Coral Growing on North Sea Oil Rigs These Installations Are Home to Thriving Colonies of an Endangered Cold-Water Coral

brief communications Coral growing on North Sea oil rigs These installations are home to thriving colonies of an endangered cold-water coral.

his summer the coral Lophelia pertusa The occurrence of the coral raises ques- was found growing on oil platforms in tions about how to deal with this species, Tthe North Sea and on the Brent Spar which is listed under the Convention on oil-storage buoy during its decommission- International Trade in Endangered Species ing. The findings indicate that Lophelia has a (CITES), when platforms are decommis- wider distribution and a more rapid rate of sioned. At a meeting in Sintra in 1998 of growth than previously believed. The dis- countries belonging to the Oslo–Paris covery also has implications for the debate (Ospar) convention on protecting the over oil exploration in the Atlantic Ocean marine environment, Ospar decision 98/3 and the perceived benefits of onshore dis- indicated that the ‘footings’ of large plat- mantling of deep-water platforms. forms (jacket weight of more than 10,000 When Brent Spar was raised out of the tonnes) might be left in place. Such an sea in stages for dismantling (Fig. 1), option would preserve existing colonies colonies of Lophelia of up to 20 cm linear Figure 1 The top of Brent Spar being removed from the Heerema and might allow Lophelia to spread in the growth were found on the sides and bot- heavy-lift barge during its dismantling in Yrkefjorden, Norway. North Sea. tom, which had been at depths of 60–109 Niall Bell, Jan Smith m. Lophelia has recently been filmed at European Commission’s Species Habitat Cordah Environmental Management Consultants, depths of 100–129 m on two platforms in Directive (92/43/EEC). Kettock Lodge, the North Sea about 140 km east of the It has been suggested that Lophelia is Aberdeen Science and Technology Park, Shetland Islands. These domed colonies susceptible to disturbance from increased Aberdeen AB22 8GU, UK were up to 54 cm long, and were found on sedimentation and from the toxicological e-mail: [email protected] 9,10 installations that have been producing oil effects of drilling discharges . However, 1. Mikkelsen, N., Erlenkauser, H., Killingley, J. S. & Berger, W. H. since the late 1970s. Annual average linear the apparently healthy colonies of Lophelia Boreas 5, 163–171 (1982). growth rates of 25 mm have previously on platforms in the North Sea have been 2. Mortensen, P. B. & Rapp, H. T. Sarsia 83, 433–446 (1998). 1 3. Wilson, J. B. J. Mar. Biol. Assoc. UK 59, 149–164 (1979). been suggested for this coral , but more exposed to agreed quality standards of 4. Zibrowius, H. Mem. Inst. Oceanogr. Monaco 11, 126–130 recent estimates of 5.5–6.0 mm have been operational discharges, such as oily water, (1980). proposed2. The size of the colonies on these drilling muds and chemicals, and contami- 5. Strømgren, T. Det Kon. Nor. Vid. Sel. Skr. 6, 1–9 (1971). 6. Hovland, M., Mortensen, P. B., Brattegard, T., Strass, P. & 20-year-old platforms means that the aver- nants that may leak from the cuttings piles Rokoengen, K. Palaios 13, 189–200 (1998). age growth rate must have been at least (E. Breuer, personal communication) that 7. Pearce, F. New Sci. 164(2211), 16 (1999). 26 mm per year. If the colonies found on lie 10–15 m below some of the colonies. 8. Turrell, W. R. & Slesser, G. Marine Laboratory Aberdeen Report No. 1/99 (1999). Brent Spar had grown at a similar rate, This indicates that it is not obviously affec- 9. Rogers, A. New Sci. 154(2087), 53 (1997). then Lophelia must have settled on the ted by discharges from oil platforms. 10.Edwards, R. New Sci. 154(2085), 10 (1997). structure when it was in the Brent Field in the North Sea. Lophelia is widespread in the Atlantic at depths of 150–1,500 m, but is particularly Evolution by virtue of its homology to the doublesex common on the upper continental slope at gene from Drosophila and mab-3 from 200–600 m (refs 3,4). It has also been found Conservation of a Caenorhabditis elegans, which encode puta- at a depth of 50 m in Scandanavian fjords5, sex-determining gene tive transcription factors with a conserved off the coast of Norway6, and on the Beryl DM domain that is thought to bind DNA4. platform7. Its occurrence on oil installations Vertebrates exhibit a surprising array of sex- DMRT1 maps to the minimal region of the is the first recorded instance of live colonies determining mechanisms, including X- and small arm of human chromosome 9, which of this species in the North Sea. It has been Y-chromosome heterogametes in male is deleted in patients with XY male-to- shown2 