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Journal of Environmental Science and Health, Part B: Pesticides, Food Contaminants, and Agricultural Wastes Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/lesb20
Screening differentially expressed genes in an amphipod (Hyalella azteca) exposed to fungicide vinclozolin by suppression subtractive hybridization a
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Yun H. Wu , Tsung M. Wu , Chwan Y. Hong , Yei S. Wang & Jui H. Yen a
Department of Agricultural Chemistry, National Taiwan University, Taipei, Taiwan Published online: 04 Sep 2014.
To cite this article: Yun H. Wu, Tsung M. Wu, Chwan Y. Hong, Yei S. Wang & Jui H. Yen (2014) Screening differentially expressed genes in an amphipod (Hyalella azteca) exposed to fungicide vinclozolin by suppression subtractive hybridization, Journal of Environmental Science and Health, Part B: Pesticides, Food Contaminants, and Agricultural Wastes, 49:11, 856-863, DOI: 10.1080/03601234.2014.938556 To link to this article: http://dx.doi.org/10.1080/03601234.2014.938556
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Journal of Environmental Science and Health, Part B (2014) 49, 856–863 Copyright © Taylor & Francis Group, LLC ISSN: 0360-1234 (Print); 1532-4109 (Online) DOI: 10.1080/03601234.2014.938556
Screening differentially expressed genes in an amphipod (Hyalella azteca) exposed to fungicide vinclozolin by suppression subtractive hybridization YUN H. WU, TSUNG M. WU, CHWAN Y. HONG, YEI S. WANG and JUI H. YEN
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Department of Agricultural Chemistry, National Taiwan University, Taipei, Taiwan
Vinclozolin, a dicarboximide fungicide, is an endocrine disrupting chemical that competes with an androgenic endocrine disruptor compound. Most research has focused on the epigenetic effect of vinclozolin in humans. In terms of ecotoxicology, understanding the effect of vinclozolin on non-target organisms is important. The expression profile of a comprehensive set of genes in the amphipod Hyalella azteca exposed to vinclozolin was examined. The expressed sequence tags in low-dose vinclozolin-treated and -untreated amphipods were isolated and identified by suppression subtractive hybridization. DNA dot blotting was used to confirm the results and establish a subtracted cDNA library for comparing all differentially expressed sequences with and without vinclozolin treatment. In total, 494 differentially expressed genes, including hemocyanin, heatshock protein, cytochrome, cytochrome oxidase and NADH dehydrogenase were detected. Hemocyanin was the most abundant gene. DNA dot blotting revealed 55 genes with significant differential expression. These genes included larval serum protein 1 alpha, E3 ubiquitin-protein ligase, mitochondrial cytochrome c oxidase, mitochondrial protein, proteasome inhibitor, hemocyanin, zinc-finger–containing protein, mitochondrial NADH-ubiquinone oxidoreductase and epididymal sperm-binding protein. Vinclozolin appears to upregulate stress-related genes and hemocyanin, related to immunity. Moreover, vinclozolin downregulated NADH dehydrogenase, related to respiration. Thus, even a non-lethal concentration of vinclozolin still has an effect at the genetic level in H. azteca and presents a potential risk, especially as it would affect non-target organism hormone metabolism. Keywords: Endocrine disrupter chemical, vinclozolin, Hyalella azteca, suppression subtractive hybridization, differential expression.
Introduction The dicarboximide-type fungicide vinclozolin [3-(3,5dichlorophenyl)-5-methyl-5-vinyl-1,3-oxazolidine,4-dione] was broadly introduced to control various vegetable and orchard diseases in the world. It is used for eradication of disease and protection against many fungal diseases, especially those caused by Botrytis cinerea, Sclerotinia sclerotiorum and Monilinia sp. infection in grapes, fruits and vegetables.[1] Because of its wide use, it is found as a residue in the environment and also acts as an endocrine disruptor chemical.[1,2] Moreover, vinclozolin is an environmental antiandrogen and influences gonad Address correspondence to Yei-Shung Wang and Jui-Hung Yen, Department of Agricultural Chemistry, College of Bioresources and Agriculture, National Taiwan University, R317, No. 1, Sec. 4, Roosevelt Road, Taipei, 10617 Taiwan (R.O.C); E-mail: [email protected] and [email protected]. Received April 30, 2014. Color versions of one or more of the figures in this article can be found online at www.tandfonline.com/lesb.
development and fertility.[3–7] It induces transcriptional alteration during gonadal sex determination and early testis development.[8] Moreover, it was found to act transiently during embryonic sex determination to promote a spermatogenic cell defect in the F1 generation as well as subfertility in males.[8] Vinclozolin was detected in underground water, lakes, rivers and seawater in Greece.[9] However, few studies have investigated the ecotoxicity or environmental toxicity of vinclozolin, especially in aquatic ecosystems. As such, the effect on non-target organisms of vinclozolin needs to be evaluated. The amphipod Hyalella azteca is a representative invertebrate in aquatic ecosystems. It was widely used in ecotoxicological evaluations of sediment because of its availability, easy culture in the laboratory and sensitivity to contaminants.[10] Furthermore, knowledge of the reproductive physiology of H. azteca is adequate, and both acute and chronic effects have been studied for many environmental contaminants.[11] In recent years, most studies of reproduction in aquatic species have focused on endocrine disruptor chemicals with estrogenic action.[12] Comparatively little is known
Suppression subtractive hybridization for screening differentially expressed genes in Hyalella azteca exposed to vinclozolin 857
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about chemicals that might interact with androgens because the functions of androgen receptors are largely unknown.[13] Suppression subtractive hybridization (SSH) is a powerful procedure that allows comparison of two mRNA populations by PCR-based cDNA subtraction.[14] SSH has been successfully used to identify differentially expressed genes in Neocaraidina denticiculata,[15] Penaeus japonicas,[16–18] Daphnia magna[19] and Litopenaeus vannamei.[20] In this study, we used SSH to identify the genes with differential expression in the amphipod H. azteca after exposure to vinclozolin for a comprehensive understanding of the mechanism of vinclozolin exposure in the amphipod.
Materials and methods Chemicals and reagents Vinclozolin (99.4% purity, CAS no. 50471-44-8; Chem Service, USA) was dissolved in acetone to establish a 10,000 mg L¡1 stock solution.
Amphipod culture H. azteca has been used extensively for standardized water and sediment toxicity testing.[10] Test organisms were procured from the Environmental Analysis Laboratory EPA (Taoyuan, Taiwan). Long-term cultures were maintained under static conditions in 1-L glass dishes with photoperiod 16:8 h (light/dark), 24 § 1 C and pH 6.8 § 0.2. Half the volume of overlying water in the aquaria was changed weekly by siphoning and adding fresh water. The culture diet consisted of a mixture of 0.5 g shrimp food and amphipods were fed once a week.
After exposure, the vinclozolin concentration was analyzed by high-performance liquid chromatography (HPLC) with UV detection. The conditions for HPLC were (1) C18 column (Merck, LiChrospher 100RP-18-e); (2) UV detector with absorption λmax 202 nm; (3) mobile phase: acetonitrile: H2O D 60:40; and (4) flow rate: 1 mL min¡1. Under these conditions, the retention time of vinclozolin was 10.2 min, and the detection limit was 0.20 mg L¡1. Preparation of poly (A)C RNA Total RNA was extracted by the TRIzol RNA reagent method (Invitrogen, CA, USA). After exposure, amphipods were collected for total RNA extraction, frozen by use of nitrogen (l) and, then, 2 mL TRIzol RNA reagent was added and mixed completely. After the addition of phenol-chloroform, NaCl and sodium citrate, the homogenate was centrifuged at 12,000 £ g for 10 min at 4 C. The supernatant was extracted with the use of isopropanol, and the total RNA was precipitated by ethanol and dissolved in diethylpyrocarbonate (DEPC) -treated water. Poly (AC) RNA was extracted by use of the Oligotex mRNA Kit (Quiagen, Valencia, CA, USA). Briefly, 0.25 mg total RNA was added to 250 mL Buffer OBB and 15 mL Oligotex Suspension. The mixture was incubated at 70 C for 3 min and cooled at room temperature for 10 min; then, the homogenate was centrifuged at 16,000 £ g for 2 min and the supernatant was removed. The pellet was resuspended in 400 mL Buffer OW2 by vortexing and homogenate was centrifuged at 16,000 £ g for 1 min. In total, 400 mL Buffer OW2 was added to the column, and the homogenate was centrifuged at 16,000 £ g for 1 min and the flow-through was discarded. Then, 20 mL hot (70 C) Buffer OEB was added to the column and centrifuged at 16,000 £ g for 1 min.
