Thiotaurine and Hypotaurine Contents in Hydrothermal-Vent Polychaetes Without Thiotrophic Endosymbionts: Correlation with Sulﬁde Exposure Ã PAUL H

JOURNAL OF EXPERIMENTAL ZOOLOGY 311A:439–447 (2009)

A Journal of Integrative Biology

Thiotaurine and Hypotaurine Contents in Hydrothermal-Vent Polychaetes Without Thiotrophic Endosymbionts: Correlation With Sulﬁde Exposure Ã PAUL H. YANCEY1 , JOANNE ISHIKAWA1, BRIGITTE MEYER1, 2 3 PETER R. GIRGUIS , AND RAYMOND W. LEE 1Biology Department, Whitman College, Walla Walla, Washington 2Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, Massachusetts 3School of Biological Sciences, Washington State University, Pullman, Washington

ABSTRACT Invertebrates at hydrothermal vents and cold seeps must cope with toxic H2S. One proposed protection mechanism involves taurine derivatives: At vents and seeps, many animals have high levels of hypotaurine and thiotaurine (a product of hypotaurine and HS), originally found in animals with thiotrophic endosymbionts. To further test the role of these compounds, we analyzed them in vent polychaetes without endosymbionts: Paralvinella sulfincola, P. palmiformis and P. pandorae (paralvinellids) and Nicomache venticola (maldanid). P. sulfincola were collected from a high temperature (42–681C) and a warm site (21–351C). P. palmiformis and pandorae were from cool sites (12–181C) and N. venticola were from a cold site (41C). H2S concentrations in vent effluent largely correlate with temperature. Some specimens were frozen; other ones were kept alive in laboratory chambers, with and without sulfide. Tissues were analyzed for taurine derivatives and other solutes that serve as organic osmolytes. The major osmolyte of all species was glycine. Thiotaurine contents were significantly different among all species, in the order P. sulfincola hot4P. sulfincola warm4P. pandorae4P. palmiformis4N. venticola. P. sulfincola also had high levels of sarcosine; others species had none. Sarcosine and hypotaurine contents of P. sulfincola’s branchiae were higher, while glycine contents were lower, than in main body. In P. palmiformis kept in pressure chambers with sulfide, thiotaurine contents were higher and hypotaurine lower than in those kept without sulfide. These results support the hypothesis that conversion of hypotaurine to thiotaurine detoxifies sulfide in vent animals without endosymbionts. J. Exp. Zool. 311A:439–447, 2009. r 2009 Wiley-Liss, Inc.

How to cite this article: Yancey PH, Ishikawa J, Meyer B, Girguis PR, Lee RW. 2009. Thiotaurine and hypotaurine contents in hydrothermal-vent polychaetes without thiotrophic endosymbionts: correlation with sulﬁde exposure. J. Exp. Zool. 311A:439–447.