that Lophelia occurs at tempera- mammals, Z- and W-chromosome hetero- female sex reversal4–6. A related gene, tures of 4–8 °C. At the original Brent Spar gametes in female birds, and a temperature- DMRT2, has also been mapped within this site (61° 03፱ N, 01° 40፱ E), the water tem- dependent mechanism in many reptiles1. region7. Human genetic data indicate that perature at the depth where the shallowest The Y-chromosome-linked SRY gene initi- DMRT1, alone or together with DMRT2, Lophelia were found can exceed 10 °C (ref. ates male development in mammals2,3, but may operate in a dose-dependent fashion 8); in the area of the two production plat- other vertebrates lack SRY and the genes within the male (testis)-determining path- forms, the maximum water temperature controlling sex determination are largely way. The chicken DMRT1 homologue is at depths of 100–129 m varies between 7.6 unknown. Here we show that a gene impli- located on the Z sex chromosome8. and 9.7 °C. cated in human testis differentiation, We used regions mainly or totally out- The presence of Lophelia on oil-produc- DMRT1, has a gonad-specific and sexually side the DM domain of the published ing platforms has implications for the dimorphic expression profile during human and chicken DMRT1 complemen- licensing of oil exploration. Greenpeace embryogenesis in mammals, birds and a tary DNA sequences to design primers for contends that the British Department of reptile (Alligator mississippiensis). Given the amplification, by the polymerase chain Trade and Industry failed to consider different sex-determining switches in these reaction with reverse transcription (RT- whether licensed oil-producing areas that three groups, this gene must represent an PCR), of the mouse, chicken and alligator may contain Lophelia should be designated ancient, conserved component of the ver- homologues. Sequence analysis confirmed as potential special areas of conservation tebrate sex-determining pathway. the identity of the PCR products (GenBank and, as such, is in contravention of the The human DMRT1 gene was isolated accession numbers for mouse and alligator

DMRT1 are AF192561 and AF192560, Bioenergetics respectively). Specific mouse and chicken PCR fragments were used as probes for Proton pumping by whole-mount in situ hybridization analysis of DMRT1 expression during urogenital cytochrome c oxidase development. These fragments detected Proton pumping by cytochrome c oxidase1 single bands on Southern blots of genomic was thought to be restricted to the oxidative DNA, excluding the possibility of cross- part of its catalytic cycle2, but this has been hybridization with DMRT2. For the alli- questioned3. New results4 were interpreted gator, specific primers were used for as an indication that two protons are RT-PCR analysis of whole urogenital tissues pumped during the oxidative phase, and or gonads from embryos incubated at 30 °C two during a subsequent reductive phase, (female-producing), 33 °C (male-producing) and that this latter pumping is energetically and 34.5 °C (female-producing). coupled to the oxidative phase. Here I re- Expression of DMRT1 was sexually evaluate these results and draw an alterna- dimorphic in all three species, and was tive conclusion. stronger in male gonads than in female ones Figure 3 of ref. 4 shows that only about (Fig. 1). In mouse embryos at 11.5 days 44% of the electric-field generation (caused post-coitum (d.p.c.), when the testis-deter- by proton pumping, electron transfer and mining Sry gene is activated in males and proton uptake) occurs during oxidation of just before testicular differentiation, the fully reduced enzyme. However, the DMRT1 was already being expressed in the claim4 that four protons (two during oxida- gonads of both sexes. By 14.5 d.p.c., tion and two during re-reduction) are DMRT1 was expressed more strongly in dif- pumped per catalytic cycle is inconsistent ferentiating testes than in ovaries (Fig. 1a). with the other experiment reported at the Taken together with human gene results Figure 1 Sexually dimorphic expression of DMRT1 in embryonic same time (Fig. 2 of ref. 4). implicating DMRT1 in XY sex reversal, gonads. a, b, Whole-mount in situ hybridization of embryonic There are four indications that a molar these observations indicate that DMRT1 is mouse (a) and chicken (b) urogenital systems during develop- excess of oxygen over active, correctly ori- necessary for testis differentiation, presum- ment, showing stronger expression (purple staining) in male than entated cytochrome c oxidase has been used ably lying downstream of the master male- in female gonads (scale bar, 0.5 mm). c, RT-PCR analysis of (www.biophys.mpg.de/michel/public/cox- determinant