Exposure tests H. azteca was exposed to one vinclozolin treatment and one water control under laboratory conditions. Vinclozolin treatment was with vinclozolin, 3 mg L¡1, dissolved in water with stock solutions of vinclozolin in acetone to obtain 3 mg L¡1 vinclozolin. The concentration is the maximum solubility in water. The exposure test was performed in glass dishes (20 cm length, 20 cm wide and 6 cm high). A 100-g porcelain tile was placed on the bottom of dishes, and 200 adult H. azteca, approximately 1 cm in length, from laboratory cultures were placed in each dish together with 1 L solution. The exposure lasted 14 days, and 10% volume of overlying water in the aquaria was changed every 24 h by siphoning and adding fresh solution. The exposure test was maintained under static conditions in a 1-L glass dishes with a photoperiod of 16:8 h (light/dark) at 24 § 1 C and pH 6.8 § 0.2.
Suppression subtractive hybridization (SSH) SSH was performed to generate the subtracted cDNA library with vinclozolin treatment and the water control involved the use of the PCR-Select cDNA Subtraction Kit (Clontech, Palo Alto, CA, USA). In the forward cDNA library, vinclozolin treatment was the tester and water control the driver, then in the reverse cDNA library, water control was the tester and vinclozolin treatment the driver. Briefly, 2 mg mRNA was mixed with 1 mL cDNA Synthesis Primer. The mixture was incubated at 70 C for 2 min, cooled on ice for 2 min and, then, briefly centrifuged. Thereafter, 2 mL 5X First-Strand Buffer, 1 mL dNTP, 1 mL sterile H2O, 1 mL DTT and 1 mL SMARTScribe Reverse Transcriptase were added to the mixture and incubated at 42 C for 1.5 h. To this, 48.4 mL sterile H2O, 16 mL 5X Second-Strand Buffer, 1.6 mL dNTP, and 4 mL 20X Second-Strand Enzyme Cocktail were added to
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858 the first-strand synthesis reaction tubes and incubated at 16 C for 2 h. In total, 2 mL T4 DNA polymerase was added for incubation at 16 C for 30 min. Then, 4 mL of 20X EDTA/Glycogen Mix and 100 mL phenol: chloroform:isoamyl alcohol (25:24:1) was added, mixed thoroughly and centrifuged at 16,000 £ g for 10 min. The top aqueous layer was placed in a fresh 0.5-mL microcentrifuge tube and 100 mL chloroform: isoamyl alcohol (24:1) was added. Then, 40 mL of 4 M NH4OAc and 300 mL of 95% ethanol was added and the mixture was centrifuged at 16,000 £ g for 20 min. Double-stranded cDNA was precipitated by ethanol and dissolved in 50 mL sterile H2O. Briefly, tester and driver cDNA populations were digested with RsaI and equally divided into tester populations, respectively. Each cDNA was ligated with two adaptors (adaptor 1 and adaptor 2R). After the mixture of cDNA and adaptors was incubated at 16 C overnight, tester cDNA was ligated with adaptor 1 and adaptor 2R separately, which then underwent hybridization. Briefly, Rsa I-digested driver cDNA was mixed with adaptor 1-ligated tester cDNA or adaptor 2R-ligated tester cDNA and the mixture was denatured at 98 C for 90 s. The tester cDNA was hybridized with excess driver cDNA at 68 C for 8 h. After the first hybridization, the two products (adaptor 1 and adaptor 2R) were mixed and immediately hybridized again with an excess of fresh denatured driver cDNA overnight at 68 C. After secondary hybridization, tester cDNA with different adaptors on each end was generated. Then, differentially expressed sequences were selectively amplified by two rounds of PCR, with primers specific to the two adaptors, in the first round of PCR only using PCR primer 1 5ʹ-CTAATACGACTCACTATAGGGC-3ʹ and, in the second round of PCR, using Nested PCR primer 1 5ʹ-TCGAGCGGCCGCCCGGGCAGGT-3ʹ and Nested PCR primer 2R 5ʹ-AGCGTGGTCGCGGCCGAGGT3ʹ, resulting in the suppression of sequences common to the two cDNA populations and exponential amplification of the differentially expressed sequences. To evaluate the subtraction efficiency, the relative amount of the constitutively expressed b-actin was compared in subtracted and unsubtracted cDNA by PCR. The PCR reactions involved the b-actin–specific primers, forward 5ʹ-CCCAGAGCAAGAGAGGTA-3ʹ, and reverse, 5ʹ- GCGTATCCTTCGTAGATGGG-3ʹ. The PCR products were examined on a 2.0% agarose/EtBr gel. The subtracted PCR products were cloned separately into the yT&A Cloning Vector (Yeastern Biotech, Taipei) and transformed into Escherichia coli JM109 (Yeastern Biotech), then plated onto agar plates containing 20 mg mL¡1 ampicillin, X-gal (5-bromo-4-chloro-3-indolyl-b-dgalactopyranoside) and isopropyl-b-d-thiogalactopyranoside (IPTG) to create the forward- and reverse-subtracted cDNA libraries. The DNA of colonies were amplified by PCR. The PCR reactions involved the nested PCR primer, forward, 5ʹ-TCGAGCGGCCGCCCGGGCAGGT-3ʹ,
Wu et al. and reverse, 5ʹ-AGCGTGGTCGCGGCCGAGGT-3ʹ. The PCR products were examined on a 2.0% agarose/ EtBr gel. Transformation efficiency was calculated and white colonies were selected for further DNA sequence analysis. Screening genes with differential expression For screening gene fragments with differential expression, probes were generated by random priming with use of a DIG High Prime DNA Labeling and Detection Starter Kit (Roche, Mannheim, Germany). Briefly, after four kinds of probes were generated: forward, reverse and unsubtracted water control and vinclozolin treatment. The DNA-probe-labeling efficiency was determined by the use of DIG-labeled control DNA. If those four probes had the same efficiency, then we would use them on DNA dot blotting. Briefly, 108 mL sterilized water and 112.5 mL 0.6 M NaOH was added to 4.5 mL PCR product; then, 50 mL of the mixture was placed on a Hybond-N Nylon membrane (GE Healthcare, Piscataway, NJ, USA), with four repeats. DNA was fixed onto the Hybond-N Nylon membrane by use of the UV crosslink CL-E508.M (UVitec, Cambridge, UK) under 120 mJ for 5 min. The four kinds of probes were denatured at 100 C for 5 min and rapidly cooled on ice. The denatured four kinds of probes were then added to the DIG Easy Hyb buffer at 70 C, and 50 mL of the probe mixture was added to the hybridization buffer. The blots were incubated with probes for 16 h at 42 C with 2 rpm. Membranes were washed and incubated with DIGAP antibody with blocking solution for 30 min; the wash cycle was repeated once and expression was detected by the use of detection buffer and CSPD-ready to use, and the results photographed by using G:Box (Syngene, Cambridge, UK) for 10–20 min. DNA sequence analysis DNA sequencing involved the use of the ABI 3730xl DNA Analyzer (Perkin Elmer Applied Biosystems, USA) and was performed by Genomics Biotechnology Co. (Taipei). The sequences underwent a BLASTX search against the NCBI GenBank database (http://www.ncbi.nlm.nih.gov/ BLAST) with default parameters and non-redundant databases.[21]
Results and discussion Exposure of H. azteca Survival of H. azteca during the exposure test was 97.7% with control treatment and 93.7% with vinclozolin treatment; US EPA shows that the survival of longterm exposure should be greater than 88%.[10] In this study, the result shows that the exposure was an
Suppression subtractive hybridization for screening differentially expressed genes in Hyalella azteca exposed to vinclozolin 859
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Fig. 1. Subtraction efficiency of the subtracted cDNA in Hyalella azteca exposed to vinclozolin. PCR with b-actin–specific primers performed with subtracted (lanes 1 and 2) and unsubtracted (lanes 3 and 4) cDNA from water control and vinclozolin treatment in H. azteca.
appropriate concentration selection within the range of chronic contamination. Vinclozolin concentration measured in solution before exposure was 2.37 § 0.91 mg L¡1. After 24 h, the concentration was 1.29 § 0.59 mg L¡1 in solution, which explained the need to replace solutions daily.