Marine invertebrates at hydrothermal vents external bacteria and detritus in sulfide-laden and cold seeps must cope with potentially toxic waters, and may therefore also be exposed to toxic levels of hydrogen sulfide (Somero et al., ’89), sulfide (Juniper and Martineu, ’95). In addition, which can bind to iron and disrupt various animals without thiotrophic endosymbionts do not processes including mitochondrial function benefit from the sulfide-oxidizing capacity of (Bagarinao, ’92; Arp et al., ’95; Fisher, ’98). Some symbionts, which can detoxify sulfide. Such of these animals—vestimentiferan (siboglinid) polychaetes, vesicomyid clams, and bathymodiolin Grant sponsors: Perry Grant, Whitman College; National Science mussels—cannot avoid sulfide exposure because Foundation;Ã Grant number: OCE-0623554. Correspondence to: Paul H. Yancey, Biology Department, Whitman they need it for their sulfide-oxidizing (thio- College, Walla Walla, WA 99362. E-mail: [email protected] trophic) bacterial endosymbionts. Other vent and Received 12 January 2009; Revised 20 March 2009; Accepted 23 March 2009 seep animals such as other polychaetes and Published online 28 April 2009 in Wiley InterScience (www. gastropods have no endosymbionts, but feed on interscience.wiley.com). DOI: 10.1002/jez.541 r 2009 WILEY-LISS, INC. 440 P.H. YANCEY ET AL. animals include the paralvinellid polychaetes reversible (Pruski and Fiala-Me´dioni, 2003). Thus, (Terebellida, Alvinellidae), Paralvinella sulfincola thiotaurine might serve to store sulfide nontoxi- (sulfide worms), P. palmiformis (palm worms), and cally within cells, and release it as the endosym- P. pandorae, which are among the most abundant bionts deplete free sulfide, thus acting as a animals at vents of the Juan de Fuca Ridge sulfide ‘‘buffer’’. Laboratory studies show that (Levesque et al., 2003). These congeners form a thiotaurine contents increase during sulfide model system for testing adaptations to tempera- exposure in symbiont-bearing tissues of vesico- ture and sulfide, because P. sulfincola lives in myids, bathymodiolins, vestimentiferans (Pruski much warmer and more sulfide-laden waters than and Fiala-Me´dioni, 2003), and shallow-living sole- the other two species (Sarrazin et al., ’99). myid clams (Joyner et al., 2003). For protection against sulfide toxicity, various Thiotaurine was originally found at high levels mechanisms have been proposed and tested. These in tissues with endosymbionts (trophosome in include the following: sulfide-binding proteins vestimentiferans, gills in bivalves), but only at (e.g., modified hemoglobins) in body fluids of low levels in other tissues, hemolymph or blood. endosymbiont-bearing animals, for transporting These findings suggest that the solutes are located sulfide nontoxically (e.g., Arp et al., ’87; Childress primarily intracellularly (Yin et al., 2000), and led et al., ’93; Kraus, ’95); external tubes and mucus of to a proposal that thiotaurine is a general marker some polychaetes, which may reduce diffusion of of thiotrophic endosymbiosis (Pruski et al., sulfide into the animal; microbes living on the 2000b). However, we have found that two species tubes, which oxidize sulfide before it can enter the of gastropods (snail and limpet) from hydrother- animal (Juniper and Martineu, ’95); specialized mal vents of the Juan de Fuca Ridge contain sulfide-oxidizing organelles in epidermal tissue in substantial contents of both thiotaurine and some polychaetes from sulfide-rich habitats (Arp hypotaurine (Rosenberg et al., 2006), even though et al., ’95; Menon et al., 2003); conversion to they lack endosymbionts. Recently, hypotaurine thiosulfate or elemental sulfur (Vetter, ’85; Powell has been found to protect erythrocytes of a and Somero, ’86; Arp et al., ’95; Arndt et al., 2001); polychaete without thiotrophic symbionts from and binding to intracellular metals, protein, and sulfide toxicity because of the solute’s sulfide- glutathione (Vismann, ’91). Among vent animals scavenging ability (Ortega et al., 2008). These without endosymbionts, P. palmiformis has been findings suggest that the two solutes are used in reported to have high (but variable) sulfide levels sulfide detoxification but not strictly for symbiosis. in the blood along with a sulfide-binding hemoglo- The ratio of thiotaurine to total hypotaurine bin-like protein (Martineu et al., ’97). Both plus thiotaurine (hereafter called the ‘‘exposure P. palmiformis and P. sulfincola have enzymatic indicator ratio’’) has been proposed to be an sulfide-oxidizing activity in their tissues, which is indicator of the level of sulfide exposure (Pranal considerably higher in P. sulfincola (Juniper and et al., ’95). Indeed, we and others have found that Martineu, ’95), the worm from warmer, higher- the ratio tends to be higher in animals with higher sulfide vents. exposures in situ and in the laboratory in seep and In recent years, two compounds called thiotaur- vent species (Pruski et al., 2000a; Brand et al., ine and hypotaurine have been proposed to serve 2007), including the vent gastropods without in protection from and/or transport of sulfide endosymbionts (Rosenberg et al., 2006). We intracellularly. Hypotaurine reacts with sulfide also found evidence that, regardless of differing to produce thiotaurine (Pruski et al., 2000b): exposure indicator ratios between populations of a species, the total of hypotaurine plus thio- ð Þþ þ hypotaurine NH3 CH2 CH2 SO2 SH taurine tends to be consistent in and to correlate ! ð Þ NH3 CH2 CH2 SO2 SH thiotaurine with a species’ maximum sulfide exposure (Brand et al., 2007). These unusual amino acids were first reported Thiotaurine and hypotaurine are concentrated in seep and vent vestimentiferans and bivalves enough in these animals to serve another function, with thiotrophic endosymbionts (Alberic, ’86; namely that of an organic osmolyte. Like most Pranal et al., ’95; Pruski et al., 2000a). The source other marine invertebrates, vent and seep of SH could be a free radical, a ligand donated by a invertebrates are osmoconformers that must protein R as R-SSH, or a moiety donated by balance their osmotic pressure with that of the glutathione-SSH. Synthesis of thiotaurine from ocean. In cells of most marine invertebrates, this is hypotaurine appears to be enzymatic and is done with organic osmolytes, primarily free

