Function of the Anal Sacs and Mid-Gut in Mitochondrial Sulphide

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Marine Biology Research Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/smar20 Function of the anal sacs and mid-gut in mitochondrial sulphide metabolism in the echiuran worm Urechis unicinctus Yu-Bin Ma a b , Zhi-Feng Zhang a , Ming-Yu Shao a , Kyoung-Ho Kang c , Li-Tao Zhang a , Xiao-Li Shi a & Ying-Ping Dong a a Key Laboratory of Marine Genetics and Breeding of Ministry of Education, Ocean University of China, Qingdao, China b Shandong Provincial Key Laboratory of Energy Genetics, Key Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, China c Department of Aquaculture, Chonnam National University, Yeosu, South Korea Version of record first published: 26 Sep 2012.

To cite this article: Yu-Bin Ma, Zhi-Feng Zhang, Ming-Yu Shao, Kyoung-Ho Kang, Li-Tao Zhang, Xiao-Li Shi & Ying-Ping Dong (2012): Function of the anal sacs and mid-gut in mitochondrial sulphide metabolism in the echiuran worm Urechis unicinctus , Marine Biology Research, 8:10, 1026-1031 To link to this article: http://dx.doi.org/10.1080/17451000.2012.707320

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SHORT REPORT

Function of the anal sacs and mid-gut in mitochondrial sulphide metabolism in the echiuran worm Urechis unicinctus

YU-BIN MA1,2, ZHI-FENG ZHANG1*, MING-YU SHAO1, KYOUNG-HO KANG3, LI-TAO ZHANG1, XIAO-LI SHI1 & YING-PING DONG1

1Key Laboratory of Marine Genetics and Breeding of Ministry of Education, Ocean University of China, Qingdao, China, 2Shandong Provincial Key Laboratory of Energy Genetics, Key Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, China, and 3Department of Aquaculture, Chonnam National University, Yeosu, South Korea

Abstract Sulphides are naturally occurring and widely distributed, poisonous substances and sulphide:quinone oxidoreductase (SQR) has been identified to be responsible for the initial oxidation of sulphide in mitochondria. In a previous study, we found that in the sulphide-adapted species Urechis unicinctus, SQR mRNA concentrations in the mid-gut and in the anal sacs were higher than in the body wall and in the hindgut. To investigate the function of the mid-gut and anal sacs and mitochondrial sulphide metabolism in U. unicinctus, we determined the SQR protein expression in different tissues and the SQR protein expression and enzyme activity after sulphide exposure (25, 50 and 150 mM) in the anal sacs and in the mid- gut. The results showed the highest SQR expression was in the anal sacs, followed by the body wall and the hindgut, and finally the mid-gut. During exposure to 50 mM sulphide, the SQR expression in the anal sacs was significantly increased up to 2 h, reaching a maximum at 24 h and then decreasing up to 48 h. In the anal sacs, SQR enzyme activity was increased significantly up to 6 h and continued to 48 h during exposure to 50 mM sulphide, whereas in mid-gut, the SQR expression and enzyme activity did not increase significantly. We conclude that the anal sacs act as an important organ while the mid- gut only acts as an ‘assistant’ organ for mitochondrial sulphide metabolism in U. unicinctus.

Key words: Sulphide adaptation, sulphide:quinone oxidoreductase (SQR), anal sacs, mid-gut, Urechis unicinctus

Introduction metabolism (Bagarinao 1992), and oxidative damage to RNA and DNA (Joyner-Matos et al. 2010). A variety of animals living in different habitats such Previous studies indicate that mitochondrial sulphide as mudflats, marshes, cold seeps, and hydrothermal oxidation is an important mechanism for reducing vents can be periodically or continuously exposed 2 sulphide toxicity in sulphide-adapted animals to sulphide (the sum of H2S, HS , and S ) (Grieshaber & Vo¨lkel 1998). (Grieshaber & Vo¨lkel 1998). Sulphide is a well- Recently, sulphide:quinone oxidoreductase (SQR) known toxin which can cause potential harm to has been shown to be the first enzyme in the organisms by, for example, reversible inhibition of mitochondrial sulphide oxidation enzyme system cytochrome c oxidase (Evans 1967; Nicholls 1975), (Hildebrandt &Grieshaber 2008; Tiranti et al. decreased haemoglobin oxygen affinity (Carrico 2009). In the polychaete worm, Arenicola marina, et al. 1978), sulph-haemoglobin formation (Bagar- SQR has been shown to convert sulphide to persul- inao 1992; Kraus et al. 1996), mitochondrial depo- phides (Theissen & Martin 2008). Subsequently, a