Suppression subtractive hybridization The concentration and quality of the initial RNA is crucial for the qualified SSH library. In this study, the total RNA of H. azteca was 3.08 mg mL¡1 in control and 3.09 mg mL¡1 in the vinclozolin treatment group, whereas quality
was OD260/280 1.87 and 1.94 and OD230/280 was 1.58 and 1.79 with water control and vinclozolin treatment, respectively. Integrity was confirmed by agarose electrophoresis for producing double-stranded cDNA. The efficiency of subtraction was evaluated by PCR performed on the tester (unsubtracted) and subtracted cDNA with primers against the b-actin gene. b-actin was detected in the unsubtracted, but not in the subtracted, cDNA library, which suggests great reduction of highly abundant cDNA (Fig. 1). From the forward-subtracted cDNA library, suggesting upregulated gene expression with vinclozolin, we randomly selected 297 clones for sequencing; the fragment length was from 150 to 1000 bp. From the reverse-subtracted cDNA library, suggesting downregulated gene expression with vinclozolin, we randomly selected 197 clones for sequencing; the fragment length was from 150 to 900 bp. We identified 27 known genes in the forwardsubtracted cDNA library and 14 in the reverse-subtracted cDNA library. Differentially expressed genes were identified as being related to respiration (Clone ID. R10 and R62), stress and defense (Clone ID. F2, F212, and F61) and energy production and conversion (Clone ID. F29, F31 and F91; Tables 1 and 2). These genes included hemocyanin (Clone ID. F2 and F212), NADH dehydrogenase (Clone ID. R10 and R62), cytochrome (Clone ID. F29), cytochrome oxidase (Clone ID. F31 and F91) and heatshock protein (Clone ID. F61).
Table 1. Putative genes upregulated in Hyalella azteca with vinclozolin treatment. Clone ID
GenBank Accession no.
F2 F212
CAI78901.1 CAI78901.0
F29 F31 F91
AAT69317.1 ABF00487.1 ACD99584.1
F61
ACF98297.1
F291
ABN58714.1
F16 F22 F23 F11 F39 F62 F87 F123 F151 F240 F272
XP_001028745.1 EFX60192.1 XP_002723895.1 EFX77118.1 XP_002723895.1 XP_002723895.1 YP_064252.1 XP_001028745.1 XP_002723895.1 XP_001028745.1 CBA31922.1
Description of putative protein
Species
Defense-related proteins hemocyanin subunit 1 Gammarus roeseli hemocyanin subunit 1 Gammarus roeseli Energy-production and conversion-related proteins cytochrome b Parhyale hawaiensis cytochrome c oxidase subunit I Hyalella azteca cytochrome oxidase subunit 1 Hyalella sp. Stress-related proteins heatshock protein 70 Eriocheir sinensis Other proteins pol-like protein Biomphalaria glabrata Hypothetical proteins hypothetical protein TTHERM_02141640 Tetrahymena thermophila hypothetical protein DAPPUDRAFT_125127 Daphnia pulex hypothetical protein Oryctolagus cuniculus hypothetical protein DAPPUDRAFT_305875 Daphnia pulex hypothetical protein Oryctolagus cuniculus hypothetical protein Oryctolagus cuniculus hypothetical protein DP0516 Desulfotalea psychrophila LSv54 hypothetical protein TTHERM_02141640 Tetrahymena thermophila hypothetical protein Oryctolagus cuniculus hypothetical protein TTHERM_02141640 Tetrahymena thermophila hypothetical protein Csp_D29540 Hydra magnipapillata
E value
Length (bp)
2.00E-31 5.00E-36
382 568
3.00E-61 5.00E-36 2.00E-36
506 390 391
2.00E-67
427
1.00E-36 2.00E-10 6.00E-12 1.00E-13 8.00E-11 1.00E-13 1.00E-13 5.40E-03 8.00E-09 8.00E-11 4.00E-75 3.00E-73
149 475 675 422 629 644 302 148 541 144 683
860
Wu et al.
Table 2. Putative genes downregulated in H. azteca with vinclozolin treatment.
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Clone ID
Accession no.
R10 R62
ACC63589.1 CAX15419.1
R14
ZP_08451640.1
R7 R55 R58 R65 R69 R94 R117 R124
CAM36311.1 XP_002109707.1 EFX73237.1 CAM36311.1 EFX73237.1 CAM36311.1 XP_003329069.1 XP_001637750.1
Description of putative protein Respiration-related proteins NADH dehydrogenase subunit 2 NADH dehydrogenase subunit 2 Other proteins putative SNF2/helicase domain-containing protein Hypothetical proteins hypothetical protein hypothetical protein hypothetical protein DAPPUDRAFT_93183 hypothetical protein hypothetical protein DAPPUDRAFT_93183 hypothetical protein hypothetical protein PGTG_10809 predicted protein
Species
E value
Length (bp)
Gammarus duebeni celticus Parhyale hawaiensis
4.00E-15 5.00E-22
433 434
Streptomyces sp. Thermobia domestica Trichoplax adhaerens Daphnia pulex Thermobia domestica Daphnia pulex Thermobia domestica Puccinia graminis f. sp. Nematostella vectensis
439 4.00E-04 1.00E-15 4.00E-04 1.00E-15 4.00E-04 1.40E-03 2.00E-06
285 526 326 345 325 345 670 667
Fig. 2. Differential screening of the forward- and reverse-subtracted cDNA library by DNA dot blotting. F, R, WC and VC indicate the four kinds of probes. The guidelines for interpreting different combinations of hybridization for each of the four probes followed the manufacturer’s instructions in the DIG High Prime DNA Labeling and Detection Starter Kit (Roche, Mannheim, Germany). Clone No. is from F1 to F96, with marker A1 being clone F1, B1 clone F2, C1 clone F3, A2 clone F9 etc. According to the manufacturer’s instructions, those highlight were the differential expression genes.
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Table 3. Genes with significant differential expression in H. azteca with vinclozolin treatment. Clone I.D.
Accession no.