SRY. embryonic alligator urogenital systems (developmental stages disc). For example, complete oxidation In chickens and other birds, the mecha- 20–23) or isolated gonads (stages 24–27) showed more upregu- leads to pumping of only about 1.2 protons nism of sex determination is unknown. Sex lation of DMRT1 expression at the male-determining temperature per enzyme. Re-reduction would then lead may be controlled by a dominant ovary- (33 °C) than at either female-producing temperature. ȋ-Actin gel- to pumping of 2.4 protons per turnover, determining gene carried on the W chromo- loading controls are also shown. assuming that the same number of protons some, or it may depend on Z-chromosome were pumped during re-reduction as dur- dosage (two doses for male development The sexually dimorphic pattern of ing oxidation. Four protons per enzyme can and one for female). DMRT1 is Z-linked, DMRT1 expression in mouse, chicken and then be pumped, if a molar excess of oxygen and its expression profile implicates it in alligator embryos is consistent with a con- is present, leading partially to a second avian sex determination. As in the mouse, served role in vertebrate sex determination. turnover. chicken embryos showed gonad-specific We suggest that high DMRT1 expression is Assuming an optical pathway of roughly expression of DMRT1, with much stronger necessary for testicular differentiation, 1 cm (ref. 5) and using published spectra6, expression in developing male than in whereas lower expression is compatible we calculate that the concentration of oxi- female gonads (Fig. 1b). This difference was with ovarian differentiation. Our observa- dized cytochrome c oxidase (Fig. 2a, bot- evident before and during the time of tions support the idea that core compo- tom, of ref. 4) is roughly 0.70 ȖM, rather Ȗ Ȗ gonadal sex differentiation (developmental nents of the vertebrate sex-determining than 1.25 M. The addition of 1.25 M O2 stages 25–30; days 4.5–6.5 of incubation). pathway have been conserved during evolu- was superstoichiometric. Another possibility In birds, the Z chromosome does not tion. As Drosophila and C. elegans also have is that the optical path length might have seem to undergo inactivation, as the X related genes involved in sexual develop- been shorter (0.56 cm), but calculations for chromosome does in mammals. Although ment4, the DM-domain genes are the first to Fig. 2 of ref. 5 indicate a longer path length the sexually dimorphic expression of show sexually dimorphic expression across (0.73 cm) for the device used. DMRT1 in chicken embryos may simply both vertebrate and invertebrate phyla. The observation that four protons have reflect this lack of dosage compensation, Craig A. Smith, Peter J. McClive, already been pumped when only 40% of the expression in the male is clearly more than Patrick S. Western, Kirsty J. Reed, haem groups have been re-reduced was twice that seen in females and suggests Andrew H. Sinclair interpreted4 as an indication that input of strong upregulation in ZZ embryos. This Department of Paediatrics and Centre for Hormone only one electron may be sufficient to elicit observation, together with the fact that Research, University of Melbourne, pumping of two protons, and was presented expression is confined to the gonads, points Royal Children’s Hospital, Melbourne, as support for the existence of an energy- to an active role for DMRT1 in avian Victoria 3052, Australia rich state, O~. However, this result was gonadal development. In the alligator, 1. Bull, J. J. Evolution of Sex-determining Mechanisms (Benjamin caused by the excess of oxygen, so there is DMRT1 expression was initially detectable Cummings, Menlo Park, California, 1983). no evidence that four protons are pumped by RT-PCR in the urogenital systems of 2. Sinclair, A. H. et al. Nature 346, 240–244 (1990). per turnover when starting from the fully 3. Koopman, P., Gubbay, J., Vivian, N., Goodfellow, P. N. & embryos incubated at both male- and Lovell-Badge, R. Nature 351, 117–121 (1991). reduced enzyme, or for the existence of the female-producing temperatures (Fig. 1c). 4. Raymond, C. S. et al. Nature 391, 691–695 (1998). O~ state. The fully reduced state is not part However, gonadal expression subsequently 5. Fleijter, W. L. et al. Am. J. Hum. Genet. 63, 794–802 (1998). of the natural reaction cycle of cytochrome 6. Guioli, S. et al. Am. J. Hum. Genet. 63, 905–908 (1998). became higher in developing male embryos 7. Raymond, C. et al. Hum. Mol. Genet. 8, 989–996 (1999). c oxidase. This artificial cycle tells us little than in female embryos (Fig. 1c). 8. Nanda, I. et al. Nature Genet. 21, 258–259 (1999). about proton pumping in the natural cycle,