Downloaded by [Qingdao Institute of Biomass Energy and Bioprocess Technology] at 01:39 28 November 2012 larization (Julian et al. 2005), coelomocyte death, putative sulphur dioxygenase oxidizes a persulphide decreased cell proliferation (Hance et al. 2008), molecule into sulphite and a second persulphide is inhibition of almost 20 enzymes involved in aerobic added to sulphite by a sulphur transferase-rhodanese,

*Correspondence: Zhi-Feng Zhang, Key Laboratory of Marine Genetics and Breeding of Ministry of Education, Ocean University of China, Qingdao 266003, China. E-mail: [email protected] Published in collaboration with the University of Bergen and the Institute of Marine Research, Norway, and the Marine Biological Laboratory, University of Copenhagen, Denmark

(Accepted 6 June 2012; Published online 24 September 2012; Printed 4 October 2012) ISSN 1745-1000 print/ISSN 1745-1019 online # 2012 Taylor & Francis http://dx.doi.org/10.1080/17451000.2012.707320 Function of the anal sacs and mid-gut in Urechis unicinctus 1027

250 producing the end-product -thiosulphate 25 µM (Hildebrandt & Grieshaber 2008). More recently, 50 µM 200 the mitochondrial sulphide oxidation pathway has 150 µM been validated in mammals which utilize SQR, ethylmalonic encephalopathy 1 as a sulphur dioxy- 150 genase and rhodanese (Tiranti et al. 2009). In a previous study, we cloned the SQR full-length 100 cDNA and validated its function in vitro for the 50 echiuran worm Urechis unicinctus (von Drasche, concentration(µM) Sulfide 1881) (Ma et al. 2011). This worm is mainly distributed around China, Korea, Russia and Japan, and 0 0123456 inhabits marine sediments, especially intertidal and Sulfide exposure (h) subtidal mudflats (Li et al. 1994). The worm has a thick, muscular body wall, abundant coelomic fluid, Figure 1. Sulphide addition time determination by measure metanephridial gonoducts, and an elaborate digestive sulphide variation during sulphide exposure in the experimental system. Data as mean9S.E.M, n 5. tract leading to a specialized respiratory hindgut region and terminating in a cloaca communicating natural seawater as a control. For each sulphide with paired excretory structures termed anal sacs concentration (control, 25, 50 and 150 mM), 5 tanks (Arp et al. 1995). We found that the levels of SQR were used. For each tank, 8 worms were placed in a mRNA in the mid-gut and in the anal sacs were higher 15-l water tank containing 12 l seawater. During the than in the body wall and in the hindgut (Ma et al. experiment, a relatively constant sulphide concentra- 2011). The mid-gut and anal sacs had been shown to tion was maintained by adding the stock solution act as a digestive organ and an excretory organ, every 2 h based on the measurement results of respectively, in echiuran worms (Harris & Jaccarini sulphide concentration in each tank, approximately 1981; Seto et al. 1993; Arp et al. 1995; Menon & Arp 10% variation every 2 h (Figure 1). Sulphide 1998). Due to the high expression of the key sulphide concentration was determined spectrophotometri- oxidation enzyme SQR, whether these organs play cally using a methylene blue method (Cline 1969). important role in sulphide detoxification in echiuran Dissolved oxygen variation in the experimental sys- worms requires further investigation. tem was measured during sulphide exposure using a In this study, we attempted to validate the func- YSI Model 55 Handheld Dissolved Oxygen meter tion of the anal sacs and mid-gut of U. unicinctus in (Figure 2). At 0, 2, 6, 12, 24 and 48 h after initiation sulphide metabolism. SQR protein expression in of sulphide exposure, one worm was removed from different tissues and SQR protein expression and each tank (n5). The anal sacs and mid-gut of the enzyme activity in the anal sacs and mid-gut after worms were excised, frozen in liquid nitrogen, and exposure to sulphide were investigated. stored at 808C for subsequent analysis.

