chain reaction (RT-qPCR) on six target genes (Fcc00966, Fcc04911, Fcc03521, Fcc01821, Fcc01655, Fcc01574) and two reference genes (YWHAZ and
SDHA (de Boer et al., 2009)) were performed for the total RNA samples from springtails which were exposed for 7 days using the same exposure method as in the microarray experiment. Reverse transcription reaction was performed with about 1 μg total RNA using FastQuant RT Kit (Cat. No. KR106, Tiangen Biotech, China) according to the manufacturer’s protocol. The obtained cDNA was diluted 10 times and 2 μL was used in the 20 μL qPCR volume. The qPCR reaction was performed with SuperReal PreMix Plus (SYBR Green) Kit following the manufacturer’s protocol (Cat. No. FP205, Tiangen Biotech, China) using the LightCycler 480 system (Roche, Switzerland). The qPCR conditions were as follows: initial denaturation at 95 oC
for 15 min; 40 cycles of 95 oC denaturation for 10s, 60 oC annealing for 20s and
72oC elongation for 20s. Detection of melting curves was performed after amplification. Primer sequences and efficiencies were summarized in Table S8.
2.3 Data analysis
To verify the effect of BDE209 (0.81 mg/kg) on reproduction of F. candida after 28-day exposure, juvenile numbers in the BDE209-polluted soil, solvent control soil or control soil were compared using one-way ANOVA followed by Tukey test. The scanned images of microarrays were analyzed by Feature Extraction software 10.7 (Agilent technologies, USA), and the detected and undetected genes were determined by this software. The data are accessible in NCBI's Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE137740). The raw data obtained were normalized by quantile algorithm using “limma” package in R
software. The significantly differentially expressed genes were determined by the Significance
Analysis
of
Microarrays
(SAM)
method
(http://statweb.stanford.edu/~tibs/SAM/) (Tusher et al., 2001). Genes with q-value (false discovery rate-corrected p-value) < 0.05 were considered as significantly differentially expressed genes. Annotations of all genes were performed using BLASTN (e-value < 10-5) against the F. candida online genomic database (https://collembolomics.nl/collembolomics/folsomia/index.php). GO term enrichment analysis was performed for significantly differentially expressed genes using Fisher’s exact test (p < 0.05) in Blast2GO (version 4.1.9) (Conesa et al., 2005). From the RT-qPCR data, relative expression values of the six target genes were calculated according to Pfaffl (2001) and the values were log2 transformed. The log2 fold change values of the six target genes measured by RT-qPCR and microarray were analyzed with Pearson correlation in OriginPro 2015.
3. Results
3.1 Effects of BDE209 on reproduction and gene expression
To verify that BDE209 had a significant effect on the reproduction of F. candida at the reported EC50 concentration (0.81 mg/kg), we performed a 28-day reproduction test using BDE209-polluted soil, solvent control soil and control soil. No significant difference was found in adult mortality and juvenile numbers between the solvent control and control group. Compared with the solvent control, the juvenile
numbers in BDE209-polluted soil reduced significantly by 37% (Fig. S1), and no significant adult mortality was observed, indicating that the reduction of juvenile numbers was not caused by the presence of fewer adults. Statistical analysis of gene expression changes in F. candida exposed to BDE209-polluted soil identified 16 and 771 significantly differentially expressed genes after 2 and 7 days of exposure, respectively (Table S1-3). No significantly differentially expressed genes were found at 14 days. At the 2 and 7 day time points, there were no common regulated genes (Fig. 1). To validate the microarray data, we performed RT-qPCR on six target genes and two reference genes. The expression changes of the six target genes measured by microarray and RT-qPCR were similar (Fig. 2), and values of the two tests were significantly correlated (Pearson 0.92, p < 0.05, Fig. S2).
3.2 Functional analysis of differentially expressed genes
BDE209 significantly affected many biological processes and molecular functions (GO terms) in F. candida at 2 and 7 days (Tables S4-7). At 2 days, ten biological process terms and eight molecular function terms were up-regulated (Table S4), and one biological process term and one molecular function term were down-regulated (Table S5). A transcript encoding cuticlin-1 (Fcc00252), a component of the cuticles, was up-regulated (Table S2). At 7 days, 53 biological process terms and 15 molecular function terms were up-regulated (Table S6), and 13 biological process terms and 11 molecular function
terms were down-regulated (Table S7). The biological process term “translation” was up-regulated, indicating that translation was induced (Table S6). Four biological process terms related to apoptosis, including “programmed cell death”, “regulation of apoptotic process”, “regulation of programmed cell death” and “apoptotic process” (Table S6), suggesting apoptosis was induced by BDE209. Six transcripts related to moulting were up-regulated, including nuclear hormone receptor E75 (Fcc04094), ecdysteroid-regulated 16 kDa protein (Fcc04359), chitinase (Fcc00789), cuticle protein (Fcc05439 and Fcc05711) and cuticlin-1 (Fcc01731) (Table S3). Five transcripts related to nervous system were affected, including four up-regulated transcripts: neurotransmitter-gated ion-channel ligand-binding domain (Fcc00888), gamma-aminobutyric acid receptor (GABA receptor, Fcc02667), dopamine N-acetyltransferase (DAT, Fcc03190) and neuroendocrine convertase 1 (Fcc01701), and one down-regulated transcript: dopamine N-acetyltransferase (Fcc04605) (Table S3). Four transcripts (Fcc00299, Fcc01329, Fcc05556 and Fcc04311) encoding phase I detoxification enzyme cytochrome P450s (CYPs) were up-regulated (Table S3). The transcripts encoding phase II detoxification enzymes UDP-glucuronosyltransferase (UGT, Fcc05333) and glutathione S-transferase (GST, Fcc01166) were up-regulated (Table S3).
