This article was downloaded by: [Qingdao Institute of Biomass Energy and Bioprocess Technology] On: 28 November 2012, At: 01:39 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK 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 PLEASE SCROLL DOWN FOR ARTICLE Full terms and conditions of use: http://www.tandfonline.com/page/terms-and-conditions This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. The publisher does not give any warranty express or implied or make any representation that the contents will be complete or accurate or up to date. The accuracy of any instructions, formulae, and drug doses should be independently verified with primary sources. 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Marine Biology Research, 2012; 8: 1026Á1031 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 dis- tributed 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).