F9 F10 F15 F40 F64 F93 F101 F113 F114 F116 F143 F152 F169 F170 F171 F174 F175
Unknown ECQ49745.1 Unknown Unknown Unknown EBX99237.1 EBX99237.1 Unknown ECK87989.1 Unknown Unknown Unknown Unknown EBL19604.1 G63995.1 EBO57669.1 EBO57669.1
F176
ECS69823.1
hypothetical protein GOS_3682226
F178 F213 F219 F220 F222
Unknown ECX98331.1 Unknown Unknown ECS69823.1
hypothetical protein GOS_3682226
Cytochrome c oxidase subunit I
7.00E-33
448 276 322 297 354
F227 F235
Unknown EDF50390.1
pothetical protein GOS_943747
5.00E-06
251 427
F245 R9 R10
Unknown Unknown EDF50390.1
proteasome (prosome, macropain) inhibitor subunit 1 mitochondrial NADHubiquinone oxidoreductase
6.00E-05
278 394 427
R12 R21 R26 R29
Unknown Unknown Unknown ECG27471.1
7.00E-13
394 224 229 355
R32 R35 R39 R66 R95 R103 R104 R109 R110 R111 R118
Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown EBN21900.1
1.00E-12
224 224 203 203 277 237 278 254 229 237 744
Description
Function
E value
Length (bp)
hypothetical protein GOS_5418419
4.00E-77
hypothetical protein GOS_6321307 hypothetical protein GOS_6321307
5.50E-03 5.20E-03
hypothetical protein GOS_5601959
4.00E-36
hypothetical protein GOS_8608655 hypothetical protein GOS_9399287 hypothetical protein GOS_8056947 hypothetical protein GOS_8056947
6.00E-83 5.00E-04 8.00E-59 8.00E-59
271 707 515 276 233 147 140 473 467 236 251 354 212 457 389 299 299
7.00E-33
356
E3 ubiquitin-protein ligase Cytochrome c oxidase subunit I
hypothetical protein GOS_2439418
hypothetical protein GOS_943747 [marine metagenome]
3.00E-28
hypothetical protein GOS_6423281 [marine metagenome]
hypothetical protein GOS_8282476 [marine metagenome]
862
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Screening genes with differential expression To confirm the results of SSH, all clones were selected for DNA dot blotting. We found 55 genes with significant differential expression (Fig. 2). These genes included larval serum protein 1 alpha, E3 ubiquitin-protein ligase, mitochondrial cytochrome c oxidase, mitochondrial protein, proteasome inhibitor, hemocyanin, zinc finger-containing protein, mitochondrial NADH-ubiquinone oxidoreductase and epididymal sperm-binding protein (Table 3). Few studies have investigated the effect of environmental hormones on freshwater invertebrates, especially at the gene level. Some have investigated the effect of polychlorinated biphenyls (PCBs) on Gammarus pulex and atrazine on H. azteca by proteomics.[22,11] The exposure of G. pulex to PCBs may affect the metabolism of carbohydrates, energy, amino acids and nucleotides.[22] Besides this, energy metabolism was affected in H. azteca exposed to atrazine. We found differential regulation of genes in H. azteca exposed to the fungicide vinclozolin. Vinclozolin appears to upregulate stress-related genes and hemocyanin, which is related to immunity, and downregulates NADH dehydrogenase, related to respiration. Heatshock protein was upregulated in this study; this protein is a stress-related protein. Amphipods exposed to xenobiotics often show upregulated expression of heatshock protein. Examples are G. pulex collected in rivers contaminated with polychlorinated biphenyl, polycyclic aromatic hydrocarbon and other pollutants, H. azteca exposure in atrazine and G. pulex exposure in PCBs.[11,22,23] The heatshock protein may be induced by exposure to atrazine, a known endocrine disruptor. Moreover, hormetic responses may be due to an induction of heatshock proteins or other changes at the molecular level that may stimulate growth.[24] The identification of this protein in the present study suggests that H. azteca exposed to vinclozolin upregulates heatshock protein. Hemocyanin is copper-based respiratory protein involved in the transport of O2 in the body fluids of many arthropod species, including crustaceans, especially in the liver.[25,26] The changed concentration of hemocyanin represents a general physiological response to environmental variations of oxygen concentration.[27] It can also be related to the molting cycle. Palinurus elephas hemocyanin has periodic variation in its expression over years, and Cancer magiste hemocyanin has periodic variation in its expression over months.[28,29] This protein has multiple functions in the immune system.[30] Hemocyanin expression was upregulated in H. azteca exposed to atrazine, G. pulex exposed to PCBs and Neocaridina denticulate exposed to nonylphenol.[11,22,31] In this study, H. azteca exposed to vinclozolin showed upregulated hemocyanin expression: with affected metabolism, the demand for oxygen differed. We found cytochrome c oxidase upregulated in H. azteca exposed to vinclozolin. This protein is part of a
Wu et al. large transmembrane protein complex found in mitochondria and located in the mitochondrial membrane. Its function is to provide an efficient mechanism for dioxygen reduction coupling the free energy of water formation to the generation of a transmembrane electrochemical gradient to drive ATP synthesis.[32] Cytochrome oxidase activity was suggested to be associated with epigenetic activity, causing genetic variation without gene mutation.[33,34] Vinclozolin-upregulating transcription programs during gonadal sex determination and early testis development was examined; it was found to have an epigenetic effect.[8] We found upregulation of cytochrome c oxidase in H. azteca exposed to vinclozolin,; therefore, the oxidase might be a biomarker of vinclozolin or other chemicals having an epigenetic effect. We found that vinclozolin may downregulate NADH dehydrogenase in H. azteca. Vinclozolin may affect respiration in H. azteca, which may explain the increased mortality with vinclozolin. NADH dehydrogenase is the first enzyme (Complex I) of the mitochondrial electron transport chain and is one of the entry enzymes of oxidative phosphorylation in mitochondria.[35] Daphnia is closely related phylogenetically to H. azteca. More studies have investigated daphnia rather than H. azteca at the gene level. However, similarly, few studies have investigated hormone-controlling genes. Therefore, unknown functional genes we found may affect the endocrine disruptor function in H. azteca with vinclozolin but needs further investigation.
Conclusions Overall, we used subtractive hybridization libraries to identify genes with differential expression in H. azteca exposed to vinclozolin. Vinclozolin treatment upregulated hemocyanin, heatshock protein, cytochrome b and cytochrome c oxidase in H. azteca and downregulated NADH dehydrogenase. This study offers data for further study of xenobiotic effects in amphipods.
Acknowledgments The authors are grateful to the Environmental Analysis Laboratory, EPA (Taoyuan, Taiwan), for providing H. azteca.
References [1] Clark, T.; Watkins, D.A.M. Photolysis of vinclozolin. Chemosphere. 1984, 13(12), 1391–1396. [2] Boccelli, R.; Molinari, G.P.; Delre, A. Pesticide-residues in soil gas-chromatographic determination of vinclozolin. J. Agr. Food. Chem. 1982, 30(6), 1233–1236.