Materials and methods Quantitative analysis of SQR protein expression Animals and exposure to sulphide An indirect competitive enzyme link immunoassay Individuals of Urechis unicinctus (mean fresh mass of (ELISA) method was established to determine the 2897.4 g), collected from a coastal intertidal flat during August 2011 in Yantai, China were main- 4.00 control tained for 1 week in an aquarium with aerated, 3.50 recirculating seawater at 20918C, pH 8.2590.02, 3.00 25 µM salinity 25 and were fed with microalgae (Chlorella 2.50 50 µM vulgaris and Nitzschia closterium). Feeding was sus- 2.00 150 µM pended 24 h prior to experimentation. Five worms Downloaded by [Qingdao Institute of Biomass Energy and Bioprocess Technology] at 01:39 28 November 2012 1.50 were excised to determine the wet weight of different 1.00 tissues (body wall, hindgut, anal sacs and mid-gut). Tissue samples for protein isolation were excised, Dissolved oxygen (mg/L) 0.50 frozen in liquid nitrogen, and stored at 808C. 0.00 The sulphide concentration of the incubation 0 6 12 24 30 36 48 medium in each tank was maintained at 25, 50 and Sulfide exposure (h) 150 mM, respectively, by dilution of the stock Figure 2. Dissolved oxygen variation during sulphide exposure in sulphide solution (10 mM Na2S, pH 8.0), and using the experimental system. Data as mean9S.E.M, n 5. 1028 Y.-B. Ma et al.

SQR expression in different tissues, and in the anal mid-gut. Mitochondria were isolated from the dif- sacs and mid-gut after sulphide exposure. The ferent tissues according to the methods of Schroff & special rabbit polyclonal antibody for SQR with a Scho¨ttler (1977) with slight modifications: the iso- titre of 96,000 was previously prepared using re- lation medium consisted of 0.0584 M saccharose, combinant SQR expression in Escherichia coli. The 0.1402 M glycine, 40 mM Tris (pH 7.4), 2 mM specificity of SQR polyclonal antibody has been EGTA, and 0.2% BSA. SQR enzyme activity was assessed by Western blotting (Ma et al. 2011). measured at room temperature (258C). A 0.3 ml Approximately 100 mg tissue (body wall, hindgut, reaction mixture was produced containing 0.02 M anal sacs, mid-gut) was homogenized in 1 ml of lysis TrisÁHCl (pH 8.0), 100 mM coenzyme Q10 (Sigma buffer (150 mM sodium chloride, 1% NP-40, 0.5% Chemical Co., St Louis, MO, USA), 2 mM KCN, sodium deoxycholate, 0.1% SDS, 1 mM EDTA, and isolated mitochondria. The reaction was initiated 1 mM PMSF, and 50 mM Tris, pH 7.4) on ice, with 200 mM sulphide (prepared freshly with N2- centrifuged at 12,000g for 5 min and the supernatant flushed H2O) and the decrease in 275 nm absorption collected. The assay was carried out using a 96-well followed for 3 min at 30 s intervals (modified from polystyrene ELISA microtitre plate (Corning Inc., Hildebrandt & Grieshaber 2008 and Theissen & Somerset, NJ, USA). All assays were carried out in Martin 2008). The mM extinction coefficient of duplicate. The plates were coated with 25 ng of coenzyme Q10 in the reaction buffer at 275 nm was refolded recombinant SQR in 100 ml of coating buffer 15. One unit of enzyme activity was defined as 1 nM (50 mM carbonate buffer, pH 9.6), sealed and of reduced coenzyme Q10 formed per min. Each incubated overnight at 48C. This was followed by enzyme activity assay was performed in triplicate. blocking with 300 ml of 2% bovine serum albumin (BSA) in PBST (PBS containing 0.05%Tween 20, Protein assay pH 7.4; PBS, 137 mM NaCl, 2.7 mM KCl, 10 mM sodium phosphate dibasic, 2 mM potassium phos- Protein concentrations were determined by the phate monobasic) for 2 h at 378C. Serial dilutions of Bradford method using BSA as a standard (Bradford standard antigen (refolded recombinant SQR, range 1976). The tissues from each of the five animals per from 1 to 2500 ng well1) were prepared in micro- time point were tested in duplicate. centrifuge tubes with phosphate buffer saline (PBS) containing 0.1% BSA and incubated for 2 h at 378C Statistical analysis after adding equal volumes of specific primary anti- serum (1 : 20,000). The tissues samples were also Data are presented as the mean9standard error of diluted (1 : 10) and incubated simultaneously with the mean of five samples. Significant differences the standard antigen. The wells were blocked with between means were tested using one-way analysis of 300 ml of 2% BSA in PBST for 2 h at 378C, followed variance followed by least significant difference tests, by incubation with 100 ml of 1 : 8000 diluted horse using the SPSS statistical package (version 13.0; radish peroxidase-conjugated goat anti-rabbit immu- SPSS Inc., Chicago, IL, USA) at a significance level noglobulin G (IgG) (Jackson ImmunoResearch, West of p B0.05. Grove, PA, USA) for 2 h at 378C. For colour develop- ment, the wells were filled with 100 mlofsolubleTMB Results (tetramethylbenzidine) solution. After incubation for 10minat378C, 50 mlof2MH2SO4 was added to The percentage of different tissues in total wet terminate the enzymatic reaction and the absorbance weight were determined (Table I); the results showed of each well was determined by a microtitre plate the body wall (39.8292.91%) was the highest, reader (SpectraMax 190, Molecular Devices Co., San followed by mid-gut (10.8490.49%) and hindgut Francisco, CA, USA) at 450 nm. The microtitre plate wasshakendryandwashed5timesaftereach Table I. Different tissue wet weight and percentage of total wet incubation procedure with 330 mlofPBST. weight in Urechis unicinctus. Different tissues Wet weight (g) Percentage wet weight Downloaded by [Qingdao Institute of Biomass Energy and Bioprocess Technology] at 01:39 28 November 2012