J. Exp. Zool. THIOTAURINE IN PARALVINELLIDS 441 amino acids and methylamines such as sarcosine MATERIALS AND METHODS (methylglycine) and betaine (trimethylglycine). Organic osmolytes are often called ‘‘compatible Sample collection and temperature solutes’’, because (unlike salt ions) they usually measurements do not disrupt protein function at high concentra- Species used in this study were collected at tions and thus can be safely accumulated to raise hydrothermal vents of the Juan de Fuca Ridge, cellular osmotic pressure. Other organic osmolytes Endeavour Segment, using the R/V Atlantis and are called ‘‘counteracting’’ because they can DSRV Alvin. Sites of collection (all around 2,200 m offset the effects of some factors that perturb depth) included the Dante edifice at the Main proteins such as temperature and hydrostatic Endeavour field, Clam Bed field, the Faulty pressure; other organic osmolytes may be meta- Towers complex in the Mothra field, and a new bolic cytoprotectants such as antioxidants (Yancey small field found 700 m north of the Dante edifice. et al., ’82; Yancey, 2005). In various invertebrates Temperatures of vent fluids around the worms from shallow waters, taurine, betaine, and were taken for periods greater than 10 sec with an glycine are the dominant organic osmolytes. external probe using the Alvin manipulator, However, in deep-sea invertebrates studied to before the worms were removed with a suction date, there is less taurine and more methylamines device. Upon recovery, most specimens were plus inositols and organic phosphates. These may rapidly frozen and stored aboard the ship at counteract the effects of pressure (Fiess et al., 701C, and then transported on dry ice for storage 2002; Yancey et al., 2002). And of course there are at 801C to the home laboratory. high levels of thiotaurine and hypotaurine in vent A subset of freshly collected P. palmformis and seep species that contribute to osmotic worms collected from the Roane (Faulty Towers) balance, but these may primarily be for sulfide chimney in the Mothra field were kept alive on detoxification. board ship. Upon recovery, these worms were To further test the possible roles of hypotaurine rapidly placed into ice-cold seawater, and then and thiotaurine in adaptation to sulfide stress in pressurized to 3,500 PSI (238 atm or 24 MPa) in a vent animals without symbionts, and to continue 1.1 L titanium pressure vessel. Thirty minutes investigating other osmolytes that might relate to later, the vessel was flushed with seawater habitat stresses other than osmotic, we have containing 95 mM oxygen, 235 mM sulfide, pH 7.0, analyzed the three Paralvinella species from cool at 301C as described in Girguis and Childress to hot hydrothermal diffuse flows among the vents (2006). Worms were maintained at these condi- of the Juan de Fuca Ridge. P. sulfincola live in tions for 52 hr. They were then rapidly depressur- soft mucous tubes having a high content of ized and frozen at 701C. Another group of worms elemental sulfur (Juniper et al., ’86), are found were placed into an identical vessel and were at the warmest sites (Sarrazin et al., ’99), tolerate treated as described here, but were maintained in 1 temperatures of 50–56 C in laboratory pressure flowing pressurized seawater devoid of hydrogen chambers (Lee, 2003; Girguis and Lee, 2006), and sulfide, with 120 mM oxygen and pH 8.1 for 70 hr. have the most thermostable enzymes of the three All animals were alive before freezing. Because of paralvinellids (Jollivet et al., ’95). P. palmiformis other experiments on the ship, there were insuffi- have a continuously produced mucus sheath also cient P. sulfincola and P. pandorae specimens and having a high content of elemental sulfur (Juniper pressure chambers available for similar experi- et al., ’86), are found in cooler waters (Sarrazin ments on those species. et al., ’99), and were active between 5 and 371Cin laboratory pressure chambers (Lee, 2003). Solute analysis P. pandorae (specifically P. pandorae pandorae) also have sheaths, and were found by us in cool Whole semi-thawed animals were weighed on an sites (see Results). We also analyzed the bamboo electronic balance (Mettler AE100, Columbus, OH; worm Nicomache venticola (a maldanid), which we 0.0001 g accuracy) after gentle blotting to remove found around vents but only in cold diffuse-flow surface seawater. For some P. sulfincola and areas. Specimens were collected from a number P. palmformis, branchiae were cut from the main of vent sites with temperatures ranging from body and analyzed separately. Samples were then 4to681C, and which likely exhibited various homogenized in 1,000–1,500 mLof70%coldethanol, sulfide concentrations (Juniper and Martineu, ’95; and kept at 41C overnight before centrifugation at Sarrazin et al., ’99). 15,000g for 20 min to remove proteins and cellular