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[18] Pan, D.; He, N.; Yang, Z.; Liu, H.; Xu, X. Differential gene expression profile in hepatopancreas of WSSV-resistant shrimp (Penaeus japonicus) by suppression subtractive hybridization. Dev. Comp. Immunol. 2005, 29(2), 103–112. [19] Soetaert, A.; Moens, L.N.; Van der Ven, K.; Van Leemput, K.; Naudts, B.; Blust, R.; De Coen, W.M. Molecular impact of propiconazole on Daphnia magna using a reproduction-related cDNA array. Comp. Biochem. Phys. C 2006, 142(1–2), 66–76. [20] Zhao, Z.Y.; Yin, Z.X.; Weng, S.P.; Guan, H.J.; Li, S.D.; Xing, K.; Chan, S.M.; He, J.G. Profiling of differentially expressed genes in hepatopancreas of white spot syndrome virus-resistant shrimp (Litopenaeus vannamei) by suppression subtractive hybridisation. Fish Shellfish Immu. 2007, 22(5), 520–534. [21] Altschul, S.F.; Madden, T.L.; Schaffer, A.A.; Zhang, J.; Zhang, Z.; Miller, W.; Lipman, D.J. Gapped BLAST and PSI-BLAST: A new generation of protein database search programs. Nucleic. Acids Res. 1997, 25(17), 3389–3402. [22] Leroy, D.; Haubruge, E.; De Pauw, E.; Thome, J.P.; Francis, F. Development of ecotoxicoproteomics on the freshwater amphipod Gammarus pulex: Identification of PCB biomarkers in glycolysis and glutamate pathways. Ecotox. Environ. Safe. 2010, 73(3), 343–352. [23] Triebskorn, R.; Adam, S.; Casper, H.; Honnen, W.; Pawert, M.; Schramm, M.; Schwaiger, J.; Kohler, H.R. Biomarkers as diagnostic tools for evaluating effects of unknown past water quality conditions on stream organisms. Ecotoxicology. 2002, 11(6), 451–465. [24] Smith-Sonneborn, J. The role of the “stress protein response” in hormesis; 1992; 41–52. [25] Rosenberg, D.M.; Resh, V.H. Freshwater biomonitoring and benthic macroinvertebrates; 1993; 488. [26] Van Holde, K.E.; Miller, K.I. Hemocyanins. In Advances in Protein Chemistr; 1995; Vol. 47, 1–81. [27] Giomi, F.; Beltramini, M. The molecular heterogeneity of hemocyanin: Its role in the adaptive plasticity of Crustacea. Gene. 2007, 398(1–2), 192–201. [28] Bellelli, A.; Giardina, B.; Corda, M.; Pellegrini, M.G.; Cau, A.; Condo, S.G.; Brunori, M. Sexual and seasonal variability of lobster hemocyanin. Comp. Biochem. Physiol. 1988, 91(3), 445–449. [29] Terwilliger, N.B.; Ryan, M.C.; Towle, D. Evolution of novel functions: Cryptocyanin helps build new exoskeleton in Cancer magister. J. Exp. Biol. 2005, 208(Pt 13), 2467–2474. [30] Decker, H.; Rimke, T. Tarantula hemocyanin shows phenoloxidase activity. J. Biol. Chem. 1998, 273(40), 25889–25892. [31] Liu, C.L.; Sung, H.H. Genes are differentially expressed at transcriptional level of neocaridina denticulata following short-term exposure to nonylphenol. B. Environ. Contam. Tox. 2011, 87(3), 220–225. [32] Richter, O.M.H.; Ludwig, B. Cytochrome c oxidase - structure, function, and physiology of a redox-driven molecular machine. Rev. Physiol. Biochem. Pharmacol. 2003, 147, 47–74. [33] Sookoian, S.; Pirola, C.J. DNA methylation and hepatic insulin resistance and steatosis. Curr. Opin. Clin. Nutr. Metab. Care. 2012, 15(4), 350–356. [34] Sookoian, S.; Rosselli, M.S.; Gemma, C.; Burgueno, A.L.; Gianotti, T.F.; Castano, G.O.; Pirola, C.J. Epigenetic regulation of insulin resistance in nonalcoholic fatty liver disease: Impact of liver methylation of the peroxisome proliferator-activated receptor gamma coactivator 1 alpha promoter. Hepatology. 2010, 52(6), 1992–2000. [35] Adachi, K. Study on the reduced pyridine nucleotide dehydrogenase of bovine erythrocytes: I. Crystallization and properties of the reduced pyridine nucleotide dehydrogenase of bovine erythrocytes. Biochim. Biophys. Acta. 1972, 289(2), 262–268.
Journal of Environmental Science and Health, Part B: Pesticides, Food Contaminants, and Agricultural Wastes Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/lesb20
Screening differentially expressed genes in an amphipod (Hyalella azteca) exposed to fungicide vinclozolin by suppression subtractive hybridization a
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Yun H. Wu , Tsung M. Wu , Chwan Y. Hong , Yei S. Wang & Jui H. Yen a
Department of Agricultural Chemistry, National Taiwan University, Taipei, Taiwan Published online: 04 Sep 2014.
To cite this article: Yun H. Wu, Tsung M. Wu, Chwan Y. Hong, Yei S. Wang & Jui H. Yen (2014) Screening differentially expressed genes in an amphipod (Hyalella azteca) exposed to fungicide vinclozolin by suppression subtractive hybridization, Journal of Environmental Science and Health, Part B: Pesticides, Food Contaminants, and Agricultural Wastes, 49:11, 856-863, DOI: 10.1080/03601234.2014.938556 To link to this article: http://dx.doi.org/10.1080/03601234.2014.938556
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Journal of Environmental Science and Health, Part B (2014) 49, 856–863 Copyright © Taylor & Francis Group, LLC ISSN: 0360-1234 (Print); 1532-4109 (Online) DOI: 10.1080/03601234.2014.938556
Screening differentially expressed genes in an amphipod (Hyalella azteca) exposed to fungicide vinclozolin by suppression subtractive hybridization YUN H. WU, TSUNG M. WU, CHWAN Y. HONG, YEI S. WANG and JUI H. YEN
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Department of Agricultural Chemistry, National Taiwan University, Taipei, Taiwan
Vinclozolin, a dicarboximide fungicide, is an endocrine disrupting chemical that competes with an androgenic endocrine disruptor compound. Most research has focused on the epigenetic effect of vinclozolin in humans. In terms of ecotoxicology, understanding the effect of vinclozolin on non-target organisms is important. The expression profile of a comprehensive set of genes in the amphipod Hyalella azteca exposed to vinclozolin was examined. The expressed sequence tags in low-dose vinclozolin-treated and -untreated amphipods were isolated and identified by suppression subtractive hybridization. DNA dot blotting was used to confirm the results and establish a subtracted cDNA library for comparing all differentially expressed sequences with and without vinclozolin treatment. In total, 494 differentially expressed genes, including hemocyanin, heatshock protein, cytochrome, cytochrome oxidase and NADH dehydrogenase were detected. Hemocyanin was the most abundant gene. DNA dot blotting revealed 55 genes with significant differential expression. These genes included larval serum protein 1 alpha, E3 ubiquitin-protein ligase, mitochondrial cytochrome c oxidase, mitochondrial protein, proteasome inhibitor, hemocyanin, zinc-finger–containing protein, mitochondrial NADH-ubiquinone oxidoreductase and epididymal sperm-binding protein. Vinclozolin appears to upregulate stress-related genes and hemocyanin, related to immunity. Moreover, vinclozolin downregulated NADH dehydrogenase, related to respiration. Thus, even a non-lethal concentration of vinclozolin still has an effect at the genetic level in H. azteca and presents a potential risk, especially as it would affect non-target organism hormone metabolism. Keywords: Endocrine disrupter chemical, vinclozolin, Hyalella azteca, suppression subtractive hybridization, differential expression.
Introduction The dicarboximide-type fungicide vinclozolin [3-(3,5dichlorophenyl)-5-methyl-5-vinyl-1,3-oxazolidine,4-dione] was broadly introduced to control various vegetable and orchard diseases in the world. It is used for eradication of disease and protection against many fungal diseases, especially those caused by Botrytis cinerea, Sclerotinia sclerotiorum and Monilinia sp. infection in grapes, fruits and vegetables.[1] Because of its wide use, it is found as a residue in the environment and also acts as an endocrine disruptor chemical.[1,2] Moreover, vinclozolin is an environmental antiandrogen and influences gonad Address correspondence to Yei-Shung Wang and Jui-Hung Yen, Department of Agricultural Chemistry, College of Bioresources and Agriculture, National Taiwan University, R317, No. 1, Sec. 4, Roosevelt Road, Taipei, 10617 Taiwan (R.O.C); E-mail: [email protected] and [email protected]. Received April 30, 2014. Color versions of one or more of the figures in this article can be found online at www.tandfonline.com/lesb.
development and fertility.[3–7] It induces transcriptional alteration during gonadal sex determination and early testis development.[8] Moreover, it was found to act transiently during embryonic sex determination to promote a spermatogenic cell defect in the F1 generation as well as subfertility in males.[8] Vinclozolin was detected in underground water, lakes, rivers and seawater in Greece.[9] However, few studies have investigated the ecotoxicity or environmental toxicity of vinclozolin, especially in aquatic ecosystems. As such, the effect on non-target organisms of vinclozolin needs to be evaluated. The amphipod Hyalella azteca is a representative invertebrate in aquatic ecosystems. It was widely used in ecotoxicological evaluations of sediment because of its availability, easy culture in the laboratory and sensitivity to contaminants.[10] Furthermore, knowledge of the reproductive physiology of H. azteca is adequate, and both acute and chronic effects have been studied for many environmental contaminants.[11] In recent years, most studies of reproduction in aquatic species have focused on endocrine disruptor chemicals with estrogenic action.[12] Comparatively little is known
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about chemicals that might interact with androgens because the functions of androgen receptors are largely unknown.[13] Suppression subtractive hybridization (SSH) is a powerful procedure that allows comparison of two mRNA populations by PCR-based cDNA subtraction.[14] SSH has been successfully used to identify differentially expressed genes in Neocaraidina denticiculata,[15] Penaeus japonicas,[16–18] Daphnia magna[19] and Litopenaeus vannamei.[20] In this study, we used SSH to identify the genes with differential expression in the amphipod H. azteca after exposure to vinclozolin for a comprehensive understanding of the mechanism of vinclozolin exposure in the amphipod.