Assay for SQR enzyme activity Body wall 9.3190.68 39.8292.91 Hindgut 0.5490.079 2.2990.34 The SQR enzyme activity after sulphide exposure in Mid-gut 2.5390.12 10.8490.49 the anal sacs and mid-gut was detected. Considering Anal sacs 0.1290.016 0.5390.069 the adaptive capability to sulphide exposure based Total wet weight 23.3793.52 on our earlier results (Zhang et al. 2003; Wang et al. Data as mean9S.E.M, n5. Each tissue’s percentage is 2010), we selected 50 mM sulphide to determine the calculated by dividing tissue wet weight by total wet weight, then effect of sulphide on SQR activity in anal sacs and multiplying it by 100. Function of the anal sacs and mid-gut in Urechis unicinctus 1029

1.6 b Discussion 1.4 New insights into sulphide detoxification of anal sacs 1.2 a from Urechis unicinctus 1 In a previous study, we found the mitochondrial 0.8 a sulphide detoxification key enzyme SQR mRNA 0.6 expression was relatively high and SQR was expressed in the whole stratified epithelium in the anal 0.4 c sacs of Urechis unicinctus (Ma et al. 2011). In this 0.2 study, SQR expression and SQR enzyme activity SQR content (ng/µg total protein) total (ng/µg content SQR 0 during sulphide exposure in the anal sacs are Body wall Hindgut Anal sacs Mid-gut determined. We conclude that in the anal sacs, Tissue SQR expression could be induced by high sulphide Figure 3. SQR expression in different tissues of Urechis unicinctus. concentrations (50 and 150 mM). At low sulphide Data as mean9S.E.M, n 5; different letters indicate signiﬁcant concentrations (25 mM), maybe the base level differences between tissues (p B0.05). expression of SQR in the mitochondria as well as other sulphide oxidation mechanisms such as sulphide-oxidizing bodies (Seto et al. 1993; Arp et al (2.2990.34%), and finally the anal sacs 1995; Zhang et al. 2003) are sufficient to deal with (0.5390.069%). sulphide. With the increase of sulphide concentra- SQR protein expression in the body wall has been tion (50 and 150 mM), the worms utilize the investigated by Western blotting (Ma et al. 2011). increased expression of SQR in the mitochondria Due to the low level of expression, the differences to deal with the higher concentration of sulphide in between different tissues could not be detected by the anal sacs. In addition, the SQR enzyme activity Western blotting. In this study, a quantitative indirect competitive ELISA method was established A 25 Anal sacs to determine the SQR expression in different tissues. control * The results showed that the highest SQR expression 25 µM 20 50 µM * was detected in the anal sacs followed by the body * 150 µM * * wall and hindgut, and, finally, the mid-gut with a * 15 * relatively low level of expression (Figure 3). SQR expression in the anal sacs and mid-gut after * * 10 sulphide exposure was determined by the quantita- * tive indirect competitive ELISA in this study. The 5 results indicated that during exposure to 25 mM SQR content (ng/µg total protein)