J. Exp. Zool. 442 P.H. YANCEY ET AL. debris. Ethanol rather than acid extraction is used to preserve thiotaurine (which breaks down to 0.02, 1.04 2.5 r b b b P 7 7

hypotaurine and sulﬁde gas in acid; Pruski et al., a a 2000b). Supernatants were dried overnight in a vacuum centrifuge and then resuspended in 600–1,500 mL of puriﬁed water. All samples were passed through with C-18 cartridges to remove 4.94.25.1 4.2 2.2 5.36 2.6 0 0 0

lipids (Varian, Inc., Palo Alto, CA) and 0.45 micron 7 7 7 7 7 ﬁlters (Millipore, Inc., Billerica, MA) as described a a a a a by Wolff et al. (’89). Samples were analyzed for amino acids, sugars, and methylamines using high performance liquid chromatography as previously described (Wolff et al., ’89; Yin et al., 2000). 4.56.72.0 18.2 5.4 20.8 1.94 16.5 20.9 14.7 7 7 7 7 7 c a a a Thiotaurine standard was synthesized according b 4.12 14.8 to Cavallini et al. (’63). 37.6 Statistical analysis 8.28.8 19.0 15.3 b b b 7 7

Data are presented as means7SD. Statistical a a signiﬁcance was determined using t-tests or ANOVA with Student–Newmann–Keuls post-tests (Instat, GraphPad Software, Inc., La Jolla, CA).

Arcsin transformations were used for statistics of 16.917.6 43.0 21.0 35.5 16.119 0 0 0 7 7 7 7 7 a a a a ratios. b

RESULTS Statistically different from others in the same column with a different letter (

In situ temperatures a,b,c,d,e 1.212.36 119 4.17 109 0.95 99.7 0.36 96.3 207 7 7 7 7 7 a a a a The Alvin temperature probe revealed fluctuat- b ing temperatures within most of the worm aggregations, with P. sulfincola (sulfide worms) experiencing the widest range. Based on temperatures, specimens were designated as follows 2.331.49 6.19 0.59 7.78 1.21 9.78 0.65 6.38 0.76

(Table 1): P. sulfincola, hot group with tempera- 7 7 7 7 7 c a a a tures measured at 42–681C; P. sulfincola, warm b group at 21–351C; P. palmiformis (palm worms), 0.74 cool at 14–181C; P. pandorae, cool at 12–141C; and N. venticola, cold at 41C. For analysis of branchiae, another group of sulfide worms was collected from 1.150.430.27 8.40 0.29 8.31 2.78 7.52 e 7 7 7 7 c a a warm site at 18–311C. b d

Body mass Thiotaurine Hypotaurine Taurine Glycine Sarcosine Betaine Other AAs Scyllo-inositol TABLE 1. Amino acid, methylamine, and polyol contents of hydrothermal vent polychaetes

Specimens of P. palmiformis weighed the most n at 0.4070.17 g. P. sulﬁncola were intermediate at 7 0.24 0.07 g and P. pandorae averaged one-tenth C) 5 0.37 SD. AAs, amino acids, primarily alanine, proline, and glutaminea. C) 6 4.36 1 1

7 7 C) 9 1.85 the size of palm worms at 0.041 0.002 g. C) 4 6.59 1 1

Glycine and other nonsulfur amino acids C) 3 0 1 (Table 1) , cold (14–18 , hot (42–68 , warm (21–35 , cold (12–14

Of the organic solutes, the most abundant , cold (4 osmolyte in paralvinellids was glycine, ranging from 96 to 119 mmol/kg wet mass (with no significant difference in concentrations between ANOVA, SNK post-test). P. pandorae N. venticola Values are mmol/kg wet mass Species, temperature P. sulfincola species). This was true for N. venticola as well, P. sulfincola P. palmiformis