4. Discussion
BDE209 is a common soil contaminant of concern, but little is known about its
effects and toxic mechanisms at the molecular level on soil invertebrates. Genomic technologies such as microarray have become effective tools for studying molecular toxic effects and mechanisms of contaminants (Qiao et al., 2015; Chen et al., 2015). Here we detected changes in the transcriptional profiles of F. candida in response to BDE209 exposure using microarray test, which can provide useful information about the molecular effects of BDE209 on F. candida. 4.1 Effects on moulting-related genes The up-regulation of chitinase gene (Fcc00789) revealed that chitin degradation was affected. Chitin is a principal component of arthropod cuticle and peritrophic membrane and chitinase degrade chitin during moulting (Merzendorfer, 2003). The peritrophic membrane is involved in digestion and located in the gut (Fountain and Hopkin, 2005). For growth and development, F. candida periodically replace cuticle and gut epithelium through the moulting process (Thimm et al., 1998; Timmermans et al., 2009). Genes encoding cuticle protein (Fcc05439 and Fcc05711) and cuticlin-1 (Fcc01731 and Fcc00252) that are also major components of cuticle were observed to be up-regulated (Sebastiano et al., 1991; Charles, 2010). From the transcriptional level changes of chitinase, cuticle protein and cuticlin-1 genes, it could be inferred that the moulting of F. candida was affected by BDE209 exposure. However, contrasting with our results, BDE209 (0.3-500μg/L) did not affect moulting frequency of the aquatic invertebrate Daphnia magna following ten-day exposure (Davies and Zou, 2012). This may have been due to the experimental conditions applied (e.g. time
intervals between observations, exposure concentration and duration of experiment). Alternatively, despite the lack of changes in moulting frequency, there may have been changes in other parameters related to moulting which were not tested, such as cuticle abnormality. Studies on the effects of other PBDEs on moulting-related genes or proteins are far more common than those on BDE209 alone. For example, in the aquatic invertebrate Gammarus pulex, the activity of chitobiase and chitinolytic enzymes differed after exposure for 96h to BDE47 and BDE99 (0.1 and 1 μg/L) (Gismondi and Thomé, 2014). Genes encoding ecdysteroid-regulated 16 kDa protein (Fcc04359) and nuclear hormone receptor E75 (Fcc04094) that were involved in the ecdysteroid signalling were up-regulated, indicating BDE209 could disrupt moulting of F. candida via endocrine disruption, specifically in the ecdysteroid pathway, a hormone pathway in arthropods which regulates moulting (Chan, 1998; Nakagawa and Henrich, 2009). Other PBDEs are known to affect genes involved in the ecdysteroid pathway. For example, in an in vitro test using epidermis of the aquatic invertebrate Callinectes sapidus, BDE47 (100 nM) induced the transcription of N-acetyl-β-glucosaminidase (NAG), an enzyme involved in ecdysteroid signalling (Booth and Zou, 2016). 4.2 Effects on nervous system We observed that five genes related to nervous system were affected, including genes
encoding
neurotransmitter-gated
ion-channel
ligand-binding
domain
(Fcc00888), GABA receptor (Fcc02667), DAT ( Fcc03190 and Fcc04605) and neuroendocrine convertase 1 (Fcc01701). GABA receptor is a kind of ligand-gated ion channel (LGIC) that participate in fast synaptic transmission (Beg and Jorgensen, 2003; Collingridge et al., 2009). Down- or up-regulation of GABA receptor transcription in F. candida exposed to BDE209 could cause protein level changes of the receptor in neuron membrane, and thus may affect neural signal transmission associated with GABA receptor. In other organisms, PBDEs affect molecules involved in signalling pathways mediated by GABA receptor. For example, the commercial PBDE mixture DE-71 was found to reduce the level of GABA(A) 2α receptor subunit and GAD67 (GABA synthesizing enzyme), and increase the level of vGAT (vesicular GABA transporter) in the frontal cortex of mice exposed to DE-71 (30 mg/kg) for 30 days (Bradner et al., 2013). Although the study used different organism and other PBDEs instead of BDE209, it also indicated that PBDEs could result in neurotoxicity probably through disrupting neurotransmitter signalling pathway, which was in line with our results. DAT catalyses the acetylation of dopamine (a neurotransmitter) and it is involved in neurotransmitter metabolism and cuticle sclerotization (Klein, 2007). The down-regulation of DAT in F. candida exposed to BDE209 indicated that BDE209 could affect dopamine metabolism, thereby disrupting dopaminergic signalling. Wang et al. (2015) studied the impact of exposure to the PBDE mixture DE-71 (0-100 μg/L, 5d) on zebrafish, and found that DE-71 reduced the level of whole-body dopamine