sulphide, the SQR expression in the anal sacs was 0 not significantly increased from that of the control 026122448 (Figure 4A); during exposure to 50 mM sulphide, the Sulfide exposure (h) SQR expression in the anal sacs was increased B Mid-gut significantly at 2 h, reaching the highest level after 0.7 exposure for 24 h, after which it decreased to the control 25 µM 0.6 control level at 48 h (Figure 4A); during 150 mM 50 µM sulphide exposure, the SQR expression in the anal 0.5 150 µM

sacs was increased at 2 h, and then, continued to 0.4 increase to 48 h sulphide exposure. For the mid-gut, 0.3 the SQR expression during sulphide exposure was not significantly increased (Figure 4B). 0.2 Downloaded by [Qingdao Institute of Biomass Energy and Bioprocess Technology] at 01:39 28 November 2012 The results indicated that the SQR enzyme 0.1 SQR content(ng/µg total protein) activity was significantly increased in the anal sacs 0 after exposure to 50 mM sulphide which increased 026122448 significantly after an initial 6 h exposure, reaching Sulfide exposure (h) the highest level after 24 h and then decreased by 48 h (Figure 5A). In the mid-gut, the SQR enzyme Figure 4. Urechis unicinctus SQR expression in anal sacs (A) and mid-gut (B) after sulphide exposure (control, 25 mM, 50 mM, 150 activity was only increased after 48 h exposure to 50 mM). Data as mean9S.E.M, n 5; *statistically signiﬁcant mM sulphide (Figure 5B). difference from control (p B0.05). 1030 Y.-B. Ma et al.

A Anal sacs Table II. Basal SQR content (SQR content without sulﬁde exposure) and highest SQR content after sulﬁde exposure for 100 * 48 h (25, 50 and 150 mM) in different tissues. * 90 * * 80 Basal SQR Highest SQR content 70 content (ng mg1 after sulfide exposure 60 Tissues total protein) (ng mg1 total protein) 50 40 Anal sacs 1.3590.08 20.2892.25 30 Hindgut 0.5590.12 14.1993.55 (Ma et al. 2012) Body wall 0.9590.10 8.1693.51 (Ma et al. 2012) 20 Mid-gut 0.2490.02 0.5690.10 10

SQR activity (U/mg mitochondria) 0 Data are given as mean9S.E.M, n5. 026122448 Sulfide exposure (h) a percentage of the total wet weight is lower compared to other tissues of the worm (Table I), B Mid-gut due to the high SQR expression as well as the special 70 filtering function, we suggest that the anal sacs have * a high sulphide detoxification ability and act as an 60 important organ in sulphide metabolic adaptation in 50 U. unicinctus.InUrechis caupo, a species related to U. 40 unicinctus, the anal sac is deemed to be an excretory 30 organ and may play a role in elimination of sulphur- containing pigment granules, sulphide detoxification 20 end-products and other waste products (Seto et al. activity (U/mg activity (U/mg mitochondria) 10 1993; Arp et al. 1995). In this study, we provide new

SQR 0 evidence of sulphide detoxification in the anal sacs of 026122448 an echiuran worm. Sulfide exposure (h)