J. Exp. Zool. THIOTAURINE IN PARALVINELLIDS 443 which had glycine concentrations of 207 mmol/kg (Table 1). The total of other amino acids (primar- 2.21 0.50 7 ily alanine, glutamine, and proline) ranged from 7 15 to 21 mmol/kg wet mass and did not differ among the species. P. palmiformis also contained substantial amounts (estimated 30–50 mmol/kg wet mass) of a compound that did not match 7.2 1.93 any amino acid, methylamine, or carbohydrate 2.4 2.00 7 standard. 7

Sarcosine, betaine, and scyllo-inositol C. (Table 1) 1 3.6 29.9 3.9 32.2 7 Sarcosine at 36–43 mmol/kg wet mass and scyllo- 7 inositol at 4–5 mmol/kg wet mass were found only in the two P. sulfincola groups (Table 1). Betaine contents were the same in two species and different in the other two species, ranging 11.4 15.0 from 38 to 4 mmol/kg wet mass in the order 3.6 17.0 7 7 P. pandorae4P. sulfincola hot 5 P. sulfincola y warm 5 P. palmiformis4N. venticola.

Thiotaurine, hypotaurine, and taurine 10.1 61.0 9.9 34.1 7 7 Thiotaurine contents were significantly differ- y ent among all species and the two P. sulfincola groups, ranging from 6.6 to 0 mmol/kg wet mass in the order P. sulfincola hot4P. sulfincola warm4P. pandorae4P. palmiformis4N. venticola 3.5 54.4 (Table 1). Hypotaurine contents were the same in 1.90 115 7 two species and higher than in the other two 7 species, ranging from 8.8 to 0.7 mmol/kg wet mass in the order P. sulfincola hot 5 P. sulfincola warm 5 P. pandorae4P. palmiformis4N. venticola (Table 1). Taurine contents were low and 1.52 12.5 1.9 8.78 7 statistically identical in all species, except for 7 y N. venticola, which had almost none. -test). t Branchiae vs. main body of P. sulfincola 0.02, r The branchiae and main body of sulfide worms P 0.46 11.3 0.74 18.1 7 had statistically identical contents of thiotaurine, 7 taurine, betaine, other amino acids, and scyllo- Thiotaurine Hypotaurine Taurine Glycine Sarcosine Betaine Other Aas Scyllo-Inositol inositol (Table 2). This batch of sulfide worms SD. AAs, amino acids, primarily alanine, proline, and glutamine. These specimens were from a warm site, 18–31 7 were collected from a warm site (18–311C), and TABLE 2. Amino acid, methylamine, and polyol contents of sulfide worm (P. sulfincola) main body vs. branchiae

had thiotaurine contents similar to or slightly n lower than those of the other warm group (Table 1). However, the branchiae had much higher contents of hypotaurine and sarcosine and much lower contents of glycine. A similar analysis of P. palmiformis found no differences

between branchiae and main body (data not body section Statistically different than in main body ( Values are mmol/kgy wet mass P. sulﬁncola Main body 3 3.65 shown). Branchiae 3 3.08