Materials and methods Chemicals and reagents Vinclozolin (99.4% purity, CAS no. 50471-44-8; Chem Service, USA) was dissolved in acetone to establish a 10,000 mg L¡1 stock solution.
Amphipod culture H. azteca has been used extensively for standardized water and sediment toxicity testing.[10] Test organisms were procured from the Environmental Analysis Laboratory EPA (Taoyuan, Taiwan). Long-term cultures were maintained under static conditions in 1-L glass dishes with photoperiod 16:8 h (light/dark), 24 § 1 C and pH 6.8 § 0.2. Half the volume of overlying water in the aquaria was changed weekly by siphoning and adding fresh water. The culture diet consisted of a mixture of 0.5 g shrimp food and amphipods were fed once a week.
After exposure, the vinclozolin concentration was analyzed by high-performance liquid chromatography (HPLC) with UV detection. The conditions for HPLC were (1) C18 column (Merck, LiChrospher 100RP-18-e); (2) UV detector with absorption λmax 202 nm; (3) mobile phase: acetonitrile: H2O D 60:40; and (4) flow rate: 1 mL min¡1. Under these conditions, the retention time of vinclozolin was 10.2 min, and the detection limit was 0.20 mg L¡1. Preparation of poly (A)C RNA Total RNA was extracted by the TRIzol RNA reagent method (Invitrogen, CA, USA). After exposure, amphipods were collected for total RNA extraction, frozen by use of nitrogen (l) and, then, 2 mL TRIzol RNA reagent was added and mixed completely. After the addition of phenol-chloroform, NaCl and sodium citrate, the homogenate was centrifuged at 12,000 £ g for 10 min at 4 C. The supernatant was extracted with the use of isopropanol, and the total RNA was precipitated by ethanol and dissolved in diethylpyrocarbonate (DEPC) -treated water. Poly (AC) RNA was extracted by use of the Oligotex mRNA Kit (Quiagen, Valencia, CA, USA). Briefly, 0.25 mg total RNA was added to 250 mL Buffer OBB and 15 mL Oligotex Suspension. The mixture was incubated at 70 C for 3 min and cooled at room temperature for 10 min; then, the homogenate was centrifuged at 16,000 £ g for 2 min and the supernatant was removed. The pellet was resuspended in 400 mL Buffer OW2 by vortexing and homogenate was centrifuged at 16,000 £ g for 1 min. In total, 400 mL Buffer OW2 was added to the column, and the homogenate was centrifuged at 16,000 £ g for 1 min and the flow-through was discarded. Then, 20 mL hot (70 C) Buffer OEB was added to the column and centrifuged at 16,000 £ g for 1 min.
Exposure tests H. azteca was exposed to one vinclozolin treatment and one water control under laboratory conditions. Vinclozolin treatment was with vinclozolin, 3 mg L¡1, dissolved in water with stock solutions of vinclozolin in acetone to obtain 3 mg L¡1 vinclozolin. The concentration is the maximum solubility in water. The exposure test was performed in glass dishes (20 cm length, 20 cm wide and 6 cm high). A 100-g porcelain tile was placed on the bottom of dishes, and 200 adult H. azteca, approximately 1 cm in length, from laboratory cultures were placed in each dish together with 1 L solution. The exposure lasted 14 days, and 10% volume of overlying water in the aquaria was changed every 24 h by siphoning and adding fresh solution. The exposure test was maintained under static conditions in a 1-L glass dishes with a photoperiod of 16:8 h (light/dark) at 24 § 1 C and pH 6.8 § 0.2.
Suppression subtractive hybridization (SSH) SSH was performed to generate the subtracted cDNA library with vinclozolin treatment and the water control involved the use of the PCR-Select cDNA Subtraction Kit (Clontech, Palo Alto, CA, USA). In the forward cDNA library, vinclozolin treatment was the tester and water control the driver, then in the reverse cDNA library, water control was the tester and vinclozolin treatment the driver. Briefly, 2 mg mRNA was mixed with 1 mL cDNA Synthesis Primer. The mixture was incubated at 70 C for 2 min, cooled on ice for 2 min and, then, briefly centrifuged. Thereafter, 2 mL 5X First-Strand Buffer, 1 mL dNTP, 1 mL sterile H2O, 1 mL DTT and 1 mL SMARTScribe Reverse Transcriptase were added to the mixture and incubated at 42 C for 1.5 h. To this, 48.4 mL sterile H2O, 16 mL 5X Second-Strand Buffer, 1.6 mL dNTP, and 4 mL 20X Second-Strand Enzyme Cocktail were added to
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858 the first-strand synthesis reaction tubes and incubated at 16 C for 2 h. In total, 2 mL T4 DNA polymerase was added for incubation at 16 C for 30 min. Then, 4 mL of 20X EDTA/Glycogen Mix and 100 mL phenol: chloroform:isoamyl alcohol (25:24:1) was added, mixed thoroughly and centrifuged at 16,000 £ g for 10 min. The top aqueous layer was placed in a fresh 0.5-mL microcentrifuge tube and 100 mL chloroform: isoamyl alcohol (24:1) was added. Then, 40 mL of 4 M NH4OAc and 300 mL of 95% ethanol was added and the mixture was centrifuged at 16,000 £ g for 20 min. Double-stranded cDNA was precipitated by ethanol and dissolved in 50 mL sterile H2O. Briefly, tester and driver cDNA populations were digested with RsaI and equally divided into tester populations, respectively. Each cDNA was ligated with two adaptors (adaptor 1 and adaptor 2R). After the mixture of cDNA and adaptors was incubated at 16 C overnight, tester cDNA was ligated with adaptor 1 and adaptor 2R separately, which then underwent hybridization. Briefly, Rsa I-digested driver cDNA was mixed with adaptor 1-ligated tester cDNA or adaptor 2R-ligated tester cDNA and the mixture was denatured at 98 C for 90 s. The tester cDNA was hybridized with excess driver cDNA at 68 C for 8 h. After the first hybridization, the two products (adaptor 1 and adaptor 2R) were mixed and immediately hybridized again with an excess of fresh denatured driver cDNA overnight at 68 C. After secondary hybridization, tester cDNA with different adaptors on each end was generated. Then, differentially expressed sequences were selectively amplified by two rounds of PCR, with primers specific to the two adaptors, in the first round of PCR only using PCR primer 1 5ʹ-CTAATACGACTCACTATAGGGC-3ʹ and, in the second round of PCR, using Nested PCR primer 1 5ʹ-TCGAGCGGCCGCCCGGGCAGGT-3ʹ and Nested PCR primer 2R 5ʹ-AGCGTGGTCGCGGCCGAGGT3ʹ, resulting in the suppression of sequences common to the two cDNA populations and exponential amplification of the differentially expressed sequences. To evaluate the subtraction efficiency, the relative amount of the constitutively expressed b-actin was compared in subtracted and unsubtracted cDNA by PCR. The PCR reactions involved the b-actin–specific primers, forward 5ʹ-CCCAGAGCAAGAGAGGTA-3ʹ, and reverse, 5ʹ- GCGTATCCTTCGTAGATGGG-3ʹ. The PCR products were examined on a 2.0% agarose/EtBr gel. The subtracted PCR products were cloned separately into the yT&A Cloning Vector (Yeastern Biotech, Taipei) and transformed into Escherichia coli JM109 (Yeastern Biotech), then plated onto agar plates containing 20 mg mL¡1 ampicillin, X-gal (5-bromo-4-chloro-3-indolyl-b-dgalactopyranoside) and isopropyl-b-d-thiogalactopyranoside (IPTG) to create the forward- and reverse-subtracted cDNA libraries. The DNA of colonies were amplified by PCR. The PCR reactions involved the nested PCR primer, forward, 5ʹ-TCGAGCGGCCGCCCGGGCAGGT-3ʹ,