Figure 5. Urechis unicinctus SQR enzyme activity in anal sacs (A) The mid-gut as an accessory organ in Urechis and mid-gut (B) during 50 mM sulphide exposure. Data as unicinctus sulphide metabolic adaptation mean9S.E.M, n 5; *statistically signiﬁcant difference from control (p B 0.05). In the previous study, we found the mitochondrial sulphide detoxification key enzyme SQR mRNA in the anal sacs is significantly increased after expression was the highest in the mid-gut compared exposed to 50 mM sulphide (Figure 5A). Further- with other tissues and that SQR was expressed in the more, we find that during 50 mM sulphide exposure whole simple columnar epithelium of the mid-gut of SQR enzyme activity only increases to a maximum of Urechis unicinctus (Ma et al. 2011). In this study, we about 2-fold while SQR expression increases to a analyse the protein expression and enzyme activity of maximum of 15-fold in the anal sacs (Figure 4A,B). SQR in the mid-gut during sulphide exposure, and We suggest that during sulphide exposure the the results reveal the SQR expression in the mid-gut increase of SQR expression is the major strategy is extremely low (Figure 3). The discrepancy in the for the anal sacs in sulphide metabolic adaptation. mid-gut mRNA and protein expression may be due As to the difference between SQR protein expression to different post-transcriptional, translational and and enzyme activity, we deduce that it may result post-translational regulation in the tissue. During from the different methods used in the detection of sulphide exposure, SQR expression is not signifi- SQR protein expression and enzyme activity or it cantly increased (Figure 4B) and SQR enzyme might be due to another unknown mechanism for activity only increase after exposed to 50 mM SQR enzyme activity regulation. Compared with sulphide for 48 h (Figure 5B). We suggest that

Downloaded by [Qingdao Institute of Biomass Energy and Bioprocess Technology] at 01:39 28 November 2012 other tissues after sulphide exposure (body wall, during sulphide exposure the inducement of SQR is hindgut and mid-gut), SQR expression is the highest not notable in the mid-gut. The basal expression of in the anal sacs (Table II). In addition, the anal sacs SQR in the mitochondria as well as other sulphide in echiuran worms have a high filtering rate as oxidation mechanisms such as sulphide-oxidizing reported in another species of echiuran worm bodies (Menon & Arp 1998) are sufficient to deal Bonellia viridis (Echiura Bonelliidae) which could with sulphide in the tissue after sulphide exposure clean extracellular substance from the coelomic fuid even up to 150 mM. We conclude that the mid-gut at 9.691.9 ml 100g1 wet weight of animal day1 only act as an accessory organ in U. unicinctus in (Harris & Jaccarini 1981). Although the anal sacs as sulphide metabolic adaptation. Function of the anal sacs and mid-gut in Urechis unicinctus 1031