J. Exp. Zool. 444 P.H. YANCEY ET AL.

Hypotaurine and thiotaurine totals and between the two P. sulfincola groups, ranging exposure indicator ratios in fresh animals from 15 to 0.7 mmol/kg wet mass in the order P. sulfincola hot 5 P. sulfincola warm4 The sum of hypotaurine and thiotaurine P. pandorae4P. palmiformis4N. venticola differed significantly among the species, but not (Table 3; Fig. 1). The exposure indicator ratio [thiotaurine:(thiotaurine1hypotaurine)] differed TABLE 3. The sum of hypotaurine, and thiotaurine, and the between the two P. sulfincola groups and was exposure indicator ratio (see text) in hydrothermal-vent higher than in the other species, ranging from polychaetes 0.4 to 0 in the order P. sulfincola hot4P. sulfincola warm4P. pandorae 5 P. palmiformis4N. venticola Species, temperature Total Th1Hy Th/[Th1Hy] (Table 3; Fig. 1). In the sulfide worm’s branchiae, P. sulfincola, hot (42–681C) 15.0a71.0 0.435a70.057 the sum of the two solutes was significantly higher P. sulfincola, warm (21–351C) 12.7a71.7 0.349b70.038 than in the main body (18 vs. 11 mmol/kg wet b c P. palmiformis, cool (14–181C) 3.15 70.46 0.123 70.090 mass), though the exposure indicator ratio was 1 c7 c7 P. pandorae, cool (12–14 C) 9.36 0.92 0.200 0.036 lower (0.14 vs. 0.24; Table 3; Fig. 1). N. venticola, cold (41C) 0.74d70.65 0d P. sulfincola, body section Main body 14.972.7 0.24470.022 Hypotaurine and thiotaurine and Branchiae 21.2y71.2 0.146y70.037 exposure indicator ratios in experimental animals Values for Total are mmol/kg wet mass7SD. Th, thiotaurine; Hy, hypotaurine. In the palm worms kept in laboratory pressure a,b,c,dStatistically different from other groups in the top section of the table with a different letter (Pr0.02, ANOVA, SNK post-test). chambers, those held 235 mM sulfide had much yStatistically different than in main body (Po0.01, t-test). higher levels of thiotaurine (0.97 vs. 0.16 mmol/kg wet mass; Po0.001) and lower levels of hypotaurine (2 vs. 3 mmol/kg wet mass; P 5 0.02) compared with those held with no sulfide (Table 4; Fig. 1). The exposure indicator ratio was considerably higher in the sulfide-treated group (0.32 vs. 0.05; Po0.001). The total of the two solutes, however, was the same in the two groups at about 3 mmol/kg wet mass.

DISCUSSION

This study on closely related polychaetes from different vent habitats greatly extends our previous finding (Rosenberg et al., 2006) that some vent animals without thiotrophic endosymbionts probably use hypotaurine to detoxify sulfide by Fig. 1. Total of hypotaurine and thiotaurine in poly- conversion to thiotaurine. Presumably, sulfide chaetes analyzed in this study, from Tables 3 and 4. P. sulf., would be removed from thiotaurine (regenerating Paralvinella sulfincola; P. palm., P. palmiformis; P. pand., hypotaurine) and released to the environment P. pandorae; N. vent., Nicomache venticola; Branch., branchiae; 1S, S: kept with sulfide or without sulfide, respec- during periods of low sulfide exposure, or the Ã tively, in laboratory chambers. Significantly higher animals may expend energy in the active transport thiotaurine than in other group(s) of the same species. (elimination) of the compound. Although in situ

TABLE 4. The sum of hypotaurine and thiotaurine, and the exposure indicator ratio (see text) in palm worms kept in shipboard pressure chambers with and without sulﬁde

Species, condition Thiotaurine Hypotaurine Total Th1Hy Th/[Th1Hy]

P. palmiformis, Chamber, no S 0.1670.12 3.0070.54 3.1670.65 0.04870.033 P. palmiformis, Chamber, 235 mM S 0.97y70.31 2.08y70.32 3.0470.40 0.317y70.091

Values (n 5 4 each) are mmol/kg wet mass7SD (except for the ratios). Th, thiotaurine; Hy, hypotaurine. yStatistically different from chamber, no S (Pr0.02, t-test).