Wu et al. and reverse, 5ʹ-AGCGTGGTCGCGGCCGAGGT-3ʹ. The PCR products were examined on a 2.0% agarose/ EtBr gel. Transformation efficiency was calculated and white colonies were selected for further DNA sequence analysis. Screening genes with differential expression For screening gene fragments with differential expression, probes were generated by random priming with use of a DIG High Prime DNA Labeling and Detection Starter Kit (Roche, Mannheim, Germany). Briefly, after four kinds of probes were generated: forward, reverse and unsubtracted water control and vinclozolin treatment. The DNA-probe-labeling efficiency was determined by the use of DIG-labeled control DNA. If those four probes had the same efficiency, then we would use them on DNA dot blotting. Briefly, 108 mL sterilized water and 112.5 mL 0.6 M NaOH was added to 4.5 mL PCR product; then, 50 mL of the mixture was placed on a Hybond-N Nylon membrane (GE Healthcare, Piscataway, NJ, USA), with four repeats. DNA was fixed onto the Hybond-N Nylon membrane by use of the UV crosslink CL-E508.M (UVitec, Cambridge, UK) under 120 mJ for 5 min. The four kinds of probes were denatured at 100 C for 5 min and rapidly cooled on ice. The denatured four kinds of probes were then added to the DIG Easy Hyb buffer at 70 C, and 50 mL of the probe mixture was added to the hybridization buffer. The blots were incubated with probes for 16 h at 42 C with 2 rpm. Membranes were washed and incubated with DIGAP antibody with blocking solution for 30 min; the wash cycle was repeated once and expression was detected by the use of detection buffer and CSPD-ready to use, and the results photographed by using G:Box (Syngene, Cambridge, UK) for 10–20 min. DNA sequence analysis DNA sequencing involved the use of the ABI 3730xl DNA Analyzer (Perkin Elmer Applied Biosystems, USA) and was performed by Genomics Biotechnology Co. (Taipei). The sequences underwent a BLASTX search against the NCBI GenBank database (http://www.ncbi.nlm.nih.gov/ BLAST) with default parameters and non-redundant databases.[21]
Results and discussion Exposure of H. azteca Survival of H. azteca during the exposure test was 97.7% with control treatment and 93.7% with vinclozolin treatment; US EPA shows that the survival of longterm exposure should be greater than 88%.[10] In this study, the result shows that the exposure was an
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Fig. 1. Subtraction efficiency of the subtracted cDNA in Hyalella azteca exposed to vinclozolin. PCR with b-actin–specific primers performed with subtracted (lanes 1 and 2) and unsubtracted (lanes 3 and 4) cDNA from water control and vinclozolin treatment in H. azteca.
appropriate concentration selection within the range of chronic contamination. Vinclozolin concentration measured in solution before exposure was 2.37 § 0.91 mg L¡1. After 24 h, the concentration was 1.29 § 0.59 mg L¡1 in solution, which explained the need to replace solutions daily.
Suppression subtractive hybridization The concentration and quality of the initial RNA is crucial for the qualified SSH library. In this study, the total RNA of H. azteca was 3.08 mg mL¡1 in control and 3.09 mg mL¡1 in the vinclozolin treatment group, whereas quality
was OD260/280 1.87 and 1.94 and OD230/280 was 1.58 and 1.79 with water control and vinclozolin treatment, respectively. Integrity was confirmed by agarose electrophoresis for producing double-stranded cDNA. The efficiency of subtraction was evaluated by PCR performed on the tester (unsubtracted) and subtracted cDNA with primers against the b-actin gene. b-actin was detected in the unsubtracted, but not in the subtracted, cDNA library, which suggests great reduction of highly abundant cDNA (Fig. 1). From the forward-subtracted cDNA library, suggesting upregulated gene expression with vinclozolin, we randomly selected 297 clones for sequencing; the fragment length was from 150 to 1000 bp. From the reverse-subtracted cDNA library, suggesting downregulated gene expression with vinclozolin, we randomly selected 197 clones for sequencing; the fragment length was from 150 to 900 bp. We identified 27 known genes in the forwardsubtracted cDNA library and 14 in the reverse-subtracted cDNA library. Differentially expressed genes were identified as being related to respiration (Clone ID. R10 and R62), stress and defense (Clone ID. F2, F212, and F61) and energy production and conversion (Clone ID. F29, F31 and F91; Tables 1 and 2). These genes included hemocyanin (Clone ID. F2 and F212), NADH dehydrogenase (Clone ID. R10 and R62), cytochrome (Clone ID. F29), cytochrome oxidase (Clone ID. F31 and F91) and heatshock protein (Clone ID. F61).
Table 1. Putative genes upregulated in Hyalella azteca with vinclozolin treatment. Clone ID
GenBank Accession no.
F2 F212
CAI78901.1 CAI78901.0
F29 F31 F91
AAT69317.1 ABF00487.1 ACD99584.1
F61
ACF98297.1
F291
ABN58714.1
F16 F22 F23 F11 F39 F62 F87 F123 F151 F240 F272
XP_001028745.1 EFX60192.1 XP_002723895.1 EFX77118.1 XP_002723895.1 XP_002723895.1 YP_064252.1 XP_001028745.1 XP_002723895.1 XP_001028745.1 CBA31922.1
Description of putative protein
Species
Defense-related proteins hemocyanin subunit 1 Gammarus roeseli hemocyanin subunit 1 Gammarus roeseli Energy-production and conversion-related proteins cytochrome b Parhyale hawaiensis cytochrome c oxidase subunit I Hyalella azteca cytochrome oxidase subunit 1 Hyalella sp. Stress-related proteins heatshock protein 70 Eriocheir sinensis Other proteins pol-like protein Biomphalaria glabrata Hypothetical proteins hypothetical protein TTHERM_02141640 Tetrahymena thermophila hypothetical protein DAPPUDRAFT_125127 Daphnia pulex hypothetical protein Oryctolagus cuniculus hypothetical protein DAPPUDRAFT_305875 Daphnia pulex hypothetical protein Oryctolagus cuniculus hypothetical protein Oryctolagus cuniculus hypothetical protein DP0516 Desulfotalea psychrophila LSv54 hypothetical protein TTHERM_02141640 Tetrahymena thermophila hypothetical protein Oryctolagus cuniculus hypothetical protein TTHERM_02141640 Tetrahymena thermophila hypothetical protein Csp_D29540 Hydra magnipapillata
E value
Length (bp)
2.00E-31 5.00E-36
382 568
3.00E-61 5.00E-36 2.00E-36
506 390 391
2.00E-67
427
1.00E-36 2.00E-10 6.00E-12 1.00E-13 8.00E-11 1.00E-13 1.00E-13 5.40E-03 8.00E-09 8.00E-11 4.00E-75 3.00E-73
149 475 675 422 629 644 302 148 541 144 683
860
Wu et al.
Table 2. Putative genes downregulated in H. azteca with vinclozolin treatment.
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Clone ID
Accession no.