Different tissues sulphide oxidation ability ranking Hance JM, Andrzejewski JE, Predmore BL, Dunlap KJ, Misiak KL, Julian D. 2008. Cytotoxicity from sulfide exposure in a Based on the weight percentage of different tissues in sulfide-tolerant marine invertebrate. Journal of Experimental total wet weight (Table I) and SQR content espe- Marine Biology and Ecology 359:102Á09. cially during sulphide exposure in different tissues Harris RR, Jaccarini V. 1981. Structure and function of the anal (Ma et al. 2012; Table II), we predict that the sacs of Bonellia viridis (Echiura: Bonelliidae). Journal of the Marine Biological Association of the United Kingdom sulphide oxidation ability of different tissues in 61:413Á30. Urechis unicinctus is highest in the body wall, followed Hildebrandt TM, Grieshaber MK. 2008. Three enzymatic by the hindgut and the anal sacs, and finally the mid- activities catalyze the oxidation of sulfide to thiosulfate in gut. Although the sulphide oxidation ability of anal mammalian and invertebrate mitochondria. Federation of sacs is lower than that of the body wall and hindgut, European Biochemical Societies Journal 275:3352Á61. Joyner-Matos J, Predmore BL, Stein JR, Leeuwenburgh C, due to its special filtering function, it is still an Julian D. 2010. Hydrogen sulfide induces oxidative damage important organ for the worm in sulphide metabolic to RNA and DNA in a sulfide-tolerant marine invertebrate. adaptation. Physiological and Biochemical Zoology 83(2):356Á65. Julian D, April KL, Patel S, Stein JR, Wohlgemuth SE. 2005. Mitochondrial depolarization following hydrogen sulfide ex- Conclusions posure in erythrocytes from a sulfide-tolerant marine invertebrate. The Journal of Experimental Biology 208:4109Á4122. In this study, we detected SQR expression in Kraus D, Doeller J, Powell, C. 1996. Sulfide may directly modify different tissues of Urechis unicinctus and SQR cytoplasmic hemoglobin deoxygenation in Solemya reidi gills. expression and enzyme activity in the anal sacs and Journal of Experimental Biology 199:1343Á52. mid-gut after sulphide exposure. Based on these Li FL, Wang W, Zhou H. 1994. Studies on the echiurans results, we conclude that the anal sacs act as an (Echiura) of the yellow sea (Huanghai) and Bohai sea. Journal important organ and the mid-gut only as an acces- of Ocean University of China 24:203Á10. Ma YB, Zhang ZF, Shao MY, Kang KH, Tan Z, Li JL. 2011. sory organ in sulphide metabolic adaptation in Sulfide:quinone oxidoreductase from echiuran worm Urechis U. unicinctus. unicinctus. Marine Biotechnology 13:93Á107. Ma YB, Zhang ZF, Shao MY, Kang KH, Shi XL, Dong YP, et al. 2012. Response of sulfide:quinone oxidoreductase to sulfide exposure in the echiuran worm Urechis unicinctus. Marine Acknowledgements Biotechnology 14:245Á51. Menon JG, Arp AJ. 1998. Ultrastructural evidence of detoxifica- We are grateful to the laboratory members for tion in the alimentary canal of Urechis caupo. Invertebrate experimental material preparation and technical Biology 117:307Á17. assistance. This work is supported by the Natural Nicholls P. 1975. The effect of sulphide on cytochrome aa3. Science Foundation of China (NSFC) (40776074 Isosteric and allosteric shifts of the reduced alpha-peak. and 31072191). Biochimica et Biophysica Acta 396:24Á35. Schroff G, Scho¨ttler U. 1977. Anaerobic reduction of fumarate in the body wall musculature of Arenicola marina (Polychaeta). References Journal of Comparative Physiology B 116:325Á36. Seto SL, Mason AZ, Arp AJ. 1993. Anal vesicle granules Arp AJ, Menon JG, Julian D. 1995. Multiple mechanisms provide accumulate sulfur and iron in Urechis caupo. American Zool- tolerance to environmental sulfide in Urechis caupo. Integrative ogist 33:84A. and Comparative Biology 35:132Á44. Theissen U, Martin W. 2008. Sulfide:quinone oxidoreductase Bagarinao T. 1992. Sulfide as an environmental factor and (SQR) from the lugworm Arenicola marina shows cyanide- and toxicant: Tolerance and adaptations in aquatic organisms. thioredoxin-dependent activity. Federation of European Bio- Aquatic Toxicology 24:21Á62. chemical Societies Journal 275:1131Á39. Bradford MM. 1976. A rapid and sensitive method for the Tiranti V, Viscomi C, Hildebrandt T, Meo ID, Mineri R, quantitation of microgram quantities of protein utilizing the Tiveron C, et al. 2009. Loss of ETHE1, a mitochondrial principle of proteinÁdye binding. Analytical Chemistry dioxygenase, causes fatal sulfide toxicity in ethylmalonic 72:248Á54. encephalopathy. Nature Medicine 15:200Á05. Carrico RJ, Blumberg WE, Peisach J. 1978. The reversible Wang SF, Zhang ZF, Cui H, Kang KH, Ma ZJ. 2010. The effect binding of oxygen to sulfhemoglobin. Journal of Biological of toxic sulfide exposure on oxygen consumption and oxidation Chemistry 253:7212Á15. products in Urechis unicinctus (Echiura: Urechidae). Journal of Cline JD. 1969. Spectrophotometric determination of hydrogen

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