and dihydroxyphenylacetic acid (DOPAC, metabolite of dopamine), the transcription of genes involved in dopaminergic neuron development and the protein levels of tyrosine hydroxylase and dopamine transporter in dopaminergic neurons. These results suggested that PBDEs can influence dopaminergic signalling by disrupting dopamine metabolism, which was similar to our results. The transcription of DAT gene in F. candida was also found to be affected by other xenobiotics. For example, Pentachlorophenol (PCP, 87 mg/kg dry soill) can repress the translation of DAT (Fcc04605) in F. candida after 1 and 2 day exposure (Qiao et al., 2015), suggesting that PCP also can disrupt dopaminergic signalling like BDE209. Neuroendocrine convertases (also called prohormone convertases and proprotein convertases, PCs) aid in activation of protein hormones and neuropeptides from their inactive precursors (Rodrigues et al., 2017). The functions of PCs in springtails are unknown, but PCs play important roles in other invertebrates. RNA interference with a PC-encoding gene in the invertebrate Schmidtea mediterranea paralyzed animals (Reddien et al., 2005). Prohormone convertase 1 (PC1) in the invertebrate Haliotis diversicolor supertexta is involved in reproductive behaviour (Zhou and Cai, 2010). The up-regulation of PC1 in F. candida exposed to BDE209 could affect F. candida reproduction and behaviour through hormone and neuropeptide regulation processes. 4.3 Effects on the detoxification system The detoxification enzyme genes CYP (Fcc00299, Fcc01329, Fcc05556 and Fcc04311), UGT (Fcc05333) and GST (Fcc01166) were up-regulated, indicating that
they were involved in the metabolism of BDE209. Effects of PBDEs on detoxification enzymes were also found in other organisms. For example, Usenko et al. (2013) found that the transcriptions of some CYPs and UGTs in zebrafish were affected by PBDEs. Zhang et al. (2014) reported that the GST activity of earthworm was repressed by exposure to BDE209. Other xenobiotics also can affect the transcription of the detoxification enzyme genes in F. candida. For example, PCP (87 mg/kg dry soil) induced the CYP gene (Fcc00299) after 1 and 7 day exposure (Qiao et al., 2015). Diclofenac (50 mg/kg dry soil) also induced the CYP gene (Fcc00299) after 2 day exposure (Chen et al., 2015). 4.4 Comparison among exposure durations The transcriptional responses of F. candida to BDE209 differed greatly at 2, 7 and 14 days. The number of significantly differentially expressed genes at 2 day was far fewer than that at 7 day. Qiao et al. (2015) studied the transcriptional profile of F. candida exposed to PCP (87 mg/kg dry soil) for 1, 2 and 7 days, and found that the number of affected genes decreased with exposure time, which was different from this study. One possible reason was that the BDE209 concentration used in this study was far lower (0.81 mg/kg dry soil). Besides, the BDE209 concentration we used was close to the environmental concentration in some polluted area (Nie et al., 2015), suggesting that springtail community in these polluted area would be affected. No genes were affected by BDE209 at 14 days. BDE209 was persistent in soil (Liu et al., 2011), so the degradation of BDE209 cannot explain the result. F. candida may have
experienced acclimation after 14-day exposure to BDE209.
5. Conclusions
In this study, we demonstrated the effects of BDE209 on the gene transcription of the soil invertebrate F. candida. The transcriptional profiles provided insights into the toxic mechanism of BDE209 on F. candida. We found that BDE209 affects the transcription of genes related to moulting, neural signal transmission and detoxification. It seems that BDE209 could disrupt moulting of F. candida via the ecdysteroid
pathway,
and
cause
neurotoxicity
through
disrupting
some
neurotransmitter signalling pathways. Our study could give useful information when assessing the environmental risk of BDE209 and other similar chemicals in soil.
Acknowledgements
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Fig. 1. Venn diagrams of differentially expressed genes between springtails (Folsomia candida) cultured in BDE209-polluted soil and control soil for 2 and 7 days (SAM, q-value (%) < 5). Numbers in red represent up-regulated genes, and numbers in green represent down-regulated genes.
Fig. 2. Comparison of Folsomia candida gene expression measured using microarray and RT-qPCR. Values shown are average log2 fold changes and standard error (n = 3).
Transcription responses of F. candida differed greatly at 2, 7 and 14 days.
BDE209 affected moulting, nervous and detoxification system.
BDE209 could disrupt moulting of F. candida via the ecdysteroid pathway. BDE209 caused neurotoxicity by disrupting some neurotransmitter signaling pathways.
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