J. Exp. Zool. THIOTAURINE IN PARALVINELLIDS 445 sulfide data were not available in this study, two were more thermostable in P. pandorae than previous work has shown that sulfide levels in the palm worms (though less so than in sulfide correlate with vent fluid temperatures (Sarrazin worms). Finally, P. pandorae tend to colonize et al., ’99; work done on Juan de Fuca sites with open vent sites earlier than P. palmiformis these paralvinellids). The detoxification hypoth- (Levesque et al., 2003), which could subject them esis is supported by the higher contents of to warmer fluids. thiotaurine (Tables 1 and 4) and higher exposure Hypotaurine may play another protective role in indicator ratios (Tables 3 and 4) in (1) P. sulfincola vent habitats. Oxygen radicals may be generated compared with its congeners from cooler sites, (2) during the spontaneous oxidation of sulfide all paralvinellids compared with the maldanid (Tapley et al., ’99), and hypotaurine can scavenge worm from the coldest site, (3) the P. sulfincola some oxygen radicals, becoming taurine (Aruoma from hot waters compared with those from warm et al., ’88). This might explain the pattern of much waters, and (4) the P. palmiformis in laboratory higher taurine contents in the paralvinellids chambers exposed to high sulfide compared with compared with the maldanid. those kept with no sulfide. The occurrence of sarcosine only in the hot- The total of hypotaurine plus thiotaurine adapted species (P. sulfincola) (Table 1) and the (Tables 2 and 4; Fig. 1) is consistent with our considerably higher contents of sarcosine and hypothesis that this is relatively consistent within hypotaurine in branchiae compared with main a species and indicates the maximum likely sulfide body of these worms (Table 2) are intriguing. One exposure for a species (Brand et al., 2007). The or both of these differences could be due to sulfide worms had a higher total than the other specialized metabolic reactions in sulfide worms species, but did not differ among those from the (especially in the branchiae) that are not related to hot and the warm sites (including Main Body; environmental stresses. However, one or both Fig. 1). Also, the palm worms from different might also be adaptations to exposure to hotter, sulfide exposures in laboratory chambers had the more sulfide-laden waters. In particular, bran- same total (Fig. 1; Table 4), since hypotaurine chiae probably have a greater exposure than the apparently declined while thiotaurine apparently main body because they have a much higher increased during sulfide exposure. surface area:volume ratio and also frequently The only pattern that might not fit the protrude from the tube. If their exposure is higher, sulfide-exposure hypothesis is that seen for they might need more hypotaurine to react with P. palmiformis compared with P. pandorae. Both any surges in sulfide diffusing into them (although are found in similar thermal (and presumably we did not find a difference in thiotaurine in sulfide) habitats (Tsurumi and Tunnicliffe, 2001), this particular group). As evidence of a higher yet the latter species had more thiotaurine and environmental exposure for branchiae, Marie hypotaurine than the former (Table 1 and the sum et al. (2006) found that, in oxygenated water in in Table 3). However, it is also possible that the a laboratory pressure chamber, the branchiae of two species differ in their strategies for sulfide P. grasslei exhibited greater oxidative damage tolerance. P. pandorae in this study were much than the body wall. In addition, the aerobic smaller, averaging 10% of the body mass of enzyme citrate synthase in the branchiae is more P. palmiformis (possibly a result of competition thermostable than the same enzyme from the between the two species; Tsurumi and Tunnicliffe, body wall (Rinke and Lee, 2009). 2001; Levesque et al., 2003). Thus, P. pandorae Scyllo-inositol may also relate to habitat condi- have a much higher surface-area:volume ratio. As tions, since it was only found in the sulfide worms a consequence, they may find it more difficult to (Tables 1 and 2). However, it was relatively low in exclude sulfide diffusion into their bodies and may content, and did not differ between branchiae and therefore need more hypotaurine. The palm main body. Also, it has been reported at moderate worms also have external bacteria in their mucus levels in other deep-sea (but not shallow) marine sheaths that might oxidize sulfide (Juniper et al., invertebrates from cold sites (Yancey et al., 2002, ’86; Juniper and Martineu, ’95), although 2004). This polyol, but not other inositols, has P. pandorae also have a mucus sheath that been found to prevent b-amyloid formation asso- presumably provides some protection. Transient ciated with Alzheimer’s disease (McLaurin et al., temperatures (and thus sulfide exposures) could 2006). Thus, it may affect protein structure in also be higher for P. pandorae: of three enzymes useful ways. However, the role of this solute in examined in paralvinelleds by Jollivet et al. (’95), deep-sea animals remains unknown.

J. Exp. Zool. 446 P.H. YANCEY ET AL.

Sarcosine could also be hypothesized to relate to Cavallini D, Mondovi B, DeMarco C. 1963. Hypotaurine and greater exposure factors in sulfide worms (again, thiotaurine. Biochem Prep 10:72–75. especially in branchiae). A possible factor is Childress JJ, Fisher CR, Favuzzi JA, Arp AJ, Oros DR. 1993. The role of a zinc-based, serum-borne sulfide-binding temperature, since there is no known relationship component in the uptake and transport of dissolved between sarcosine and sulfide metabolism. Sarco- sulfide by the chemoautotrophic symbiont-containing clam sine has been used in a number of biochemical Calyptogena elongata. J Exp Biol 179:131–158. studies as a thermostabilizer for various prokar- Fiess JC, Hom JR, Hudson HA, Kato C, Yancey PH. 2002. yotic and eukaryotic proteins. In one study on Phosphodiester amine, taurine and derivatives, and other bovine RNaseA, it was a slightly better thermo- osmolytes in vesicomyid bivalves: correlations with depth and symbiont metabolism. 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