R10 R62
ACC63589.1 CAX15419.1
R14
ZP_08451640.1
R7 R55 R58 R65 R69 R94 R117 R124
CAM36311.1 XP_002109707.1 EFX73237.1 CAM36311.1 EFX73237.1 CAM36311.1 XP_003329069.1 XP_001637750.1
Description of putative protein Respiration-related proteins NADH dehydrogenase subunit 2 NADH dehydrogenase subunit 2 Other proteins putative SNF2/helicase domain-containing protein Hypothetical proteins hypothetical protein hypothetical protein hypothetical protein DAPPUDRAFT_93183 hypothetical protein hypothetical protein DAPPUDRAFT_93183 hypothetical protein hypothetical protein PGTG_10809 predicted protein
Species
E value
Length (bp)
Gammarus duebeni celticus Parhyale hawaiensis
4.00E-15 5.00E-22
433 434
Streptomyces sp. Thermobia domestica Trichoplax adhaerens Daphnia pulex Thermobia domestica Daphnia pulex Thermobia domestica Puccinia graminis f. sp. Nematostella vectensis
439 4.00E-04 1.00E-15 4.00E-04 1.00E-15 4.00E-04 1.40E-03 2.00E-06
285 526 326 345 325 345 670 667
Fig. 2. Differential screening of the forward- and reverse-subtracted cDNA library by DNA dot blotting. F, R, WC and VC indicate the four kinds of probes. The guidelines for interpreting different combinations of hybridization for each of the four probes followed the manufacturer’s instructions in the DIG High Prime DNA Labeling and Detection Starter Kit (Roche, Mannheim, Germany). Clone No. is from F1 to F96, with marker A1 being clone F1, B1 clone F2, C1 clone F3, A2 clone F9 etc. According to the manufacturer’s instructions, those highlight were the differential expression genes.
Suppression subtractive hybridization for screening differentially expressed genes in Hyalella azteca exposed to vinclozolin 861
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Table 3. Genes with significant differential expression in H. azteca with vinclozolin treatment. Clone I.D.
Accession no.
F9 F10 F15 F40 F64 F93 F101 F113 F114 F116 F143 F152 F169 F170 F171 F174 F175
Unknown ECQ49745.1 Unknown Unknown Unknown EBX99237.1 EBX99237.1 Unknown ECK87989.1 Unknown Unknown Unknown Unknown EBL19604.1 G63995.1 EBO57669.1 EBO57669.1
F176
ECS69823.1
hypothetical protein GOS_3682226
F178 F213 F219 F220 F222
Unknown ECX98331.1 Unknown Unknown ECS69823.1
hypothetical protein GOS_3682226
Cytochrome c oxidase subunit I
7.00E-33
448 276 322 297 354
F227 F235
Unknown EDF50390.1
pothetical protein GOS_943747
5.00E-06
251 427
F245 R9 R10
Unknown Unknown EDF50390.1
proteasome (prosome, macropain) inhibitor subunit 1 mitochondrial NADHubiquinone oxidoreductase
6.00E-05
278 394 427
R12 R21 R26 R29
Unknown Unknown Unknown ECG27471.1
7.00E-13
394 224 229 355
R32 R35 R39 R66 R95 R103 R104 R109 R110 R111 R118
Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown EBN21900.1
1.00E-12
224 224 203 203 277 237 278 254 229 237 744
Description
Function
E value
Length (bp)
hypothetical protein GOS_5418419
4.00E-77
hypothetical protein GOS_6321307 hypothetical protein GOS_6321307
5.50E-03 5.20E-03
hypothetical protein GOS_5601959
4.00E-36
hypothetical protein GOS_8608655 hypothetical protein GOS_9399287 hypothetical protein GOS_8056947 hypothetical protein GOS_8056947
6.00E-83 5.00E-04 8.00E-59 8.00E-59
271 707 515 276 233 147 140 473 467 236 251 354 212 457 389 299 299
7.00E-33
356
E3 ubiquitin-protein ligase Cytochrome c oxidase subunit I
hypothetical protein GOS_2439418
hypothetical protein GOS_943747 [marine metagenome]
3.00E-28
hypothetical protein GOS_6423281 [marine metagenome]
hypothetical protein GOS_8282476 [marine metagenome]
862
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Screening genes with differential expression To confirm the results of SSH, all clones were selected for DNA dot blotting. We found 55 genes with significant differential expression (Fig. 2). These genes included larval serum protein 1 alpha, E3 ubiquitin-protein ligase, mitochondrial cytochrome c oxidase, mitochondrial protein, proteasome inhibitor, hemocyanin, zinc finger-containing protein, mitochondrial NADH-ubiquinone oxidoreductase and epididymal sperm-binding protein (Table 3). Few studies have investigated the effect of environmental hormones on freshwater invertebrates, especially at the gene level. Some have investigated the effect of polychlorinated biphenyls (PCBs) on Gammarus pulex and atrazine on H. azteca by proteomics.[22,11] The exposure of G. pulex to PCBs may affect the metabolism of carbohydrates, energy, amino acids and nucleotides.[22] Besides this, energy metabolism was affected in H. azteca exposed to atrazine. We found differential regulation of genes in H. azteca exposed to the fungicide vinclozolin. Vinclozolin appears to upregulate stress-related genes and hemocyanin, which is related to immunity, and downregulates NADH dehydrogenase, related to respiration. Heatshock protein was upregulated in this study; this protein is a stress-related protein. Amphipods exposed to xenobiotics often show upregulated expression of heatshock protein. Examples are G. pulex collected in rivers contaminated with polychlorinated biphenyl, polycyclic aromatic hydrocarbon and other pollutants, H. azteca exposure in atrazine and G. pulex exposure in PCBs.[11,22,23] The heatshock protein may be induced by exposure to atrazine, a known endocrine disruptor. Moreover, hormetic responses may be due to an induction of heatshock proteins or other changes at the molecular level that may stimulate growth.[24] The identification of this protein in the present study suggests that H. azteca exposed to vinclozolin upregulates heatshock protein. Hemocyanin is copper-based respiratory protein involved in the transport of O2 in the body fluids of many arthropod species, including crustaceans, especially in the liver.[25,26] The changed concentration of hemocyanin represents a general physiological response to environmental variations of oxygen concentration.[27] It can also be related to the molting cycle. Palinurus elephas hemocyanin has periodic variation in its expression over years, and Cancer magiste hemocyanin has periodic variation in its expression over months.[28,29] This protein has multiple functions in the immune system.[30] Hemocyanin expression was upregulated in H. azteca exposed to atrazine, G. pulex exposed to PCBs and Neocaridina denticulate exposed to nonylphenol.[11,22,31] In this study, H. azteca exposed to vinclozolin showed upregulated hemocyanin expression: with affected metabolism, the demand for oxygen differed. We found cytochrome c oxidase upregulated in H. azteca exposed to vinclozolin. This protein is part of a
Wu et al. large transmembrane protein complex found in mitochondria and located in the mitochondrial membrane. Its function is to provide an efficient mechanism for dioxygen reduction coupling the free energy of water formation to the generation of a transmembrane electrochemical gradient to drive ATP synthesis.[32] Cytochrome oxidase activity was suggested to be associated with epigenetic activity, causing genetic variation without gene mutation.[33,34] Vinclozolin-upregulating transcription programs during gonadal sex determination and early testis development was examined; it was found to have an epigenetic effect.[8] We found upregulation of cytochrome c oxidase in H. azteca exposed to vinclozolin,; therefore, the oxidase might be a biomarker of vinclozolin or other chemicals having an epigenetic effect. We found that vinclozolin may downregulate NADH dehydrogenase in H. azteca. Vinclozolin may affect respiration in H. azteca, which may explain the increased mortality with vinclozolin. NADH dehydrogenase is the first enzyme (Complex I) of the mitochondrial electron transport chain and is one of the entry enzymes of oxidative phosphorylation in mitochondria.[35] Daphnia is closely related phylogenetically to H. azteca. More studies have investigated daphnia rather than H. azteca at the gene level. However, similarly, few studies have investigated hormone-controlling genes. Therefore, unknown functional genes we found may affect the endocrine disruptor function in H. azteca with vinclozolin but needs further investigation.
Conclusions Overall, we used subtractive hybridization libraries to identify genes with differential expression in H. azteca exposed to vinclozolin. Vinclozolin treatment upregulated hemocyanin, heatshock protein, cytochrome b and cytochrome c oxidase in H. azteca and downregulated NADH dehydrogenase. This study offers data for further study of xenobiotic effects in amphipods.
Acknowledgments The authors are grateful to the Environmental Analysis Laboratory, EPA (Taoyuan, Taiwan), for providing H. azteca.
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